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Characterization of portohepatic glucosensors in sympathoadrenal counterregulation

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Characterization of portohepatic glucosensors in sympathoadrenal counterregulation

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CHARACTERIZATION OF PORTOHEPATIC GLUCOSENSORS
IN SYMPATHOADRENAL COUNTERREGULATION
by
Andrea Lynn Hevener
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Exercise Science)
May 1998
Copyright 1998 Andrea Lynn Hevener
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
Andrea Lynn Hevener
under the direction of h.$F....... Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School, in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Dean of Graduate Dean of Graduate Studies
Date
DISSERTATION COMMITTEE
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CHARACTERIZATION OF PORTOHEPATIC GLUCOSENSORS
IN SYMPATHOADRENAL COUNTERREGULATION
Traditionally ascribed to the central nervous system, glucosensors involved in
sympathoadrenal counterregulation against hypoglycemia have now been constrained
to the portohepatic region. The purpose of the current investigations was to further
localize glucosensors to either the liver or hepatic vasculature, and ascertain neural
mediation of this sensing mechanism. Animals were cannulated in the portal vein either
adjacent to (POR-ADJ) or distal from (POR-DIST) the liver. Animals participated in
one of three hyperinsulinemic-hypoglycemic clamp protocols distinguished by the site
of glucose infusion. Whole body hypoglycemia was induced with either liver and portal
vein glucose normalization (POR-DIST), or liver glucose normalization alone (POR-
ADJ). Despite cerebral hypoglycemia, a significant suppression in the sympathoadrenal
response was observed during liver and portal vein glucose normalization. In contrast,
liver glucose normalization alone, resulted in a full sympathoadrenal response, not
significantly different from whole body hypoglycemia. These findings are consistent
with localization of glucosensors to the portal vein, not the liver.
As a portal glucosensor locus attaches significance to pre-hepatic glycemia, we
sought to determine the presence of glucosensors in the hepatic artery. Animals were
cannulated in the hepatic artery allowing for hepatic artery and liver glucose
normalization during systemic hypoglycemia. Unlike the previous findings for the
portal vein, normalization of hepatic artery glycemia had no impact on the
sympathoadrenal response to systemic hypoglycemia. These findings indicate an
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apparent lack of glucosensors in the hepatic artery, and confirm findings regarding a
lack of glucose sensing by the liver.
The importance of portal vein afferents in hypoglycemic detection and
sympathoadrenal counterregulation was assessed following phenol induced portal
denervation. The catecholamine response to whole body hypoglycemia was
significantly blunted in portally denervated animals (PDN). In contrast to findings in
SHAM operated animals, in PDN, portal glucose normalization had no impact on the
sympathoadrenal response to systemic hypoglycemia when compared to whole body
hypoglycemia. That is, no suppression in the sympathoadrenal response to systemic
hypoglycemia was observed during portal vein glucose normalization for PDN. Results
indicate that portal vein afferents are critical for hypoglycemic detection and normal
sympathoadrenal counterregulation.
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TABLE OF CONTENTS
I. INTRODUCTION 1
Hypotheses 6
Significance 10
II. BACKGROUND
A. Glucose Homeostasis 12
1. Insulin Dependent Diabetes Mellitus 13
2. Hypoglycemia 15
B. Glucose Counterregulation 16
1. Efferent Limb 16
2. Afferent Limb 23
III. EXPERIMENTS
A. Portal Vein Glucosensor Locus 37
1. Research Design and Methods 37
2. Results 42
3. Discussion 48
B. Portal Vein Glucosensor Locus Confirmation 52
1. Research Design and Methods 52
2. Results 58
3. Discussion 63
C. Portal Vein Denervation 66
1. Research Design and Methods 66
2. Results 71
3. Discussion 78
IV. SUMMARY AND CONCLUSIONS 83
V. REFERENCES 89
ii
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LIST OF FIGURES
Figure 1. Schematic representation of autonomic nervous
connections between the hypothalamus and the primary
counterregulatory organs
Figure 2. Schematic diagram of the putative Porto-Sympathoadrenal
Neural Reflex
Figure 3. Normal glucose counterregulation in humans
Figure 4. Glucose counterregulatory effect of epinephrine on liver,
pancreas, muscle, and adipose
Figure 5. Schematic representation of the brain clamp experiment
and graphic depiction of the hormonal response for the three
experimental conditions during steady state
Figure 6. Values are expressed as means ± SE for arterial glucose,
hepatic glucose, epinephrine, and norepinephrine
concentrations at basal and during experimental
sampling periods
Figure 7. Schematic diagram of the three experimental conditions
distinguished by the site of glucose infusion
Figure 8. Glucose infusion rates expressed as means + SEM for the
three glucose infusion conditions during the
hyperinsulinemic-hypoglycemic clamp
Figure 9. Values are expressed as means + SEM for arterial glucose
concentration (A), estimated hepatic glucose concentration (B),
and estimated portal vein glucose concentration (C) at basal
and during the hyperinsulinemic-hypoglycemic clamp
Figure 10. Epinephrine (A) and norepinephrine (B) concentrations
at basal and during sustained hypoglycemia, expressed
as mean values + SEM
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Figure 11. Schematic representation of the 55
hyperinsulinemic-hypoglycemic clamp protocol
Figure 12. Schematic representation of the two glucose infusion 56
conditions
Figure 13. Glucose infusion rate expressed as mean + SE for the 60
two infusion conditions during hyperinsulinemic-hypoglycemic
clamp
Figure 14. Values are expressed as means + SE for arterial (A), 61
estimated hepatic glycemia (B), estimated portal vein
glycemia (C), and estimated hepatic artery glycemia (D)
as a function of time
Figure 15. Average values (means + SE) for epinephrine (A) and 62
norepinephrine (B) concentration at basal and during
progressive hypoglycemia
Figure 16. Ventral view of the hepatic neural and vascular architecture 64
Figure 17. Schematic diagram of the four experimental conditions 70
distinguished by the site of glucose infusion and the surgical
manipulation
Figure 18. Data are expressed as means + SE for glucose infusion rate 72
(GINF) during the hyperinsulinemic-hypoglycemic clamp
Figure 19. Data are expressed as means + SE for arterial glucose 73
concentration (A), and estimated portal vein glucose
concentration (B) at basal and during the
hyperinsulinemic-hypoglycemic clamp
Figure 20. Epinephrine (A) and norepinephrine (B) concentrations 74
at basal and during sustained hypoglycemia for SHAM
animals, expressed as mean values + SE
Figure 21. Epinephrine (A) and norepinephrine (B) concentrations 75
at basal and during sustained hypoglycemia for peripheral
glucose infusion experiments (PDN and SHAM), expressed
as mean values + SE
iv
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Figure 22. Epinephrine (A) and norepinephrine (B) concentrations
expressed as means + SE for basal and the final two
sampling periods (90 & 105 min) of sustained hypoglycemia
Figure 23. Values are expressed as means + SE for adrenal epinephrine
(A) and norepinephrine (B) for SHAM operated (open bars)
and hepatic denervated (HPDN; closed bars) animals at basal
and following 45 minutes of deep hypoglycemia (50 mg/dl)
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I. INTRODUCTION
Glucose homeostasis is the process by which the body, via negative feedback
system, attempts to maintain blood glucose within narrow limits protecting the central
nervous system from potentially harmful excursions in glycemia. Inherent in this
negative feedback system is an efferent limb, responsible for directing neural-hormonal
output re-establishing glucose homeostasis, and an afferent or sensory limb responsible
for glycemic detection. The efferent portion of counterregulation is well characterized
in the literature. Following a meal, elevations in glycemia are challenged by enhanced
insulin secretion. Whereas insulin is the dominant glucose lowering hormone, during
decrements in glycemia, the body relies on a host of redundant neural an hormonal
counterregulatory factors including: catecholamines, glucagon, cortisol, and growth
hormone, stimulated in a hierarchical fashion. The prevention and correction of
hypoglycemia is imperative for glucose is the predominant fuel source of CNS
metabolism. As the brain is unable to synthesize, store, or quickly elevate glucose
extraction from the circulation, maintenance of a constant cerebral glucose supply is
achieved via neural stimulation of the liver fortified by counterregulatory hormone
secretion synergistically employed for rapid augmentation of hepatic glucose
production.
While the efferent limb of counterregulation has been studied since the turn of the
century, by comparison relatively little is known about the afferent or sensory limb,
wherein blood glucose is detected. Since the classical observations by Bernard, (1849),
1
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glycemic detection and hormonal counterregulation have been believed the exclusive
domain of the CNS. [17], Evidence supporting CNS control of efferent output has
emerged primarily from studies characterizing changes in peripheral metabolism
induced by electrical or chemical stimulation, and or lesioning of various aspects within
the hypothalamus [25, 160, 162, 163]. Subsequent investigations employing
microdialysis and microinjection of glucose, 2-deoxyglucose, and gold thioglucose,
suggest that the ventral medial and lateral portions of the hypothalamus possess
important glucose sensitive neurons [24, 26], Manipulations o f hypothalamic glycemia
were shown to modulate neural output to the liver, pancreas, and adrenals, reinforcing
the predominant view of CNS exclusivity in glycemic detection and hormonal
counterregulation. That the CNS has an absolute requirement for glucose, it is not
surprising that the nervous system plays a role in glucose homeostasis. The prevailing
belief is that the hypothalamus, replete with glucosensors and autonomic neural
connections of glucoregulatory organs, form the architecture for which moment to
moment fluctuations in glycemia can be rectified.
Alternatively, peripheral glucosensors, particularly those in the portohepatic region,
have been observed to profoundly affect hormonal counterregulation and may
predominate over those in the CNS during progressive hypoglycemia. In vivo studies
from our laboratory have shown that portohepatic hypoglycemia is essential to
engender a full sympathoadrenal response. In addition during portal vein glucose
infusion, yielding portohepatic normoglycemia, a significant suppression in
2
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sympathoadrenal counterregulation is observed despite systemic (including cerebral)
hypoglycemia [62, 63]. As only the portal vein and liver glucose concentrations were
elevated, portohepatic glucosensors are strongly implicated in the regulation of
sympathoadrenal counterregulation. In congruence, selective portohepatic
hypoglycemia was shown to override the inhibitory effect of CNS normoglycemia on
sympathetic output. That is, significant elevations in adrenal catecholamine secretion
were observed despite cerebral normoglycemia [175]. Modulation of sympathoadrenal
counterregulation via peripheral glucose sensors, either present in the liver or portal
vein, is strongly suggested by these investigations.
Previous neurophysiologic studies have delineated the requisite neural circuitry for
negative feedback loops originating in the portohepatic region, thus implicating a
neurally mediated portohepatic glucose sensing mechanism. Hepatic vagal afferents
have been shown to be sensitive to intraportal glucose infusion. Specifically in situ,
elevations in portohepatic glucose concentration significantly suppressed vagal afferent
firing rates. Concomitant reductions in splanchnic nerve activity affecting adrenal
catecholamine and pancreatic insulin secretions were also observed [132, 133, 134,
139,160, 164], In addition, suppression of efferent discharges recorded from fibers of
the hepatic branch of the splanchnic nerve resulted in reductions in hepatic glucose
output [137, 139, 160, 161]. Sectioning of the hepatic vagus nerve eliminated changes
brought about by intraportal glucose infusion, indicating maintenance o f glucose
homeostasis via portohepatic glucose sensitive vagal afferents which neurally modulate
3
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counterregulatory efforts by the adrenals, pancreas, and liver. These neural loops have
previously been identified as the hepato-hepatic, hepato-pancreatic, and hepato-adrenal
reflexes [137, 175],
For over a century great efforts have been made to constrain the locus of glycemic
detection in the CNS, however little attention has been focused on the importance of
portohepatic glucosensing. While portohepatic glucosensors have generally been
presumed to reside within the liver, the precise locus has remained obscure. Recently
ascertaining the precise locus for hypoglycemic detection has taken on renewed
interest. Vagal afferents observed emanating from the portal vein and hepatic artery,
provide strong foundation for a hepatic vascular, as opposed to liver, locus.
This dissertation, comprised of three investigations, was undertaken to determine
the specific site o f glycemic detection in the portohepatic region, and discern neural
mediation of this sensing mechanism. To constrain these sensors to either the liver or
the supporting vasculature (i.e. portal vein and or hepatic artery), a local irrigation
approach perfected by our laboratory was employed for all three investigations. The
liver irrigation technique permits the hepatic region to be exposed to varying metabolic
signals as compared to the systemic circulation. That is, the infusion vessel (portal vein
or hepatic artery) and liver are exposed to elevated glucose concentrations, while the
arterial glucose concentration (including the CNS) is clamped at a deep hypoglycemic
level. This approach allows for determination of specific tissues possessing glucose
sensitive afferents involved in modulation of hormonal counterregulation.
4
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CEREBRUM
HYPOTHALAMUS
□ VMH
§ LH
CEREBELLUM
LIVER
CELIAC GANGLION
Glucose
Glucagon
PANCREAS
Insulin
ADRENAL MEDULLA
Epinephrine
Figure 1 . Schematic representation of presumed autonomic nervous connections
between the hypothalamus and the primary couterregulatory organs. The solid line
indicates the VMH-splanchnic nerve pathway and the dotted line illustrates the LH-
vagus pathway. Reproduced from [156],
5
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Portohepatic G lucosensor Locus. Ia vivo experiments were performed to test the
hypothesis that glucosensors involved in the modulation o f sympathoadrenal
counterregulation exist exclusively in the portal vein, not the liver. A variation of the
local glucose irrigation technique was employed, involving cannulation of the portal
vein either several centimeters from, or immediately adjacent to, the liver. Cannula
position allowed for the maintenance of either portal vein and liver normoglycemia, or
liver normoglycemia alone. The magnitude of the sympathoadrenal response to
hypoglycemia was dictated by the glucose concentration in the portal vein and liver, but
not the liver alone. This finding is consistent with the localization of glucosensors to
the portal vein, not the liver.
H epatic A rtery L ocus. As a portal locus for hypoglycemic detection attaches
significance to pre-hepatic glycemia, experiments to determine if sensors reside in the
hepatic artery were conducted. A negative finding for the hepatic artery would also
confirm a lack of glucosensing by the liver. A modified approach of the liver irrigation
technique implementing cannulation of the hepatic artery for glucose infusion, instead
of the portal vein, was employed. Hepatic artery glucose infusion allowed for the
maintenance of hepatic artery and liver normoglycemia, during concomitant portal vein
and systemic hypoglycemia. That is, under this experimental design the portal vein
glucose concentration was decreased in concert with the systemic circulation such that
there were no significant differences in portal vein and systemic glucose concentrations
between the two glucose infusion conditions, hepatic artery vs. peripheral.
6
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If glucosensors were located in the hepatic artery, the sympathoadrenal response to
systemic hypoglycemia would have been significantly suppressed during hepatic artery
glucose infusion, as the hepatic artery glucose concentration was normalized. If
sensors were present exclusively in the portal vein, the sympathoadrenal response to
systemic hypoglycemia during both glucose infusion conditions would have been
identical (i.e. full response), as portal vein hypoglycemia was achieved during both
protocols. That the magnitude of the sympathoadrenal response was unaffected by the
glucose concentration in the hepatic artery and liver, (a full response was observed
during both infusion conditions), suggests a lack of glucosensors in these loci. Thus,
this negative finding during hepatic artery glucose infusion is consistent with the
previous investigation, confirming the lack of hypoglycemic detection by the liver p er
se.
Portal Vein A ffe re n t Innervation. That the site of glycemic detection was
constrained to the portal vein, not the liver, would presuppose a porto-adrenal, as
opposed to a hepato-adrenal, neural reflex. The purpose of the third investigation was
to test the hypothesis that portal vein glucosensors are neurally mediated, and
innervation of these sensors is critical for normal sympathoadrenal counterregulation.
In addition, findings from this investigation would yield information with respect to the
portal vein sensor detection limits (e.g. hypoglycemia, hyperglycemia).
If the null hypothesis were correct, hepatic afferents play no role in the modulation
of the sympathoadrenal counterregulation, the characteristic responses to the portal
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vein and peripheral glucose infusion protocols would have been observed (i.e. a full
blown sympathoadrenal response to whole body hypoglycemia, and a significant
suppression in the sympathoadrenal response to systemic hypoglycemia during
portohepatic normoglycemia).
In contrast, a blunted sympathoadrenal response to whole body hypoglycemia was
shown in portally denervated animals. In addition, portal vein euglycemia had no
impact on sympathoadrenal counterregulation during systemic hypoglycemia. That is,
there was no significant difference in the epinephrine and norepinephrine responses
between the two glucose infusion protocols for portally denervated animals.
These findings are consistent with hypoglycemic detection in the portal vein by a
neurally mediated glucosensor. The latter findings provide evidence for a mechanism
by which termination of hormonal counterregulation could be effected in order to
potentiate glycogen deposition when exogenous glucose becomes available. In
combination these data indicate a neurally mediated glucose sensor responsible for
modulation of sympathoadrenal counterregulation according to the absolute glucose
concentration in the portal vein.
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PORTO-SYMPATHOADRENAL NEURAL REFLEX
EFFERENT \
LIMB
BRAIN
VMH
ADRENAL
^ MEDULLA
AFFERENT LIMB
SYMPATHETIC
NERVE ENDINGS
PORTAL VEIN *
portal slucosensors
F igure 2. Schematic diagram of the putative Porto-Sym pathoadrenal Neural R eflex.
Decrements in glycemia are detected by the portal vein glucosensors, neurally
mediated by terminal afferents of the hepatic vagus nerve. Hepatic vagal afferents fire
at a rate inversely proportional to the glucose concentration within the portal vein.
The afferent signal is sent to the hypothalamus where it is integrated with other
metabolic signals so that an appropriate efferent response can be generated by the
CNS, specifically the ventral medial hypothalamus. An efferent response is sent to the
adrenal medulla and sympathetic nerve endings elevating plasma catecholamines in
attempts to counterregulate blood glucose.
9
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Significance. Deterioration of glucose homeostasis can elicit profound complications
within the body. Diabetes mellitus is the most prevalent pathogenic condition
associated with impaired glucoregulation [48, 56]. Because these patients lack the
ability to secrete insulin, which in itself is a highly regulated process controlling the
entry of glucose into the cell, exogenous insulin must be administered.
In attempts to avoid large excursions in blood glucose that are experienced during
conventional insulin therapy, more aggressive approaches to insulin replacement have
been advocated [56, 57], While such aggressive insulin therapies have been successful
at ameliorating long term complications arising from chronic exposure o f physiological
systems to high blood sugar concentrations, individuals undergoing such therapy are
placed at a three-fold greater risk for severe hypoglycemia [56, 57, 58].
Hypoglycemia currently constitutes a significant limitation in the treatment of
insulin dependent diabetes mellitus (IDDM) [46, 58], The susceptibility of IDDM
patients to hypoglycemia can in part be attributed to defective hormonal
counterregulation, o f which glucagon and epinephrine are unable to adequately
stimulate glucose production to equate the accelerated glucose disposal rate brought
about by relative excess of exogenous insulin [46, 47, 48, 85, 86], This defect in
counterregulation has been shown to be specific for glucose, as the response to other
stimuli (e.g. exercise) is apparently normal in IDDM patients [83, 86],
This observation is consistent with a defect in the afferent limb, i.e. glucose sensing.
Thus diabetes associated neuropathy may be associated with an altered sensor
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sensitivity attenuating afferent activation of sympathoadrenal counterregulation during
hypoglycemia [27, 33, 50, 51, 58], Efforts to elucidate the nature of this defect have
been constrained by our lack of understanding regarding the specific locus for glycemic
detection.
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II. BACKGROUND
A. Glucose Homeostasis
Glucose homeostasis involves complex interactions between humoral and neural
mechanisms that work in concert to tightly regulate blood sugar concentrations within
very narrow limits. The maintenance of blood glucose concentration in the normal
range is dependent upon the equation of the rate of glucose appearance from the gut
and or glucose produced by the liver and kidney, with the rate at which glucose is
removed from the blood by insulin sensitive (i.e. liver, skeletal muscle, fat) and
insensitive tissues (i.e. central nervous system).
In healthy subjects consuming a normal diet, plasma glucose is ordinarily
maintained between 70 to 140 mg/dl. As the normal vicissitudes in portal blood
glucose throughout a 24 hour time period can range from 70 to 250 mg/dl, the
homeostatic mechanisms by which the body attempts to maintain arterial blood sugar
concentrations in the normal range is appreciated. Presented with challenges in
glycemia, either from abstinence or meal consumption, the body is in a perpetual state
of modulating the secretion of glucoregulatory hormones: insulin, glucagon,
catecholamines, cortisol, growth hormone, either signaling for the storage or
production of glucose by the liver. The number and complexity of these homeostatic
mechanisms involved in glucoregulation, illustrates the gravity with which the body
attempts to avert large excursions in glycemia.
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1. Insulin D ependent D iabetes M ellitus
The most prevalent pathologic condition associated with the loss of glucoregulatoiy
capacity is diabetes m ellitus. Insulin dependent diabetes mellitus afflicts over one
million Americans, most of which are under the age of twenty years. Diabetes costs the
U.S. taxpayer over $100 billion dollars a year in treatment alone, making it the most
expensive disease on a cost per patient basis. It is generally accepted that diabetes is
not a single disease, but a group of metabolic disorders characterized by high blood
sugar concentrations. Fasting hyperglycemia in the IDDM population is due to
compromised removal of glucose from the blood either as a result of a relative or
absolute lack of insulin [22, 47, 56],
In order to simplify the classifications of this complex group of metabolic disorders,
diabetes has been distilled to two primary categories. Even though the etiology of
these metabolic disorders varies widely, the two classifications of diabetes are referred
to as non insulin dependent (NIDDM) and insulin dependent (IDDM), diabetes
mellitus. Although the latter only afflicts 10% of the diabetic population, unlike
NIDDM, IDDM cannot be reversed by dietary, drug, or exercise manipulations [56, 57,
58]. Insulin dependent diabetes mellitus is an autoimmune disease that destroys the
pancreatic beta cell rendering the diabetic patient incapable o f producing sufficient
endogenous insulin [22, 57, 77], The diabetic patient must rely on exogenous insulin
replacement in order to control the glucose concentration in the blood. IDDM patients
not only suffer from the inability to produce endogenous insulin, but the deterioration
1 3
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of the glucagon secreting alpha cell, glucagon being the primary counterregulatory
hormone involved in the production of glucose, is also hallmark o f this disease [22, S6,
57, 58, 86], The declension o f the glucoregulatory reflex mechanisms, coupled with
the imprecision of exogenous insulin administration, exacerbates the already
problematic task of glycemic regulation for this disease population.
Due to imprecise means o f insulin replacement, the diabetic patient often suffers
from an insulinopenic state, permissive of hyperglycemia. Chronic hyperglycemia is
known to result in such anomalies as retinopathies leading to blindness, cardiovascular
disease, neuropathy leading to amputation, renal failure, and diabetic coma. The
importance of strict glycemic regulation has recently come to light through the work o f
the Diabetes Control and Complications Trials (DCCT) [56, 57, 58]. It is formally
recognized that the widespread pathogenesis of this disease results from chronic
exposure of various physiological systems to elevated blood sugar concentration [57],
Aggressive insulin therapies have been prescribed in attempts to ameliorate the
effects of chronic hyperglycemia so as to improve the quality and duration of life.
However, while reducing the prevalence and progression of hyperglycemic associated
disease by 35 to 75% in most patients, this type of insulin treatment has significantly
increased the frequency and severity of hypoglycemic episodes [58]. Hypoglycemia is
currently the primary limiting factor in the treatment if IDDM [46, 58].
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2. H ypoglycem ia
The precise definition of hypoglycemia remains in dispute specifically in
quantitative terms, however qualitatively, hypoglycemia is viewed as a clinical
syndrome characterized by a reduction in plasma glucose concentration to levels
sufficient to produce symptoms that revert upon restoration of normal glucose
concentration [171], Hypoglycemia is potentially dangerous as the central nervous
system is unable to store or synthesize glucose to an appreciable extent, and must
therefore rely exclusively on glucose supplied by the blood to fuel metabolism and
maintain neural integrity.
Typically individuals undergoing conventional therapies experience one
symptomatic episode of hypoglycemia per week, while this number increases 3 fold
during intensive therapy [56, 58], In addition, these patients experience at least one
severely debilitating hypoglycemic episode per year (e.g. seizure, coma) where medical
treatment is necessitated [58], The clinical manifestations of hypoglycemia are divided
into two major categories: adrenergic symptoms including palpitations, sweating,
tremor, and anxiety, (which may be causally related to the triggering of the
sympathoadrenal counterregulatory system) and neuroglycopenic symptoms, including
fatigue, confusion, headache, seizures, and coma [45, 57, 58]. Interestingly, the
adrenergic symptoms of hypoglycemia have been proposed by some investigators to
constitute the early warning signs of an impending hypoglycemic state, as it has been
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observed that adrenergic stimulation, i.e. sympathoadrenal counterregulation,
commonly precedes impairment in cerebral function [33,47, 57, 58],
Pathogenesis in the afferent limb of EDDM patients significantly increases the risk
for more severe hypoglycemia, and is associated with three clinical syndromes
collectively termed hypoglycem ia-associated autonom ic fa ilu re [58], These
syndromes are characterized by defective glucose counterregulatory capacity (deficient
secretory capacity for glucagon and epinephrine), hypoglycemia unawareness (deficient
autonomic and neurogenic symptoms), and reduced glycemic thresholds (more severe
hypoglycemia) for symptoms and counterregulatory activation [34, 47, 48, 50, 51, 85,
86, 95, 115, 147], As 4% o f deaths in EDDM patients result from hypoglycemia, the
mechanisms involved in the pathogenesis of the autonomic nervous system require
elucidation.
B. Glucose Counterregulation
1. E fferent Lim b
The normal counterregulatory response to hypoglycemia is well characterized with
respect to the glucose concentration that elicits the secretion of counterregulatory
hormones: glucagon, epinephrine, norepinephrine, cortisol, and growth hormone
(Figure 3). Elevated plasma insulin concentrations resulting from aggressive insulin
therapy cause elevated glucose disposal rates in insulin sensitive tissues and marked
suppression of hepatic glucose production (HGP). It is the imbalance in the rate of
glucose production vs. glucose disappearance brought about by excess insulin, that
initiate decrements in blood glucose concentration [22,44,45].
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The body responds to decrements in glycemia through a host of neuroendocrine
responses including elevated circulating counterregulatory hormones and neural
stimulation of the liver [137, 161]. Under conditions o f rapidly decreasing arterial
glucose, direct neural stimulation of the sympathetic nerves innervating the liver allows
for immediate degradation of hepatic glycogen, liberating glucose into the systemic
circulation. Rapid increases in phosphorylase (300%) and glucose-6-phosphatase
activity (40%), two key regulatory enzymes required for glucose production by the
liver, were also observed following hepatic splanchnic nerve stimulation [161]. As the
duration of hypoglycemia is prolonged neural stimulation of the liver is also responsible
for elevating the rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxy
kinase (PEPCK), while inhibiting the key glycolytic enzyme pyruvate kinse (PK), thus
promoting continued glucose production [161], Furthermore, sympathetic stimulation
is also responsible for the reductions in insulin sensitivity, an important mechanisms for
reducing the disposal rate of blood glucose [12, 59, 69, 89, 141, 150],
In addition to the adrenal source, neural stimulation of sympathetic nerve endings
results in elevations of ganglionic norepinephrine [121]. Norepinephrine of this nature
is responsible for stimulating peripheral lipolysis which becomes increasingly important
as hypoglycemia is prolonged [36, 69, 85, 121]. Elevations in lipolytic rate provides an
alternative fuel source and allows the sparing of glucose.
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NORMAL GLUCOSE COUNTERREGULATION
|------------------------------► f INSULIN (8 3 * 3 m g /d l} ---------------------------------- ^
f GLUCOSE ^GLUCOSE
! ► I G LU C A G O N ( « 8 t 2mg/dL) \
► ♦ EPINEPHRINE {6» * 2 m g /d L )-------
( f B rain G lu c o t* U plak*. -A T ing/dl.)
♦ GROW TH HORMONE ( M t2 m g /d l)
4 CORTISOl (3B*Jm g/dl)
(S rm p io m i. • '3 4 m g / d I ; | Cognilian, — 4 ? m g /d l)
■ — GLUCOSE AUTO REGULATION («50. > J0m g/dL )
OTHER H O R M O N ES. NEUROTRANSMITTERS.
OTHER SUBSTRATES
F igure 3. Normal glucose counterregulation in humans. Values are expressed as
means ± SE for arterialized glycemic thresholds of various physiological responses to
decrements in blood glucose concentration. Compiled from data reported by [44, 126,
157].
18
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I
LIVER
4 EPINEPHRINE
I _________
1 -----------
PANCREATIC
ISLETS
t Insulin
♦ Glyeogenolysis 1
♦ Gtuconeogenesisj ^
4 GLUCOSE PRODUCTION
I ____________
t Glucogon
4 Glycolysis
4 Lactate & j S
Alanine
MUSCLE
Pi
4 Glucose
Transport
A
PAT
Pt.Pl
4 Lipolysis
, 1
4 Glycerol
f NEFA
}
t GLUCOSE
VX
4 GLUCOSE UTILIZATION
Figure 4. Glucose counterregulatory effect of epinephrine on liver, pancreas, muscle,
and adipose. Compiled from data reported by [16, 45, 59, 69, 149].
19
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Since neuronal norepinephrine typically exerts its effect and is degraded locally,
only 15% of norepinephrine secreted from preganglionic nerve endings is released into
the systemic circulation [6S]. While stimulation of hepatic efferents is critical for rapid
corrections in glycemia, neural stimulation of visceral organs that secrete
counterregulatory hormones acting systemically is essential for sustaining long term
glucose production.
The hormonal milieu is not only important for fortification of the glycogenolytic
response produced by direct hepatic neural stimulation, but many o f these hormones
(glucagon, epinephrine, norepinephrine, growth hormone, cortisol) also stimulate de
novo glucose production via regulation of key gluconeogenic enzymes and expansion
of the gluconeogenic precursor pool. As gluconeogenesis can account for up to 88%
of glucose production during hypoglycemia of an extended duration, the importance of
hormonal counterregulation is illustrated [36, 37, 73, 74, 117].
Although glucagon is considered the primary controller of hepatic glucose
production [38, 44, 73, 74, 81, 83, 84, 85, 119], circulating catecholamines become
critical to efficacious counterregulation during severe hypoglycemia, especially in
IDDM patients with defective a-cell secretory capacity [59, 74, 78, 84, 85, 89, 127,
150, 154]. During hyperinsulinemia in type I diabetes, a cell responsiveness to
hypoglycemia may become severely impaired [22, 47, 108, 119, 125, 141, 155].
Unlike glucagon which exerts its effect exclusively on the liver, epinephrine in addition
has a direct antagonist effect on insulin stimulated glucose uptake by peripheral tissues
20
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[36, 42, 69, ISO], as well as an antagonistic effect (in normal patients) on insulin
secretion from pancreatic beta cells via the o 2 adrenoreceptor [12, 78, 85, 141]. In
addition, it was shown that during physiologic insulin infusion (0.7 mU^kg'^mm1 ),
epinephrine was considerably more effective than glucagon at counteracting
decrements in glycemia [59]. Furthermore, it was demonstrated that prolonged
glucagon infusion only raised plasma glucose levels transiently, while prolonged
epinephrine infusion sustained increases in plasma glucose [65, 153]. Although
epinephrine has a limited temporal effect on hepatic glucose production, inhibition of
insulin stimulated glucose disposal was shown to be sustained [42, 59, 67, 170].
Inhibition of glucose disposal is thought to result from direct effects o f the hormone on
the insulin receptor as well as indirect effects mediated through FFA.
In addition to limiting glucose disposal via inhibition of transport, elevations in FFA
have also been shown to limit the rate of carbohydrate oxidation by skeletal muscle,
providing further means for minimizing carbon loss [7, 23, 36]. Thus, epinephrine
stimulated elevations in FFA concentration allows for recycling of glucose precursors
to spare glucose for oxidation by the CNS [36, 44], Epinephrine may therefore play a
greater role in counterregulation than once believed, and fill a particularly important
compensatory role for glucagon in IDDM.
In addition to reductions in glucose disposal, and stimulation of hepatic
glycogenolysis and peripheral lipolysis, catecholamines play a major role in stimulating
the expansion of the gluconeogenic precursor pool (lactate, glycerol, and alanine) [63,
21
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65, 69, 70]. As glycogenolysis is more readily suppressed than gluconeogenesis during
periods of relative insulin excess, the re-synthesis of glucose via lactate can account for
as much as 50% c f the life sustaining glucose produced [41, 73], Unlike the transient
three fold increase in glucagon, the catecholamine expansion in response to progressive
hypoglycemia is substantially larger (16 fold increase) and more robust. For the
evidence aforementioned, the sympathoadrenal response to slow progressing
hypoglycemia was utilized as the dependent measure in the investigations presented
herein.
Studies in which the increments in glucagon and epinephrine were prevented
demonstrated an accessory role for cortisol and growth hormone in combating
hypoglycemia [23, 44, 84, 85, 88]. In addition it was shown that these
counterregulatory hormones become progressively more important during prolonged
hypoglycemia. As the duration of hypoglycemia is extended, it appears that cortisol
has a synergistic effect with glucagon and epinephrine, potentiating the effect on
hepatic glucose output by increasing the gluconeogenic conversion of alanine to
glucose. However, cortisol independently does not appear to have a direct effect on
hepatic glucose production p er se [64]. Similarly, studies with growth hormone
deficiency had no demonstrable effect on glucose counterregulation [84]. Although
both hormones are known to stimulate gluconeogenesis [88] there is limited support
for a substantive role in glucose counterregulation.
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2. Afferent lim b
Traditionally the brain has been implicated as the primary locus for glycemic
detection and neurohumoral counterregulation against insulin induced hypoglycemia.
As early as 1849, it was demonstrated that lesioning specific regions of the brain (e.g.
4th ventricle) had pronounced affects upon carbohydrate metabolism [17], A
subsequent investigation by Cannon (1924), provided the first evidence for
hypoglycemia induced elicitation of sympathetic nerve activity, thus it was reasonable
for Mayer and Bates (1952), to propose the existence of glucose specific sensors in the
brain.
Many of the subsequent investigations sought support this hypothesis, however
relied on non-physiologic stimuli including microinjections of neurotransmitters [162,
163], glucose analogs [24, 149, 153, 160], and glucopenic agents [26, 100, 133], in
addition to electrical stimulation [13, 76, 87, 163] and ablation techniques [25, 61, 132,
161] to elucidate the qualitative role of the CNS in glucose detection. These
procedures while establishing the existence of glucoregulatory aspects of the brain,
failed to provide substantive evidence for CNS exclusivity in glycemic detection.
This line of research was however paramount in elucidating the efferent role of the
CNS in neuroendocrine counterregulation. Findings from these investigations targeted
the hypothalamus as integration center for afferent signals requisite in the generation of
a coordinated efferent response to fluctuations in blood glucose concentration. More
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precise investigations have shown that the ventromedial (VMH) and lateral (LH)
aspects of the hypothalamus exert reciprocal control over glucose metabolism [161].
Neuroanatomical investigations have identified hypothalamic neurons from the
ventral medial region traveling caudally through the midbrain and pons, synapsing with
neurons in the central gray substance of the brainstem, advancing through the medulla
by multi-synaptic pathways to the intermediolateral cell column of the thoraco-lumbar
spinal cord, and terminating with the splanchnic nerves innervating the liver, pancreas,
and adrenals [2, 9, 10,18, 19, 20, 38, 76, 138, 139]. In contrast, neurons of the LH
were shown traveling through the dorsal motor nucleus and the nucleus ambiguus of
the medulla and connecting with vagal nerves distributing to a wide variety of
abdominal organs also including the liver, pancreas, and adrenals.
Stimulation or ablation of the LH or VMH, and or the neural pathways from these
two distinct sites, has shown to significantly impact circulating counterregulatory
hormone concentrations [76, 161]. Lesioning of the VMH was found to cause
increased parasympathetic and reduced sympathetic tone [160]. Conversely, lesioning
of the LH increased sympathetic tone exclusively [13, 138, 161]. Alternatively,
stimulation of the VMH has been shown to decrease firing activity in the pancreatic
branch of the vagus nerve while increasing the firing rate of the splanchnic nerve [161,
162]. Splanchnic nerve stimulation was shown to elicit elevations in glucagon and
epinephrine concentration, resulting in accelerated rates of hepatic glycogenolysis,
gluconeogenesis, and peripheral lipolysis. In combination, splanchnic nerve stimulation
24
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caused increments in blood glucose concentration [161]. In contrast, stimulation of the
LH, elevated electrical activity in the vagus nerve while inhibiting activity in the
splanchnic nerve. Vagal stimulation via the LH was found to promote insulin secretion
leading to decrements in hepatic glucose output and cellular glucose sequestration[135,
161].
a. E xclusivity o f VM H vs. W idespread Brain R egions in H ypoglycem ic
Detection
Equivocal findings relating to the exclusivity of hypothalamic glucose detection
fostered the notion of a peripheral loci for important glucose sensing. In vivo
experiments conducted by Cane [34, 35] utilizing bilateral carotid artery (including the
forebrain containing the hypothalamus) or vertebral artery glucose infusions, producing
brain normoglycemia failed to impact upon glucose counterregulation (glucagon and
catecholamines) during systemic hypoglycemia. No significant differences in the
counterregulatory response between the brain clamp (vertebral or carotid glucose
infusion) and the matched systemic infusion protocols during whole-body hypoglycemia
were observed (Fig. 5).
Hypothetically, if the VMH was the primary locus for glycemic detection, then
irrigation of the forebrain (including the VMH) should have had a marked suppressive
effect on the sympathoadrenal response to systemic hypoglycemia. By the same logic,
if glucosensors were located exclusively in the hindbrain, as demonstrated by Ritter
[149] and DiRocco [61], a blunting of the counterregulatory response to systemic
hypoglycemia should have been observed during vertebral glucose infusion. These
25
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negative findings by Cane, suggest that essential glucosensors responsible for
modulating sympathoadrenal counterregulation are located elsewhere in the CNS or in
the periphery. As bilateral carotid and vertebral infusions were performed separately,
the possibility of hypoglycemic detection via widespread brain regions, could not be
eliminated.
Findings from Biggers [21] and Frizzel [75], suggest that glucose detection by
widespread brain regions is required for full counterregulation, hence counterregulation
is not directed exclusively by the hypothalamus. That is, only when glucose was
infused via both the vertebral and the carotid arteries (i.e. whole brain irrigation) was
the counterregulatory response to systemic hypoglycemia significantly suppressed
(68%). When glucose was infused either via the vertebrals or the carotid arteiy a
marked suppression in counterregulation was not observed.
In years following and in sharp contrast to the work of Cane (1988), and Frizzel
(1993), Borg et al. [24], utilizing VMH glucose perfusion (100 mM), was able to
demonstrate a 75% suppression in the glucagon response and a 90% suppression in the
epinephrine response during systemic hypoglycemia. In addition, Borg also observed
diminished glucagon, epinephrine, and norepinephrine responses (50-60% hormonal
suppression) to mild and severe hypoglycemia (75-80% hormonal suppression)
following VMH lesioning [25]. Despite the presence of peripheral euglycemia,
localized VMH glucopenia induced via 2-deoxy-glucose infusion initiated hormonal
counterregulation, including a 30-fold increase in epinephrine, and a 3.5 fold increase in
26
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norepinephrine concentration [26]. From these investigations it was concluded that the
VMH is the preeminent site for glucose detection and hormonal counterregulation, as
manipulation specifically of VMH metabolic status imposed a greater impact on
hormonal counterregulation compared to whole brain glucose perfusion.
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BRAIN CLAMP MA TCHED CLAMP
EXTRACTION
(*) BRAIN
eugtycemia
BR A IN
hypoglycemia
• G L U C O S E
INFUSION
(C ) .
artery artery
BODY
hypoglycemia
INSULIN
BODY
hypoglycemia
INSULIN
G L U C O S E IN FU SIO N
F igure 5. Schematic representation of the “brain clamp” experiment (above) and
graphic depiction of the hormonal response (below) for the three experimental
conditions during steady state. Values are expressed as means ± SE for epinephrine,
norepinephrine, and glucagon during the brain irrigation and matched infusion
protocols. Solid bars represent matched infusion and cross hatched bars represent the
brain clamp protocol. Insulin (ISO mU/min) was infused peripherally to induce
hypoglycemia, while brain euglycemia was achieved via glucose infusion in either the
carotid or vertebral artery. Glucose was infused peripherally during the matched
infusion protocol in order to establish equivalent brain and systemic hypoglycemia.
Similar responses were observed during both protocols for moderate and deep
hypoglycemia. Adapted from [34, 35].
28
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Unlike work conducted by Borg and colleagues, our laboratory has experienced
difficulty demonstrating that either region perfused by the carotid or vertebral arteries,
retains the locus for hypoglycemic detection or exerts exclusive control over integrated
counterregulation. To the contrary, we have postulated that peripheral, portohepatic,
glucosensors play a pivotal role in hypoglycemic detection and hormonal
counterregulation. From a mechanistic standpoint, it is advantageous for glucose
sensors to reside in the periphery opposed to the CNS, as they would protect the brain
from having to respond at a glycemia which is low enough to impair its own function.
The concept of a hepatic gluco sensory locus, first introduced by Russek [153], was
initially germane to the control o f satiety and feeding. Russek proposed the existence
of portohepatic glucosensors based upon the observation that portal glucose infusions
suppressed hunger relative to similar peripheral glucose infusion. That the liver is
richly innervated with both efferent and afferent fibers receptive to changes in pressure,
osmolarity, and various metabolites, it was later hypothesized that there may exist
essential glucosensors involved in modulating hormonal counterregulation as well.
Electrophysiologically investigations conducted by Niijima [133, 134, 136]
demonstrated the existence of a portohepatic glucoreceptor central mediated pathway.
Intravenous glucose injection (25 mg/kg) in vivo did not impact on the mean firing rate
of the adrenal nerve or pancreatic branch of the vagus nerve, however by comparison,
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intraportal administration of the same amount of glucose decreased the firing rate of
the adrenal nerve and increased the discharge rate of the pancreatic branch of the vagus
nerve significantly [133]. Niijima, utilizing an elegant in situ preparation, observed an
inverse relationship between the portal vein glucose concentration and the firing rate of
hepatic vagal afferents [132]. Moreover, elevations in portal vein glucose
concentration were also shown to produce a reflex inhibition of the adrenal nerve
blunting catecholamine secretion following insulin administration, a finding consistent
with the existence of a portohepatic-adrenal neural feedback loop.
The first in vivo investigations to address the existence o f a portohepatic-adrenal
axis, were conducted by our laboratory [62, 63]. We have previously demonstrated an
essential role for the portohepatic region in hypoglycemic detection and modulation of
sympathoadrenal counterregulation. Under the conditions of both moderate and deep
hypoglycemia, the sympathoadrenal response to systemic hypoglycemia was suppressed
by as much as 78% during portal vein glucose infusion. As only the portal vein and
liver were maintained normoglycemic during portal glucose infusion, it was concluded
that important glucose sensors reside in this region and that portohepatic hypoglycemia
is essential to engender a full sympathoadrenal response [63]. Lamarche and
Yamaguchi, utilizing cross perfusion of the liver during systemic euglycemia [111],
observed full sympathoadrenal counterregulation as a result of isolated porto-hepatic
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1 2 3 4 5
40 - minute time periods
1 2 3 4 5 6
40 - m inute time periods
Figure 6. Values are expressed as means ± SE for arterial glucose, hepatic glucose,
epinephrine, and norepinephrine concentrations at basal (B) and during experimental
(40 min) sampling periods (bars 1-6). Insulin was infused at 5 mU«kg* 'min‘l to induce
systemic hypoglycemia while glucose was infused to clamp the arterial glucose
concentration, either via the cephalic vein (PER) or the portal vein (POR). Solid bars
represent POR and open bars represent PER *, P<0.05. Reproduced from [63],
31
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Collectively, this work establishes a critical role for the portohepatic region as the
locus for an important sensory mechanism involved in the detection of blood glucose.
Findings suggest that this sensory limb is fundamental for transmission of metabolic
information to the CNS, modulating the sympathoadrenal response against
hypoglycemia. As transection of the splanchnic nerve was shown to obliterate the
adrenal response to hypoglycemia, the efferent neuron was functionally implicated in
this neural reflex and determined essential for counterregulation [100], In contrast, the
precise structure and locus the glucose sensor, signal transduction pathway, and the
requisite afferent nerves remain to be elucidated.
As the liver is known to be richly innervated, it has been presumed for years by
many that peripheral glucose sensors reside in the liver. Generally, hepatic nerves have
been classified into three physiological groups: efferent nerves for vasomotor
regulation of blood flow, efferent nerves to the parenchyma for increasing glucose
output, and afferent nerves responsible for chemoreception. The tracing of hepatic
efferent nerves is well documented, however due to methodological limitations there is
much debate in the literature as to the proportion of afferent innervation in this region.
Despite early reports of substantial intralobular innervation, several investigations
have been unable to identify fibers clearly terminating in the hepatic parenchyma.
These conflicting reports result from limited capacity of staining techniques to
discriminate between neural and argyrophilic connective tissue fibers. The advent of
light and laser confocal microscopy has provided the evidence necessary to confirm the
32
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existence of nerves running in the disse spaces, with unencapsulated terminals making
direct hepatocellular contact. In some rare cases these nerves were also observed
penetrating the cytoplasm and extending as far as the nuclear membrane [113, 156].
Many of the early reports however demonstrated that the vast majority of
parenchymal nerves possessed sympathetic-adrenergic endings [72, 110, 113, 114,
165]. These catecholamine containing neurons made visible by treatment with a
monoamine oxidase inhibitor (nialamide), have been shown to concentrate tritiated
norepinephrine, and degenerate following treatment with 6-hydroxydopamine. The
complete function of these nerves however, has yet to be determined [4, 113, 114],
In addition to catecholamine containing neurons, Sutherland [168], demonstrated
positive cholinesterase activity specific for acetylcholine, in livers of rat, monkey, and
guinea pig. Strong cholinesterase reactions were demonstrated in the vascular and
biliary plexuses, in addition to ganglia associated with nerves in the porta-hepatis and
parenchyma. Studies utilizing scanning electron microscopy yielded similar results,
such that Skarring and Bierring [165] suggested the existence of an extensive
cholinergic plexus throughout the lobules of rat liver. Subsequently, a non-specific
reaction used to suppress butylcholinesterase activity demonstrated a less extensive
intralobular distribution of acetylcholine-positive nerves than once proposed, with
endings found only on parenchymal cells of the portal lamina.
Although these previous findings are consistent with the presence of extensive
hepatic autonomic effector neurons, there is new evidence that a percentage these
33
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neurons may in fact be afferent or sensory nerves involved in chemoreception. The
appearance of microvesicles in nerve endings, usually indicative of effector neurons,
have also been shown for several purely sensory structures including Pacinian
corpuscles, Meissner’s corpuscles, taste buds, and the pre-synaptic elements of rods
and cones. Moreover, recent evidence has dispelled the notion that a positive
acetylcholine reaction denotes a parasympathetic effectory neuron [113], For example,
chemoreceptive glomus cells, associated with carotid bodies, also test positively for
AChe. In addition, the carotid body which is a purely sensory mechanism for P02 , was
found to closely resemble the subcellular distribution of glomus cells described for the
hepatocyte [113]. The acetylcholine present in the glomus cells may well play a role in
the generation of the chemosensory discharge, as injections o f acetylcholine into the
liver have been reported to increase the rate of hepatic afferent firing [114].
There are three types of sensory nerve endings that have been demonstrated in the
porto-hepatis, including: bare endings within the lobules and around the central vein
and bile ducts, encapsulated endings identified in the connective tissue of the septa, and
club shaped cells as well as splayed glomular endings found in association with the bile
ducts. The abdominal paraganglia have also been localized to the liver pedicle and have
been implicated in sensory aspects of metabolism. More recently the hepatic
paraganglia cells have been shown to be comprised of two types of cells: chief or type I
cells and satellite or type II cells [97]. It was observed that many large cup-shaped
afferent nerve endings make multiple synaptic contacts with chief cells. Furthermore,
34
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these large nerve endings were found to degenerate following infranodose vagotomy
[144], In addition, anterograde tracing from the nodose ganglia indicated
chemosensory vagal afiferents terminating in the gastric, thoracic, and hepatic
paraganglia [18, 19,20].
According to Berthoud (1995), the left nodose ganglia supplies 90% o f the hepatic
vagal branch (Fig. 3). In contrast to previous estimates, the more advanced techniques
utilized by Berthoud and colleagues, have allowed for the determination of a tenfold
greater presence of vagal afiferents, in the porto-hepatic region [18, 143, 144], Based
upon degenerative studies following supranodose or intracranial vagotomy, it has been
estimated that 75-90% of all fibers in the abdominal vagus are afiferents [18, 143, 144],
These findings are well supported by physiological and behavioral studies implicating
the existence of vagal hepatic afiferents sensitive to changes in portal vein metabolites
[131, 153, 117], osmolarity [1, 131], and temperature [1, 113, 131].
That requisite neural circuitry shown for the liver was supported by investigations
from our laboratory, a hepatic locus was believed to establish the foundation for the
sensory portion of the hepato-adrenal neural loop initially proposed by Niijima [137],
However, investigations utilizing laser confocal microscopy, demonstrated that hepatic
vagal afferent nerves form fine terminal aborizations running deep within the adventitia
of the portal vein [20], As afferent nerves were found intimately associated with the
portal vein, and previous investigations failed to constrain glucosensors to the liver
exclusively, a potential portal locus for this glucose sensing mechanism is now realized.
35
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It is the goal of the current investigations to test the provocative hypotheses that
neurally mediated glucose sensors are located in the portal vein, not the liver, and are
responsible for modulating sympathoadrenal counterregulation in response to glycemic
fluctuations.
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IU. EXPERIMENTS
A. PORTAL VEIN GLUCOSENSOR LOCUS
The purpose of this investigation was to test the hypothesis that important
glucosensors involved in modulating sympathoadrenal counterregulation reside in the
portal vein, not the liver.
1. Research Design and Methods
Anim als and Surgical Procedures. Experiments were conducted on male Wistar
rats (weighing 284 ± lOg, n=22) in the conscious relaxed state. All surgical and
experimental procedures were pre-approved by the University of Southern California
Institutional Animal Care and Use Committee.
One week prior to the experiment, animals were chronically cannulated under single
dose anesthesia (3:3:1 ketamine HC1, xylazine, acepromazine maleate; 0.10 cc /100 g
body weight given intramuscularly). Cannulas were placed in the portal vein (Silastic,
ID = 0.03 cm tip) for glucose infusion during liver irrigation, the carotid artery (Clay
Adams; PE-50) for arterial blood sampling, and jugular vein (dual cannula silastic, ID =
0.03 cm) for insulin and peripheral glucose infusion (PER). The portal vein cannula
was placed either adjacent, (P O R adj = 0.6 ± 0.1cm) or upstream, (P O R dist = 2.7 ± 0.1
cm) from the liver. All cannulas were tunneled subcutaneously and exteriorized at the
back of the neck and encased in silastic tubing (0.2 cm ID) sutured to the skin.
Study Design. Animals were allowed five days recovery from surgery to regain
body weight. Three days post-surgery 300 pi of blood was drawn and analyzed for
37
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plasma alanine amino transferase (ALT) activity, a sensitive measure of hepatocellular
integrity. There were no significant differences in ALT activity between animals
portaUy cannulated (PE R , PORadi, PO R dist) or those litter mates that were randomly
assigned to the blood donor group possessing only a carotid cannula (18.9 ±3.0 vs.
16.5 ± 2.4 U/L, respectively). Twenty-four hours prior to the experiment all food was
removed from the cage.
Hyperinsulinemic-hypoglycemic clamp. All animals were exposed to the same
general protocol for the induction of hypoglycemia. Prior to the experiment, animals
were placed in a modified metabolic chamber and allowed to rest for 30 min (-60 to -30
min). Basal samples were drawn at -30 and 0 min for analysis of glucose and
catecholamines. At min 0, following arterial sampling, insulin (50 mU«kg*l«min 'l) and
glucose infusions (variable) were initiated and maintained for 105 min of the
hypoglycemic clamp. Serial sampling for glucose was performed at 10 min intervals so
as to maintain the integrity of the hypoglycemic clamp and preserve the rate of fall in
blood glucose. Deep hypoglycemia (~45 mg/dl) was achieved by min 60 and sustained
for the remaining 45 min of the experiment (60 to 105 min). Arterial catecholamine
and plasma glucose samples were taken at 60, 75, 90, and 105 min of sustained deep
hypoglycemia.
Experim ental design. Each animal participated in one of three specific experimental
conditions distinguished by the site of glucose infusion (Fig. 7). Figure 7A represents
peripheral glucose infusion (PER) via the jugular vein. This protocol effectively served
38
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as the experimental control group whereby whole body hypoglycemia was induced.
Figure 7B, represents the portal vein distal experimental condition, (PO R djst), whereby
portal and liver glucose concentrations were normalized during systemic hypoglycemia.
Figure 7C, represents the portal vein adjacent glucose infusion condition (PO R adi),
whereby the cannula was advanced to the liver thus normalizing only the liver glucose
concentrations. The portal vein and peripheral glucose concentrations were lowered in
parallel to a deep hypoglycemic level. According to the working hypothesis, if the
sensors reside exclusively in the portal vein, then portal vein concentrations will dictate
the sympathoadrenal response, irrespective of liver glycemia.
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GLUCOSE INFUSION
PER
A.
H E A R T
L IV E R
K ID N E Y
H E A R T
B.
L IV E R
'ANCKEAS.
GLUCOSE INFUSION
POR-DIST
KID!
H EA RT
L IV E R
•ANCKEAS.
GLUCOSE INFUSION
POR-ADJ
KID!
Figure 7 . Schematic diagram of the three experimental conditions distinguished by the
site of glucose infusion: peripheral glucose infusion via the jugular vein, portal vein
glucose infusion upstream from the liver, and portal vein glucose infusion adjacent to
the liver. (A) peripheral glucose infusion experiment (PER), was considered the
control condition for which whole body hypoglycemia was induced; (B) glucose
infusion via portal cannula positioned upstream from the liver, (PO Rqist), allowed for
the normalization of liver and portal vein glycemia during systemic hypoglycemia; (C)
glucose infusion via portal cannula adjacent to the liver, (PO R adj), provided
normalization of liver glycemia alone.
40
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Calculations. The estimated liver glucose concentration (G l) was calculated as:
G l= G a + (G IN F por/ HPF) (1)
where G a = arterial glucose concentration (mg/dl), G IN Fpor = portal glucose infusion
rate (mg/min), and HPF = hepatic plasma flow rate (ml/min). The estimated portal vein
glucose concentration (Gpv) was calculated as:
Gi»v= G a ■ * " (G IN Fpordist/ PVPF) (2)
where G IN Fpordist = distal portal glucose infusion rate (mg/min), and PVPF = portal
vein plasma flow rate (ml/min). Hepatic plasma flow was estimated to be 1.3 ml • g'1
liver • min'1 x liver weight g, and the portal vein flow rate assumed to be 80% of the
HPF [31, 32], As experiments were preceded by a 24 hour fast, portal vein and hepatic
glycemias were assumed equal to GA in the absence of any portal glucose infusion.
Due to cannula placement during PO R adj glucose infusion, (advanced to the liver), the
portal glycemia was assumed to be equal to GA.
A nalytical procedures. Glucose was assayed utilizing the glucose oxidase method
(YSI, Yellow Springs, OH). Epinephrine and norepinephrine concentrations were
assayed using a single-isotope radioenzymatic approach [66, 146]. Basal insulin
samples were assayed via an RIA kit (Linco Research Inc. St. Charles, MO.), and
steady state hyperinsulinemic samples were assayed using a RIA according to Herbert
et al. [96]. Alanine aminotransferase was assayed spectrophotometrically [148].
Data analysis. The results are expressed as means ± SEM. Comparisons of animal
characteristics between groups were made using one-way ANOVA for independent
41
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groups. Comparisons between treatments over time were made by repeated measures
ANOVA utilizing Tukey’s test for post-hoc comparisons. Significance was set at
p<0.05.
2. Results
For all experimental protocols, arterial insulin concentrations increased from a mean
basal value of 10 ± 1 pU/ml to a hyperinsulinemic plateau of 1562 ± 300 pU/ml. No
significant differences were observed between protocols with respect to basal or
elevated insulin concentrations.
Basal arterial glucose concentrations, 129 ± 5 mg/dl, were not significantly
different between protocols. By design, there were no significant differences in arterial
glucose concentrations during deep sustained hypoglycemia, 44 + 1.8 mg/dl (p=0.80)
between the three glucose infusion protocols. Hepatic and portal vein glycemias were
also allowed to decrease to a deep hypoglycemic level during PER. In contrast hepatic
glycemia remained elevated during the liver irrigation protocols, P O R adj and PORdist
(Fig. 9). Between 60 and 105 minutes estimated liver glycemias for PO Rdist and
PO R adj were 78.4 ±5.6 mg/dl. Estimated hepatic glucose concentrations were not
significantly different between PO Radj and PO R dist (p>0.05). Under both PER and
PO R adj the portal vein was allowed to become hypoglycemic, 44+1.8 mg/dl. Only
during PO R dist was the portal vein maintained normoglycemic, averaging 99 + 5.4 mM
between 60 and 105 minutes of the hypoglycemic clamp. In order to sustain elevated
portal vein and hepatic glycemias, and allow for identical arterial glucose
42
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concentrations between the three experimental protocols, the glucose infusion rates for
POR were significantly elevated above those for PER (p<0.05), from 30 min through
the duration of the hypoglycemic clamp (Fig. 8).
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POR-ADJ POR-DIST A- PER
5 ? 15
-30 0 30 60 90 120
Time (min)
F igure 8. Glucose infusion rates expressed as means ± SEM for the three glucose
infusion conditions during the hyperinsulinemic-hypoglycemic clamp. Solid symbols ▲
and ■ represent the peripheral and P O R dist experiments respectively, while the open
symbol, O , represents PORadj. * indicates significance between infusion protocols (p<
0.05).
44
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150
A.
'3 k 120 PORADJ
-A - PER
200
^ 160 •
40
225
1 , 180
S 135
120
Time (min)
F igure 9. Values are expressed as means ± SEM for arterial glucose concentration
(A), estimated hepatic glucose concentration (B), and estimated portal vein glucose
concentration (C) at basal and during the hyperinsulinemic-hypoglycemic clamp. Solid
symbols A & ■ represent the peripheral and PO R dist experiments respectively, while
the open symbol, O , represents PO Radj. * indicates significance between infusion
protocols (p< 0.05).
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In response to whole body hypoglycemia, PER, arterial epinephrine concentrations
increased 16 fold, from basal 529 ±115 pg/ml to a mean of 8504 ± 785 pg/ml by 105
min (Fig. 3). Infusing glucose into the liver only, reestablishing hepatic euglycemia
had no affect on the maximal epinephrine response (7690 ± 615 pg/ml; p=0.73).
However during PORdist, when both the portal vein and liver were maintained
normoglycemic, there was a 67% suppression in the epinephrine response when
compared either to PER or PORa d j (p<0.001 for both).
Although the norepinephrine response was less dramatic than that of epinephrine,
2.7 vs. 16 fold above basal for PER, a similar pattern was observed between protocols.
In response to whole body hypoglycemia, (PER), norepinephrine increased from 685 ±
297 to 1837 ± 242 pg/ml (Fig. 3). During PORadj, norepinephrine levels increased to
1759 ±217 pg/ml, not significantly different from PER (p=0.84). By comparison,
there was a significant suppression in the norepinephrine concentration for PORdist
(1296 ± 184 pg/ml) when compared to PER and PORad j, during the final sampling
interval (p=0.035).
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POR-DISTAL
□ POR-ADJACENT
PERIPHERAL *
I , 2000
= 1500
S. 1000
60 75 90
Time (min)
F igure 10. Epinephrine (A) and norepinephrine (B) concentrations at basal and during
sustained hypoglycemia, expressed as mean values ± SEM. Solid bars represent
PO R dist, open bars represent PO Radj, and gray bars represent PER. * indicates
significance between PO R dist v s . PO R adj and PER (p < 0.05).
47
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3. Discussion
The current findings demonstrate that the magnitude of the sympathoadrenal
response to hypoglycemia is dictated by the blood glucose concentration present in the
portal vein, not the liver. Clamping arterial blood glucose concentrations ensured that
all tissues, except the portohepatis, were exposed to identical levels of glucose during
the three experimental protocols, i.e. PER, PORdist, PORadj- When the liver and
portal vein were allowed to become hypoglycemic along with the rest of the body, we
observed a full-blown sympathetic response that included a 16-fold increase in
epinephrine and ~3-fold increase in norepinephrine. When glucose was infUsed
adjacent to the liver (PORa d j,) such that liver glycemia, but not portal glycemia, was
normalized a similar, i.e. full-blown, sympathetic response was observed.
In sharp contrast, a significant suppression in the sympathoadrenal response to
hypoglycemia (67% for epinephrine and 50% for norepinephrine) was observed only
when the blood glucose concentrations for both the portal vein and liver were
normalized (PORdist). As the only difference between PORdist and PORadj was the
concentration of glucose in the portal vein, these findings demonstrate a portal vein
locus for glucosensors which mediate sympathoadrenal counterregulation.
While glucose infused into the portal vein significantly elevated the glucose
concentration in that region, osmolality changes were minimal, on the order of 1.2%.
Osmoreceptors believed to be present in the portal vein, have been implicated in the
release of vasopressin, however that such afferent input influences sympathoadrenal
48
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counterregulation is not substantiated. Glucose sensitive afferents decrease discharge
rate in response to increasing glucose concentration, while in contrast portal
osmoreceptor firing rate increases in response to solute elevations [131]. Furthermore,
glucose sensitive afferents in the portohepatic region were shown to be insensitive to
changes in osmolality (changes were ~17 mOsm) and specifically responsive to
alterations in D-glucose [132].
It should be recognized that the rate of glucose infused into the portal vein was
such that the maximum elevation in plasma osmolality was ~ 3 mOsm. While such
changes have been shown to be significant for central, i.e. brain, osmoreception,
receptors in the portal vein appear much less sensitive [131, 132]. The threshold for
activation of the portal osmoreceptors has been investigated and appears to respond to
elevations on the order of 15-20 mOsm, or five times the maximal elevation in the
current investigation [131], In addition, portal osmoreceptors in the rat do not respond
simply to changes in plasma osmolality [131]. That is, similar changes in osmolality
induced via sodium chloride were found to lead to substantially greater responses when
compared with other compounds such as sucrose or mannose. Such receptors appear
even less sensitive to comparable osmolality changes with glucose [132]. Based on the
evidence from previous experiments it appears highly unlikely that the small changes in
osmolality that occurred in the portal vein as a result of glucose infusion had any effect
on portal vein osmoreceptors, nor impact the sympathoadrenal response to systemic
hypoglycemia.
49
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In that glucose infusion via PORdist caused catecholamine suppression during
cerebral hypoglycemia, suggests the importance of portal glucosensors upon overall
sympathoadrenal counterregulation. Findings from other laboratories implicating the
exclusivity or predominance of the CNS in glycemic detection and modulation of
counterregulation, is not substantiated by the current investigation. In addition,
equivocal findings from the various brain clamp and VMH microdialysis investigations
cast further doubt on the prevailing concept of exclusive central glucose sensing [21,
24, 34, 35, 75],
There is however consistent and substantial neurophysiological data supporting a
portal vein glucosensor locus, as well as a link between this sensory mechanism and
hepatic vagal afferents. The portohepatic region is known to be innervated by afferent
fibers sensitive to changes in metabolite concentration [10, 20, 132], Fluorescent
staining techniques have shown hepatic vagal afferents, believed to be associated with
these portal glucosensors, terminating in the adventitia of the portal vein [20].
Glucose-sensitive afferents, localized to the portal region, were found to possess firing
rates inversely proportional to portal vein glucose concentration [132]. The portal vein
glucose concentration has also been shown to impact upon firing rates of various
aspects of the brain (lateral hypothalamus, medial hypothalamus, and nuclear tractus
solitarius) as well as sympathetic and parasympathetic fibers innervating the liver,
pancreas, and adrenals [153, 164, 137].
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While there is no conclusive data to reconcile the ongoing dispute as to the
importance of central vs. peripheral glycemic detection, it is of particular interest
however, the constancy which our laboratory has demonstrated the impact of
portohepatic glucosensors on sympathoadrenal counterregulation. Given that the
current study was conducted in the rat vs. previous investigations in the dog, the
findings are surprisingly similar. Remarkably, the percent suppression in the
catecholamine response to portal vein glucose normalization during progressive
hypoglycemia was 73% & 67%, and 67% & 50%, for epinephrine and norepinephrine
in the dog and rat respectively. These similar observations in the rat and dog models
suggest a conserved mechanism for glycemic detection in these two mammalian
species.
While these findings do not exclude other glucosensing loci, i.e. the brain, they do
suggest an important role for portal vein glucosensors in controlling sympathoadrenal
counterregulation. That similar observations have been forthcoming for two different
mammalian species greatly increases the probability that portal glucosensors are critical
for glucoregulation in other species, including humans.
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B. HEPATIC ARTERY GLUCOSE INFUSION
The purpose of this investigation was to ascertain whether hypoglycemic detection
may also occur in the hepatic artery. In addition, as normoglycemia was established in
the hepatic artery and liver during irrigation, a negative finding during hepatic artery
glucose infusion would confirm the lack of glucosensing by the liver.
1. Research Design & Methods
Anim als a n d surgical procedures. Experiments were conducted on conscious male
mongrel dogs (30.2 + 1.2 kg; n=7). Dogs were housed under controlled conditions (12
h light / 12 h dark) in the university vivarium and were fed once per day with standard
chow (25% protein, 9% fat, 45% carbohydrate; Wayne Dog Chow, Alfred Mills,
Chicago). Dogs were used for experiments only if judged to be in good health as
determined by body temperature, hematocrit, regularity of food intake, and direct
observation. All surgical and experimental procedures were approved by the University
of Southern California Institutional Animal Care and Use Committee.
One week prior to initiating the experiment, animals were chronically cannulated
under anesthesia, induced by sodium thiamylal (Biotal, Bio-Ceutic Laboratories), and
maintained with 0.5-1.0% halothane and nitrous oxide. The common hepatic artery
(HA) was cannulated directly and secured so as not to occlude flow (Wollner, et al.)
The tip o f the cannula was advanced 1.5 cm past the origin of the left gastric artery and
the gastroduodenal artery ligated. Cannulas (Tygon, ID = 0.13 cm) were also placed
in the carotid artery (sampling) and the jugular vein (insulin infusion). In addition, a
52
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femoral vein cannula advanced to the inferior vena cava, superior to the hepatic vein,
was used for sampling mixed hepatic venous blood. An inflatable cuff (Model VO-4,
Rhodes Medical) was placed around the inferior vena cava caudal to the hepatic vein.
Inflation of the cuff temporarily occludes sub-hepatic vena caval flow, allowing mixed
hepatic venous blood to be sampled from the femoral catheter. All cannulas and the
actuating tubing for the inflatable cuff were tunneled subcutaneously and exteriorized at
the back of the neck. The cannulas were filled with heparinized saline (100 U/ml),
coiled and capped, and placed in a small gauze pouch secured to the back of the neck
with athletic tape. Catheter placement was confirmed at necropsy.
Experim ental design. Each animal participated in two hyperinsulinemic-
hypoglycemic clamp experiments distinguished by the site of glucose infusion:
peripheral (cephalic vein = PER) and hepatic artery (HA)(Fig. 11). For PER,
intracatheters (19-gauge, Deseret) were acutely placed in the right cephalic vein for a
infusion of indocyanine green dye (ICG) and glucose. A constant infusion of ICG
(0.13 mg/min) was initiated at -120 min, followed by a 90-min equilibration period. A
30-min basal sampling period (-30 to 0 min) followed, during which serial samples,
arterial (glucose, insulin, ICG, epinephrine, norepinephrine, and glucagon), and hepatic
venous (ICG), were taken at 15-min intervals. At min 0, insulin infusion (5.0 mLUkg- 1
•min *') was initiated and maintained for the remaining 260 min of the experimental
period. Peripheral glucose was initiated simultaneously so as to clamp arterial glycemia
at -100 mg/dl for the next 60 min. Thereafter, the glucose infusion rate was adjusted
53
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every 10 min so as to achieve 10 mg/dl reductions in blood glucose every 40 min. This
provided for stepwise reductions in arterial glycemia, reaching a nadir of 40 mg/dl
between 220 and 260 min (Fig. 11). Serial blood samples were taken every 10 min for
glucose and insulin, and every 20 min for ICG during the 260 min experimental period.
Additional arterial blood samples were taken every 10 min during the final 20 min (i.e.
20, 30, and 40 min) at each stage for measurements of epinephrine, norepinephrine, and
glucagon.
An identical protocol was employed for HA, however glucose was infused via the
hepatic artery instead o f the cephalic vein. By design, hepatic artery glucose infusion
allowed hepatic artery and liver glycemias to remain markedly elevated above arterial
concentrations. Each animal was employed for both PER and HA with one week
interval between experiments. The experimental order of protocols was randomized so
as to avoid an order effect.
Assays. Arterial plasma was assayed on-line for glucose by the glucose oxidase
method (YSI, Yellow Springs, OH). Arterial and hepatic venous plasma ICG
concentrations were determined at 805 nm spectrophotometrically immediately upon
collection. Arterial blood samples (2 ml) for glucagon analysis were collected in chilled
culture tubes containing NaFl, heparin, and 100 pi apoprotein (Trasylol, Miles Inc.).
Prior to storage at 4°C, plasma was treated with ETOH, centrifuged, and the
supernatant evaporated and reconstituted in 1 ml of buffer for subsequent analysis.
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Progressive Hypoglycemia
i i
1----- 1
GINF (variable)
Insulin (5 mU/kg x min)
ICG (0.13 mg/min)
1 2 0 ' - 6 0 0
2 6 0
Time (min)
Figure 11. Schematic representation of the hyperinsulinemic-hypoglycemic clamp
protocol.
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A
B
GLUCOSE INFUSION
H EA R T
LIVER
LIVER
LUCtiSE INFUSION
HEPATIC ARTERY
H E P A T IC I
V E N O U S \
EFFLU EN T
K IDNEY
KIDNEY
Arterial Portal Vria Liver HtprtteAftety Arterial Portal Vtta U w r R«pnkAite«y
Figure 12. Schematic representation of the two glucose infusion conditions: (A)
peripheral (PER = cephalic vein), (B) hepatic artery (HA). Insulin was infused via
jugular vein to induce systemic hypoglycemia. Glucose was infused at a rate to clamp
the arterial glucose concentration during the progressive hypoglycemic protocol. The
occlusive cuff placed around the inferior vena cava allowed for sampling o f mixed
hepatic venous blood to determine hepatic plasma flow.
56
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Radioimmunoassays were utilized to measure insulin [68] and glucagon (Novo-Nordisk
kit 32, antisera K-5S63, Copenhagen) in duplicate. Arterial blood samples for
catecholamines (2 ml) were collected in chilled culture tubes containing heparin and
200|il of antioxidant, were centrifuged, and the plasma retained at -60°C for
subsequent analysis. Epinephrine and norepinephrine concentrations were assayed
using a single isotope radioenzymatic approach [66].
Calculations. Total hepatic plasma flow was calculated from the arterial and
venous ICG concentration difference, with the assumption of no extrahepatic the ICG
uptake. Hepatic plasma flow in dl/min was calculated
Hepatic plasma flow = I icg/ (ICGA - IC G h v ) (3)
where I ic g is the ICG infusion rate (0.13 mg/min), IC G a is the arterial ICG
concentration, and IC G hv is the hepatic-venous ICG concentration. The rate of
hepatic artery glucose infusion required to maintain the desired level of liver glycemia
during the liver irrigation protocol was calculated as
GINFp = [HPF x ( G l - G a) ] / Body Wt. (4)
where GINFp is the hepatic artery infusion (mg.kg'^min1 ), HPF is hepatic plasma flow
as determined by ICG (eq. 1), G l is desired level of liver glycemia, and G a is the
sampled arterial glucose concentration during the hypoglycemic clamp. The mean
hepatic glycemia (MHG) in mg/dl was calculated as
MHG = G a + (GINFp (m g/m in ) / HPF (dl/min)) (5)
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where Ga = the arterial glucose concentration, GINFP = the glucose infusion, and
HPF = the hepatic plasma flow. The mean hepatic artery glycemia (MHAG) in mg/dl
was calculated as
MHAG = Ga + (GINFp ( m g / m m ) / (HPF ( d i / m i n ) x 0.25)) (6)
where Ga = the arterial glucose concentration, GENFP = the glucose infusion, HPF =
hepatic plasma flow, and 0.25 = hepatic artery flow as a percentage of the total hepatic
plasma flow.
Statistical analyses. Comparisons between treatments over time were assessed
using repeated measures analysis of variance with Tukey’s test for post-hoc
comparisons. Additionally, paired t-tests were employed where appropriate for within
animal pre- post- comparisons. Statistical significance was established at the p < 0.05
level.
2. Results
For both experimental protocols, arterial insulin concentrations increased
significantly from a value of 19 ± 0.65 p.U/ml at basal, to a hyperinsulinemic plateau of
336 + 7.8 pU/ml during the 260 min insulin infusion. No significant differences were
observed between protocols with respect to basal or elevated insulin concentrations
(p>0.05). There were no significant differences between the two infusion protocols for
basal arterial glucose concentration (94 ± 1.8 mg/dl), glucose infusion rate (Fig. 13), or
glucose concentration during the six 40 min stages of progressive hypoglycemia (Fig.
14 A).
58
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Hepatic artery, liver, and portal vein glycemias were allowed to fall concomitantly
with the arterial glucose during PER, decreasing to a hypoglycemic nadir of 40 ± 1.9
mg/dl (Fig. 14). As a result of hepatic artery glucose infusion (HA), hepatic artery and
liver glycemias were significantly elevated above those for PER, from minute 40 to
termination o f the experiment at 260 min (p=0.001). The portal vein was allowed to
become hypoglycemic in parallel to the systemic circulation, with no significant
differences between the two infusion protocols, HA vs. PER (Fig. 14 C).
In response to whole body hypoglycemia, PER, arterial epinephrine concentrations
increased approximately 8-fold from basal, 102 ± 20 pg/ml, to a mean of 808 ± 271
pg/ml by the final 40 minute stage of hypoglycemia (Fig. 15 A). Infusing glucose via
hepatic artery had no significant effect on epinephrine concentrations (858 ±211 pg/ml;
p=0.74). A similar pattern was observed between infusion protocols for
norepinephrine. In response to whole body hypoglycemia, (PER), norepinephrine
increased from basal 143 + 34 to 741 ± 263 pg/ml (Fig. 15 B). During HA,
norepinephrine levels increased from 133 ± 29 to 557 ± 186 pg/ml, not significantly
different from PER.
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- a - PER
- A - HA
' I A
T /
260 140 200 20 80
Time (min)
F igure 13. Glucose infusion rate expressed as mean ± SEM for the two infusion
conditions during the hyperinsulinemic-hypoglycemic clamp. ▲, peripheral (PER)
experiment; A, hepatic artery (HA) experiment. * Significance between infusion
protocols (P<0.05)
60
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Figure 14. Values are expressed as means ± SE for arterial (A), estimated hepatic
glycemia (B), estimated portal vein glycemia (C), and estimated hepatic artery glycemia
(D) as a function of time. ▲, peripheral (PER) experiment; A , hepatic artery (HA)
experiment. * Significance between infusion protocols (P<0.05).
61
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F igure 15. Average values (means + SE) for epinephrine (A) and norepinephrine (B)
concentration at basal (bar 1) and during progressive hypoglycemia (bars 2-7).
Average values for individual animals were determined from three samples taken at 10
min intervals over the final 30 min of each sampling period. Solid bars represent PER
and open bars represent POR * Significance between infusion protocols (P<0.05).
62
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3. Discussion
The current findings demonstrate that the magnitude of the sympathoadrenal
response to hypoglycemia is unaffected by the blood glucose concentration in the
hepatic arteiy. Despite a substantial elevation in hepatic artery glucose concentration
during HA infusion, the catecholamine response to systemic hypoglycemia was not
significantly different from that during hepatic artery hypoglycemia (PER). If essential
hypoglycemic sensors were located in the hepatic arteiy, glucose infusion via the
hepatic artery should have led to a suppression in the sympathoadrenal response.
While the cannula was placed 1.5 cm distal from the origin of the left gastric artery,
it is possible that sensors may reside more distal from this location, and consequently
the surgical preparation may have posed a significant limitation for this investigation.
While the presence of such sensors distal from the hepatic artery cannula is possible, it
is highly unlikely. As glucose infusion via the hepatic artery perfused almost the entire
length of the common hepatic artery and liver, glucosensors present upstream from the
cannula tip in anatomical terms would be inconsistent with portal vein findings of a
proximal pre-hepatic locus. Although the hepatic artery only supplies 25% o f the total
hepatic blood flow, previous investigations utilizing technitium macroaggregated
albumin scans, demonstrated that common hepatic artery cannulation yielded the most
complete liver perfusion as compared to other sites that could have been employed, i.e.
gastroduodenal or splenic arteries [32].
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Liver
Hepatic Ducts
Cystic
Ducts
Portal Vein
Hepatic Artery
Bile Duct
Common Hepatic Artery
I / Anterior Plexus Gastroduodenal
Artery
Posterior
Plexus
Duodenum
F igure 16. Ventral view of the hepatic neural and vascular architecture. The liver
receives nerves via two major plexuses: the anterior hepatic plexus running from the
celiac ganglion along the trunk of the common hepatic artery, and the posterior hepatic
plexus passing to the liver along the portal vein. The arrows indicate the sites
commonly ablated for the induction of hepatic denervation. Site two was utilized for
phenol denervation by Lamarche (1995). Reproduced from [114].
64
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Ligation of the gastroduodenal artery in addition to the completeness with which the
liver was perfused (subjective observation of evans blue infusion post-mortem),
indicates that glucose distribution in the hepatic artery and liver was not limiting, nor
contributed to negative findings.
Results from the current investigation suggest the absence of important
glucosensors in the hepatic artery and liver. As the rate of glucose infusion via the
hepatic arteiy was sufficient to normalize total liver glucose during systemic
hypoglycemia, the existence of essential glucosensors within the liver would have lead
to a suppression in the sympathoadrenal response. To the contrary, it was shown that
hepatic artery and liver glucose concentration had no impact on sympathoadrenal
counterregulation. Our current findings with hepatic artery glucose infusion are
entirely consistent with a lack of essential glucosensing by the liver.
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C. Portal Vein Denervation
The purpose of this investigation was to demonstrate that afferents innervating the
portal vein are critical for hypoglycemic detection and normal sympathoadrenal
counterregulation.
1. Research Design & Methods
Anim als a n d surgical procedures. Experiments were conducted on male Wistar
rats (weight 274.8 ± 7 g, n=26) in the conscious relaxed state. All surgical and
experimental procedures were pre-approved by the University of Southern California
Institutional Animal Care and Use Committee.
One week prior to experiments, animals were chronically cannulated under single
dose anesthesia (3:3:1 ketamine HC1, xylazine, acepromazine maleate; 0.10 cc /100 g
body weight, given intramuscularly). Following a mid-line abdominal incision and
abdominal content reflection, phenol (90% w/v) was applied to a 2.5 cm region of the
portal vein proximal to the liver (PDN). Phenol was applied via cotton tip applicator.
Denervation as noted by portal vein discoloration (vessel turned white upon
application), was observed in all phenol painted animals (n=l5). Phenol was painted on
the vessel and allowed to dry for one minute. Two milliliters of a warm saline was used
on the portal vein to dilute and remove excess following each application. This
procedure was performed three times to ensure afferent destruction. The same general
procedure was performed on the remaining animals, however phenol was substituted
with 0.9% saline (SHAM). Following phenol or saline application, cannulas were
66
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placed in the portal vein (Silastic, 0.03 cm internal diameter [ID]) for glucose infusion
during liver irrigation (POR), carotid artery (Clay Adams; PE-50) for arterial blood
sampling, and jugular vein (dual cannula silastic, ID = 0.03 cm ID) for insulin and
peripheral glucose infusion (PER). All cannulas were tunneled subcutaneously and
exteriorized at the back of the neck encased in silastic tubing (0.18 cm ID) sutured to
the skin. Animals were allowed five days recovery from surgery to regain body weight.
Three days post-surgery 300 pi of blood was drawn and analyzed for plasma alanine
amino transferase (ALT) activity, a sensitive measure of hepatocellular integrity.
Comparisons in ALT activity were made between all animals portally cannulated and
those litter mates randomly assigned to the blood donor group possessing only a
carotid cannula (15.0 + 1.2 vs. 16.5 + 2.4 U/l, respectively). Twenty-four hours prior
to the experiment animals were fasted.
Hyperinsulinem ic-hypoglycem ic clamp. All animals (PDN-PER n=8, PDN-POR
n=7, SHAM-PER n=6, SHAM-POR n=5) were exposed to the same general protocol
for the induction of hypoglycemia. Prior to the experiment, animals were placed in a
modified metabolic chamber and allowed to rest for 30 min (-60 to -30 min). Basal
samples were drawn at -30 and 0 min for analysis of glucose and catecholamines. At
min 0, following arterial sampling, insulin (50 mU-kg'^min '*) and glucose infusions
(variable) were initiated and maintained for 105 min of the hypoglycemic clamp. Serial
sampling for glucose was performed at 10 min to maintain the integrity of the
hypoglycemic clamp and preserve the rate of fall in blood glucose. Arterial
67
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catecholamine and plasma glucose samples were taken at 60, 75, 90, and 10S min of
sustained deep hypoglycemia.
Experim ental design. Each animal participated in only one experiment,
distinguished by the site of glucose infusion, PER or POR (Fig. 17). Peripheral glucose
infusion (SHAM-PER) via the jugular vein served as the experimental control, whereby
whole body hypoglycemia was induced, portal vein nerves intact (Fig. 17 C). The
portal vein glucose infusion experimental condition, (SHAM-POR), whereby portal
vein and liver glucose concentrations were normalized during systemic hypoglycemia
with portal vein nerves intact, served as the experimental control for PDN-POR (Fig.
17 D). According to the working hypothesis, if portal glucosensors are mediated by
afferent nerves terminating in the portal vein, then phenol ablation would eliminate
transmission of metabolic information from this region to the CNS. Thus, portal
glycemia would have a diminished impact on sympathoadrenal counterregulation. That
is, there would be no significant difference in the catecholamine response between
PDN-POR vs. PDN-PER. In addition, assuming hypoglycemic detection by portal
glucosensors, the overall sympathoadrenal response to whole body hypoglycemia
would be blunted following portal vein denervation.
Calculations. The estimated liver glucose concentration (Gl) was calculated as:
Gt, = Ga + (G IN F por/ HPF) (7)
where GA= arterial glucose concentration (micromoles per milliliter), G IN F por = portal
glucose infusion rate (micromoles per minute), and HPF = hepatic plasma flow rate
68
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(milliliters per minute). The estimated portal vein glucose concentration (Gpv) was
calculated as
Gpv = GA + (GINF/ PVPF) (8)
where G I N F p o r d is t = distal portal glucose infusion rate (micromoles per minute), and
PVPF = portal vein plasma flow rate (milliliters per minute). Hepatic plasma flow was
estimated as 1.3 ml • g*1 liver • min'1 x liver weight g, and the portal vein flow rate
assumed as 80% o f the total HPF [105].
A nalytical Procedures. Glucose was assayed online utilizing the glucose oxidase
method (YSI, Yellow Springs, OH). Epinephrine and norepinephrine concentrations
were assayed using a single-isotope radioenzymatic approach [66, 146]. Basal rat
insulin and hyperinsulinemic porcine samples were assayed via RIA (Linco Research
Inc. St. Charles, MO). Alanine aminotransferase was assayed spectrophotometrically
[148],
Data analysis. The results are expressed as means + SE. Comparisons of animal
characteristics between groups were made using one-way ANOVA for independent
groups. Comparisons between treatments over time were made by repeated measures
ANOVA utilizing Tukey’s test for post-hoc comparisons. Data was compared
between the two denervated (PDN) experiments, whole body hypoglycemia (PDN-
PER) vs. portal vein and liver normalization (PDN-POR), as well as between the PDN
experiments and the respective control experiment, SHAM-PER and SHAM-POR
Significance was established at p<0.05.
69
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A. B.
GLUCOSE INFUSION
P E R I P H E R A L
HEART
LIVER
KIDNEY
c.
HEART
LIVER
GLUCOSE T O U IO N
PORTAL VEIN
KIDNEY
D.
GLUCOSE INFUSION
PERIPHERAL
H E A R T
LIVER
KIDNEY
HEART,
LIVER
GLUCOSE INFUSION
PORTAL VEIN
KIDNEY
Figure 17 . Schematic diagram of the four experimental conditions distinguished by
the site of glucose infusion and the surgical manipulation: (A) peripheral glucose
infusion via jugular vein in SHAM operated animals (SHAM-PER), (B) portal vein
glucose infusion with the cannula positioned upstream from the liver in SHAM
operated animals (SHAM-POR), (C) peripheral glucose infusion via jugular vein in
portally denervated animals (PDN-PER), and (D) portal vein glucose infusion in
portally denervated animals (PDN-POR).
70
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2. Results
For all experimental conditions, arterial insulin concentrations increased from a
mean basal value o f 16.6 ± 5.5 pU/ml to a hyperinsulinemic plateau o f 2677 ±313
pU/ml. No significant differences were observed between protocols for basal or
elevated insulin concentrations. Basal arterial glucose concentrations (124.8 ± 3.48
mg/dl) were not significantly different between protocols. By design, there were no
significant differences in arterial glucose concentrations during deep sustained
hypoglycemia (44.8 ± 1.64 mg/dl; p=0.89) among the four experimental glucose
infusion conditions, nor for the glucose infusion rates required to clamp arterial glucose
concentration (Fig. 18). Portal vein and hepatic glycemias were allowed to fall
concomitant with arterial concentrations during PER for both SHAM and PDN (Fig.
19). In contrast, portal vein and liver glycemias were elevated in the normal range
(89.5 ± 3.7 mg/dl, 71.3 ± 3.2 mg/dl, respectively) during liver irrigation (POR) for
both SHAM and PDN. There were no significant differences in hepatic glycemias
between SHAM-PER or PDN-PER, nor between SHAM-POR or PDN-POR, however
hepatic glycemias for both POR experiments, SHAM and PDN, were elevated
significantly above the hepatic glycemias for both PER conditions (p<0.05).
In response to whole body hypoglycemia (PER), arterial epinephrine concentrations
increased 12-fold in SHAM, from basal 687 ± 62 pg/ml to a mean of 8156 ±1115
pg/ml by 105 min (Fig. 20). However, under the same level of hypoglycemia, the mean
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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F igure 18. Data are expressed as means ± SE for glucose infusion rate (GINF) during
the hyperinsulinemic-hypoglycemic clamp. V, PDN-PER experiment; A , SHAM-PER
experiment; □, PDN-POR experiment; ■ , SHAM-POR experiment. * Significance
between infusion protocols (P<0.05).
72
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and estimated portal vein glucose concentration (B) at basal and during the
hyperinsulinemic-hypoglycemic clamp. V, PDN-PER experiment; ▲, SHAM-PER
experiment; □, PDN-POR experiment; ■ , SHAM-POR experiment. * Significance
between infusion protocols (P<0.05).
73
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sustained hypoglycemia for SHAM animals, expressed as mean values ± SE. Open
bars represent SHAM-PER, and solid bars represent SHAM-POR. * Significance
between infusion protocols (P<0.05).
74
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Figure 21. Epinephrine (A) and norepinephrine (B) concentrations at basal and during
sustained hypoglycemia for peripheral glucose infusion experiments (PDN and SHAM),
expressed as mean values + SE. Open bars represent SHAM-PER, and hatched bars
represent PDN-PER. * Significance between infusion protocols (P<0.05).
75
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Figure 22. Epinephrine (A) and norepinephrine (B) concentrations expressed as
means ± SE for basal and the final two sampling periods (90 & 105 min) of sustained
hypoglycemia. Black bars represent SHAM-POR, gray bars represent PDN-POR,
open bars represent SHAM-PER, and hatched bars represent PDN-PER. *
Significance between SHAM-PER vs. PDN-PER, PDN-POR, and SHAM-POR
protocols (P<0.05).
76
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epinephrine response was blunted by 38% in portally denervated animals (5050 ± 698
pg/ml; p=0.002; Fig. 21). When glucose was infused in SHAM-POR such that portal
and liver glycemias were normalized, there was a 50% suppression in the epinephrine
response vs. SHAM-PER, despite cerebral hypoglycemia (4068 ± 573 pg/ml; pO .O l;
Fig. 20). In contrast, portal glucose infusion in portally denervated animals, PDN-POR
(3877 + 366 pg/ml; p = 0.90), had no impact on the sympathoadrenal response to
systemic hypoglycemia (Fig. 22). That is, during portal glucose infusion, there was no
significant suppression in the epinephrine response observed for PDN-POR vs. PDN-
PER.
Although the norepinephrine response was less dramatic than the epinephrine
response, 2.6- vs. 12-fold above basal for PER, a similar pattern was observed between
the four glucose infusion conditions. In response to whole body hypoglycemia during
SHAM-PER, norepinephrine increased from 633 ± 91 to 1646 ± 144.5 pg/ml (Fig. 21).
In sharp contrast, during whole body hypoglycemia in PDN animals, there was a
significant blunting, 36%, o f the norepinephrine response (PDN-PER 1053 ±119 pg/ml
vs. SHAM-PER; p<0.01). In marked contrast to SHAM-PER, when the portal vein
glucose concentration was normalized in SHAM-POR, there was a significant, 39%,
suppression in the norepinephrine response (1001 ± 205 pg/ml; p<0.01; Fig. 20).
However, this suppression was not observed during portal glucose infusion in portally
denervated animals (PDN-POR 916 + 212 pg/ml). There was no significant difference
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
between PDN-PER vs. PDN-POR (p=0.90; Fig. 22), in the norepinephrine response to
systemic hypoglycemia.
3. Discussion
The current findings indicate that portal vein afFerents are essential for normal
sympathoadrenal counterregulation. This work illustrates that these sensors are true
glucose sensors and respond to the range o f glucose concentrations within the portal
vein. That is, these portal vein glucosensors modulate the sympathoadrenal response
according to the absolute glucose concentration in the portal vein and are not strictly
sensitive to elevations in glycemia alone.
Clamping arterial glucose concentrations ensured that all tissues except the
portohepatis, were exposed to identical levels o f glucose during the four experimental
protocols: SHAM-PER, SHAM-POR PDN-PER PDN-POR When portal vein
afFerents remained intact, and the portal vein glucose concentration was allowed to fall
concomitantly with arterial concentrations, a full-blown sympathoadrenal response was
observed, including a 12 fold increase in epinephrine and ~ 3 fold increase in
norepinephrine. In contrast, when portal vein afFerents were destroyed and whole body
hypoglycemia was induced, the epinephrine and norepinephrine responses were blunted
by 38% and 36% respectively.
When portal vein glucose concentrations were normalized in sham operated
animals, there was a significant suppression (50% epinephrine, and 39%
norepinephrine) in the sympathoadrenal response, despite cerebral hypoglycemia. This
78
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finding is consistent with our previous investigations. Sympathoadrenal suppression
during portal vein glucose normalization was not observed for portally denervated
animals. That is, contrary to the results for SHAM-PER vs. SHAM-POR, there was no
significant difference in the catecholamine response between PDN-PER vs. PDN-POR.
As a significant blunting o f the normal sympathoadrenal response to whole body
hypoglycemia (PDN-PER) was observed, and that there was no characteristic
suppression in the sympathoadrenal response to systemic hypoglycemia during portal
vein normoglycemia, reflects the importance o f afferent innervation for gluco sensor
function.
To elucidate the importance o f portal vein afferent innervation a topical application
o f 90% phenol was painted on the portal vein. In a comparative investigation o f
denervation techniques, Lautt demonstrated that denervation appeared within 20
minutes and lasted up to 14 days following phenol application [49, 116], This method
o f denervation was found to have no impact on basal glucose, insulin or glucagon
levels, hematocrit, blood pressure, or hepatic glycogen concentration. As these
parameters are indices of stress and metabolic status, phenol application appears to
maintain a localized effect. Furthermore, upon necropsy, Lautt found the only
observable change resulting from phenol application was the appearance o f adhesions
and discoloration o f painted tissues [116], The methodological investigations by Lautt
and Cucchiaro, demonstrated that phenol denervation is less traumatic, faster, and more
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certain than surgical denervation. In addition no significant re-innervation was
observed for several weeks following the denervation procedure [49,116],
Although it has been demonstrated that phenol application is a safe and effective
means for inducing denervation, the primary limitation o f the current investigation is
the inability to quantify the degree o f denervation. Given that there was pronounced
discoloration o f the vessel following each o f the three phenol applications, and that
there were significant decrements in glucosensor function, the results garnered from
this surgical procedure imply partial if not full portal vein afferent destruction.
In recent years many have focused on the liver as the primary locus for
portohepatic glucosensors, as such, complete liver denervation was employed to assess
the role of liver nerves in glycemic detection. In response to varying methodological
techniques, the effect o f hepatic denervation on sympathoadrenal counterregulation has
yielded equivocal findings. Lamarche (1995), the only investigation in which 90%
phenol was painted on the liver vasculature (including portal vein), demonstrated a
90% and 82% attenuation in maximal net response for epinephrine and norepinephrine
respectively, from the adrenal medulla. The adrenal venous catecholamine
concentrations were blunted by 68% and 53% for epinephrine and norepinephrine
respectively, following 45 min o f deep hypoglycemia (Fig. 23). Interestingly, in a
subsequent investigation showing no effect o f hepatic denervation on adrenal output,
the portal vein was surgically stripped o f nervous tissue instead of phenol painted
[106],
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Basal Hypoglycemia
Figure 23. Values are expressed as means ± SE for adrenal epinephrine (A) and
norepinephrine concentrations (B) for SHAM operated (open bars) and hepatic
denervated (HPDN; closed bars) animals at basal and following 45 minutes o f deep
hypoglycemia (50 mg/dl). *, significant difference (p<0.05) between groups. Adapted
from [110].
81
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While in the aforementioned investigation it is clear that livers were successfully
denervated (<1% o f normal liver norepinephrine) following surgical stripping o f the
hepatic vasculature, it is unclear the extent to which portal vein afFerents were
destroyed. It is not surprising that elimination o f hepatic afferent signaling via liver
denervation had no impact on hormonal counterregulation during whole body
hypoglycemia, as glucosensors have now been localized to the portal vein, not the liver
[106], That sympathoadrenal counterregulation was not impaired by liver denervation,
a lack o f essential glucosensing by the liver is again suggested.
The current investigation employing phenol denervation o f the portal vein yielded
the first in vivo evidence supporting a portal-sympathoadrenal reflex loop functionally
important for sympathoadrenal modulation in response to insulin induced decrements in
glycemia. Portal vein glucosensors, the sensory limb o f this neural reflex, are also
shown to be responsible for the suppression o f sympathoadrenal counterregulation due
to increments in glycemia that occur as exogenous glucose becomes available. Thus, it
is suggestive o f a mechanism allowing for the preservation o f glycogen stores. As the
catecholamine response to hypoglycemia was significantly impaired by portal vein
denervation, neural mediation o f portal vein glucosensors appears critical for normal
sympathoadrenal counterregulation.
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IV. SUMMARY AND CONCLUSIONS
The maintenance o f blood glucose within very narrow limits is essential for the
protection o f central nervous system function. Although efferent aspects o f
counterregulation are well characterized, little is known about the mechanism for
glucose sensing. Ascertaining the precise locus for hypoglycemic detection has taken
on renewed interest in recent years as the advent o f more aggressive therapies in the
treatment o f diabetes has significantly increased the incidence o f hypoglycemia. The
prevalence o f hypoglycemic episodes among IDDM patients has been attributed to
imprecise counterregulation, a defect shown to be specific for glucose. Such specificity
implicates a defect in the afferent limb, i.e. glucose sensing. Thus, elucidation o f the
precise locus for glycemic detection is imperative to understanding the impairment in
hormonal counterregulation.
Although glycemic detection has been traditionally attributed to the CNS, previous
investigations from our laboratory have demonstrated an important role for the
portohepatic region in hypoglycemic detection and sympathoadrenal counterregulation.
It was the purpose o f the current investigations to further constrain the locus for
glycemic detection to either the liver or supporting vasculature. In addition, the
importance o f gluco sensor afferent innervation for hypoglycemic detection and
sympathoadrenal counterregulation was ascertained.
A local liver irrigation technique during sustained systemic hypoglycemia was
performed in all three investigations with the pretense o f elevating glucose
83
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concentrations specifically in the liver, or the liver and the supporting vasculature. This
approach allowed for the further discrimination o f the glucose sensitive region(s) within
the portohepatis. As the sympathoadrenal response to systemic hypoglycemia is a
robust indicator o f metabolic stress during hyperinsulinemia and is well characterized
by this laboratory, catecholamine concentrations were utilized as the dependent
measure.
To constrain important glucosensors to either the liver or portal vein, systemic
hypoglycemia was induced via local normalization o f glucose concentrations in either
the liver (POR-ADJ), or the liver and portal vein (POR-DIST). Clamping arterial
glucose concentrations ensured that all tissues, except the portohepatis, were exposed
to identical levels o f glucose during the three experimental conditions: PER, POR-ADJ,
and POR-DIST. While both POR-ADJ and POR-DIST yielded elevated liver
glycemias during systemic hypoglycemia, only POR-DIST yielded an elevated portal
vein glucose concentration.
A significant suppression in the sympathoadrenal response was only observed when
the portal vein glucose concentration was normalized. During PER and POR-ADJ, the
portal vein glucose concentration reached a deep hypoglycemic level accordant with
the arterial circulation, thus a full sympathoadrenal response was observed for both.
That liver glycemia had no impact on the sympathoadrenal response, and that
suppression was only observed during portal vein normoglycemia, is consistent with the
localization of glucosensors to the portal vein, not the liver. These findings
84
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demonstrate that the magnitude o f the sympathoadrenal response to systemic
hypoglycemia was dictated by the glucose concentration present in the portal vein, not
the liver.
In that a portal locus suggests significance o f pre-hepatic glycemia, the purpose o f
the subsequent investigation was to determine the presence of glucosensors in the
hepatic artery. Normalization o f glucose concentrations in the hepatic artery and liver
were achieved via hepatic artery glucose infusion. Clamping systemic arterial glucose
concentrations ensured that all tissues, except the hepatic-arterial circulation, were
exposed to identical levels of glucose during the two experimental conditions:
peripheral glucose infusion and hepatic artery glucose infusion. During HA, the liver
and hepatic artery glucose concentrations were elevated well above the normal range
and significantly above the concentrations present in the carotid artery and portal vein.
By design, during the peripheral and hepatic artery glucose infusion protocols, the
portal vein glucose concentration reached a deep hypoglycemic level accordant with
the arterial circulation. Unlike the previous findings for the portal vein, normalization
o f hepatic artery glycemia had no impact on the sympathoadrenal response to systemic
hypoglycemia. These findings indicate a lack o f glucose sensing by the hepatic artery.
That there was no significant suppression in the sympathoadrenal response during
hepatic artery glucose infusion, (liver normoglycemia), is however consistent with the
previous investigation confirming glucosensors localized to the portal vein, not the
liver.
SS
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Neurophysiological data showing afferent innervation o f the portal vein adventitia,
in combination with the findings o f a portal vein glucosensor locus, compelled the
demonstration o f a neuraUy mediated portal vein glucosensing mechanism. The impact
o f phenol induced portal denervation on hypoglycemic detection and sympathoadrenal
counterregulation was assessed during systemic hypoglycemia with or without portal
vein glucose normalization. Liver and portal vein glucose normalization was achieved
via distal portal vein glucose infusion for both portally denervated and SHAM operated
animals. Clamping arterial glucose concentrations ensured that all tissues, except the
portohepatis, were exposed to identical levels o f glucose during the four experimental
conditions. PDN-PER, PDN-POR, SHAM-PER, and SHAM-POR.
Portal vein afferent destruction resulted in a significant blunting o f the
sympathoadrenal response to whole body hypoglycemia, suggesting impaired glycemic
detection. Unlike SHAM, when the portal vein glucose concentration was normalized
in portally denervated animals, no suppression in the sympathoadrenal response vs.
PDN-PER was observed. Denervation impacted detection of both portal vein hypo-
and hyperglycemia. These data are consistent with a glucose sensor responsible for
detecting a range o f glycemias, not a sensor strictly sensitive to elevations in blood
glucose. In conclusion, neural mediation o f portal vein glucosensors is critical for
hypoglycemic detection and normal sympathoadrenal counterregulation.
That glucose homeostasis is maintained via negative feedback system,
glucoregulatory reflex mechanisms controlling the modulation o f hormonal output in
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response to decrements in glycemia were previously proposed. Evidence from the final
investigation o f this dissertation illustrates for the first time in vivo, a neural link
between portal vein glucosensors and adrenomedullary output as well as the
importance o f this porto-sympathoadrenal neural reflex in hypoglycemic detection and
sympathoadrenal counterregulation.
While it has been shown that portal vein glucosensors are neurally mediated, the
specific afferent nerve responsible for transmission o f metabolic information from these
sensors to the brain remains to be elucidated. However, in situ evidence has shown an
inverse relationship between the glucose concentration within the portal vein and the
hepatic vagal afferent firing rate, suggesting a role for the hepatic vagus nerve in this
neural reflex. In addition, fluorescent microscopy has shown hepatic vagal afFerents,
believed to be associated with portal vein glucosensors, terminating in the adventitia o f
the portal vein. The specific function o f the hepatic vagus nerve in sympathoadrenal
counterregulation remains to be determined.
Defects in counterregulation in IDDM have previously been ascribed to the afferent
limb, but until now the link between deranged glucose sensing and counterregulation
had yet to be demonstrated in vivo. In that portal vein ablation, the site for
hypoglycemic detection, significantly impacted upon sympathoadrenal
counterregulation, is case indicative o f defective counterregulation specific to deranged
glucose sensing. Similar to the rat, phenol portal denervation in anesthetized dog was
found to significantly blunt sympathoadrenal counterregulation during hypoglycemia.
87
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Interestingly, in human liver transplant patients where a significant portion o f the portal
vein is removed and neural innervation destroyed, there was also a significant blunting
in the normal adrenomedullary response to systemic hypoglycemia, implicating a
conserved glucoregulatory reflex between mammalian species.
While the current findings do not exclude the importance of other gluco sensing
loci, i.e. the brain, these data collectively suggest that portal vein glucosensors are
critical for hypoglycemic detection and necessary to engender full sympathoadrenal
counterregulation. That similar observations have been forthcoming for two different
mammalian species, greatly increases the probability that portal vein glucosensors and
the porto-sympathoadrenal reflex characterized in the rat and dog, are also critical for
glucoregulation in other species including man.
Now that the peripheral locus for hypoglycemic detection is constrained to the
portal vein, and neural innervation of these sensors ascertained, it is important to
delineate the specific role o f portal vein glucosensors in integrated counterregulation.
In addition it will be as important to determine the properties of glucose sensor
function, thus allowing for the further characterization of mechanisms leading to
defective counterregulation in IDDM.
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REFERENCES
1. Adachi, A. 1974. An hepatic osmoreceptor mechanism in the rat: electrophysiological and
behavioral studies. Am. J. Physiol. 231:1043-1049.
2. Adachi, A., Shimizu, N., Oomura, Y., and M. Kobashi. 1984. Convergence of
hepatoportal glucose-sensitive afferent signals to glucose-sensitive units within the nucleus
of the solitary tract. Neurosci. Lett. 46:215-218.
3. Adkins-Marshal, B., Pagliassoti, M.J., Asher, J.R., Connolly, C.C., Neal, D.W., Williams,
P.E., Myers, S.R., Hendrick, G.K., Adkins Jr., R.B., and A.D. Cherrington. 1992. Role of
hepatic nerves in response of liver to intraportal glucose delivery in dogs. Am. J. Physiol.
262(25):E679-E686.
4. Allman, F.D., Rogers, E.L., Caniano, DA., Jacobowitz, D.M., and M.C. Rogers. 1982.
Selective chemical hepatic sympathectomy in the dog. Critical Care Medicine 2:100-103.
5. Amiel, S.A., Pottinger, R.C., Archibald, H.R., Chusney, G., Cunnah, D.T.F., Prior, P.F.,
and E.A.M. Gale. 1991. Effect of antecedent glucose control on cerebral function during
hypoglycemia. Diabetes Care 14:109-118.
6. Amiel, S.A., Simonsen, D.C., Tamborlane, W.V., DeFronzo, R.A., and R.S. Sherwin.
1987. The rate of glucose fall does not affect the counterregulatory hormone responses to
hypoglycemia in normal and diabetic man. Diabetes 36:518-522.
7. Amiel, S.A., Tamborlane, W.V., Sacca, L., and R.S. Sherwin. 1988. Hypoglycemia and
glucose counterregulation in normal and insulin-dependent diabetic subjects.
Diabetes/Metabolism Reviews 4(l):71-89.
8. Appia, F., Ewart, W.R., Pittman, B.S., and D.H. Wingate. 1986. Convergence of sensory
information from abdominal viscera in the rat brain stem. Am. J. Physiol. 251:G169-
G175.
9. Azanza, M.J. 1987. The vagal contribution to the liver innervation: a demonstration with
the cobalt impregnation method. Comp. Biochem. Physiol. 86A(2):275-279.
10. Baija, F., and R. Mathison. 1984. Sensory innervation of the rat portal vein and the
hepatic artery. J.Auton.Nerv.Syst. 10:117-125.
11. Barzilai, N., Hawkins, M., Angelov, I., Hu, M., and L. Rossetti. 1996. Glucosamine-
induced inhibition of liver glucokinase impairs the ability of hyperglycemia to suppress
endogenous glucose production. Diabetes 45:1329-1335.
89
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
12. Beard, J.C., Weinberg, C., Pfeifer, M.A., Best, J.D., Halter, J.B., and D. Porte Jr. 1982.
Interaction of glucose and epinephrine in regulation of insulin secretion. Diabetes 31:802-
807.
13. Benzo, C.A. 1983. The hypothalamus and blood glucose regulation. Life Sci. 32:2509-
2515.
14. Beigenstal, R.M., Polonsky, K., Pons, G., Jaspan, J., and A. Rubenstein. 1983. Lack of
glucagon response to hypoglycemia in type I diabetics after long-term optimal therapy with
a continuous subcutaneous insulin infusion pump. Diabetes 32:398-402.
15. Bergman, R.N., Beir, J., and P. Hourigan. 1982. Intraportal glucose infusion matched to
oral glucose absorption: lack of evidence for “gut factor” involvement in hepatic glucose
storage. Diabetes 31:27-35.
16. Berk, M.A., Clutter, W.E., Skor, D., Shah, S.D., Gingerich, R.P., Parvin, C.A., and P.E.
Cryer. 1985. Enhanced glycemic responsiveness to epinephrine in insulin-dependent
diabetes mellitus is the result of the inability to secrete insulin. J. Clin. Invest. 75:1842-
1851.
17. Bernard, C. 1849. Chiensrendus diabetiques. Compt. Rend. Soc. Biol. 1:60.
18. Berthoud, H.R., Kressel, M., and W.L. Neuhuber. 1992. An anterograde tracing study
of the vagal innervation of rat liver, portal vein and biliary system. Anat. Embryol.
186:186-431.
19. Berthoud, H.R., Carlson, N.R., and T. Powley. 1991. Topography of efferent vagal
innervation of the rat gastrointestinal tract. Am. J. Physiol. 260(29):R200-R207.
20. Berthoud, H.R., Kressel, M., and W.L. Neuhuber. 1995. Vagal afferent innervation of rat
abdominal paraganglia as revealed by anterograde Dil-tracing and confocal microscopy.
Acta. Anat. 152:127-132.
21. Biggers, D.W., Myers, S.R., Neal, D., Stinson, R., Cooper, N.B., Jaspan, J.B., Williams,
P.E., Cherrington, A.D., and R.T. Frizzell. 1989. The role of the brain in
counterregulation of insulin induced hypoglycemia in dogs. Diabetes 38:7-16.
22. Bolli, G., DeFeo, P., Compagnucci, P., Cartechini, M., Angeletti, G., Santeusanio, F.,
Brunetti, P., and J. Gerich. 1983. Abnormal glucose counterregulation in insuli-dependent
diabetes mellitus: interaction of anti-insulin antibodies and impaired glucagon and
epinephrine secretion. Diabetes 32:134-141.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
23. Bolli, G.B., DeFeo, P., Compagnucci, P., Cartechini, M.G., Angeletd, G., Santeusanio, F.,
Brunetti, P., and J.E. Gerich. 1983. Abnormal glucose counterregulation in insulin-
dependent diabetes mellitus: interaction of anti-insulin antibodies and impaired glucagon
and epinephrine secretion. Diabetes 32:134-141.
24. Bolli, G.B., Gottesman, I.S., Cryer, P.E., and J.E. Gerich. 1984. Glucose
counterregulation during prolonged hypoglycemia in normal humans. Am. J. Physiol.
247(10):E206-E214.
25. Borg, M.A., Sherwin, R.S., Borg, W.P., Tamborlane, W.V., and G.I. Shulman. 1997.
Local ventromedial hypothalamus glucose perfusion blocks counterregulation during
systemic hypoglycemia in awake rats. J. Clin. Invest. 99:361-365.
26. Borg W., During, M., Sherwin, R., Borg, M., Brines, M., and G. Shulman. 1994.
Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to
hypoglycemia. J. Clin. Invest. 93:1677-1682.
27. Borg, W., Sherwin, R., During M., Borg, M., and G. Shulman. 1995. Local ventromedial
hypothalamus glucopenia triggers counterregulatory hormone release. Diabetes 44:ISO -
184.
28. Bottini, P., Boschetti, E., Pampanelli, S., Ciofetta, M., Sindaco, P.D., Scionti, L., Brunetti,
P., and G.B. Bolli. 1997. Contribution of autonomic neuropathy to reduced plasma
adrenaline responses to hypoglycemia in IDDM. Diabetes. 46:814-823.
29. Boyle, P.J., and P.E Cryer. 1991. Growth hormone, cortisol, or both are involved in
defense against, but are not critical to recovery from, prolonged hypoglycemia in humans.
Am. J. Physiol. 260:E395-E402.
30. Boyle, P.J., Liggett, S.B., Shah, S.D., and P.E. Cryer. 1988. Direct muscarininc
cholinergic inhibition of hepatic glucose production in humans. J. Clin. Invest. 82:445-
449.
31. Boyle, P.J., Nagy, R.J., O’Connor, A.M., Kempers, S.F., Yeo, R.A., and C. Qualls. 1995.
Adaptation in brain glucose uptake following recurrent hypoglycemia. Proc. Natl. Acad.
Sci. 91:9352-9356.
32. Bowden, C.R., and R.N. Bergman. 1979. Caval occlusion technique for epatic venous
sampling: A new approach to estimating splanchnic substrate balance in conscious dogs.
Metabolism 28:562-567.
33. Burczynski, F.J., Luxon, B.A., and R.A. Weisiger. 1996. Intrahepatic blood flow
distribution in the perfused rat liver: effect of hepatic artery perfusion. Am. J. Physiol.
271(34):G561-G567.
91
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
34. Campbell, L.V., Kraegen, E.W., and L. Lazarus. 1977. Defective blood glucose counter
regulation in diabetes is a selective for of autonomic neuropathy. Br. Med J. 2:1527-
1529.
35. Cane, P., Artal, R _ , and R.N. Bergman. 1986. Putative hypothalmic glucoreceptors play
no essential role in the response to moderate hypoglycemia. Diabetes 35:268-277.
36. Cane, P., Haun, C.K., Evered, J., Youn, J.H., and R.N. Bergman. 1986. Response to deep
hypoglycemia does not involve glucoreceptors in carotid perfused tissue. Am. J. Physiol.
255(18):E680-687.
37. Caprio, S., Gelfand, R.A., Tamborlane, W.V., and R.S. Sherwin. 1989. Oxidative fuel
metabolism during mild hypoglycemia: critical role of free fatty acids. Am. J. Physiol.
256:E413-E419.
38. Caprio, S., Napoli, R., Sacca, L, Tamborlane, W., and R. Sherwin. 1992. Impaired
stimulation of gluconeogenesis during prolonged hypoglycemia in intensively treated
insulin-dependent diabetic subjects. J. Clin. Endocrinol. Metab. 75:1076-1080.
39. Carobi, C., Della, T.G., and F. Magni. 1985. Differential distribution of vagal afferent
neurons from the rat liver. Neurosci. Lett. 62:255-260.
40. Chambert, G., Kobashi, M., and A. Adachi. 1993. Convergence of gastric and hepatic
information in brain stem neurons of the rat. Brain Res. Bull. 32:525-529.
41. Cherrington, A.D., Liljenquist, J., Shulman, G., Williams, P., and W. Lacy. 1979.
Importance of hypoglycemia-induced glucose production during isolated glucagon
deficiency. Am. J. Physiol. 236(3):E263-271.
42. Chiasson, J.L., Lilgenquist, J.E., Finger, F.E., and W.W. Lacy. 1976. Differential
sensitivity of glycogenolysis and gluconeogenesis to insulin infusion in dogs. Diabetes
25:283-291.
43. Chiasson, J., Shikama, H., Chu, D., and J. Exton. 1981. Inhibitory effect of epinephrine
on insulin-stimulated glucose uptake by rat skeletal muscle. J. Clin. Invest. 68:706-713.
44. Cryer, P.E. 1992. Iatrogenic hypoglycemia as a cause of hypoglycemia-associated
autonomic failure in IDDM: a vicious cycle. Diabetes 41:255-260.
45. Cryer, P.E. 1993. Glucose counterregulation: prevention and correction of hypoglycemia
in humans. Am. J. Physiol. 264(27):E149-E155.
92
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
46. Cryer, P.E. 1993. Hypoglycemia begets hypoglycemia in IDDM. Diabetes 42:1691-
1693.
47. Cryer, P.E. 1994. Hypoglycemia: The limiting factor in the Management of IDDM.
Diabetes 43:1378-1389.
48. Cucchiaro, G., Yamaguchi, Y., Mills, E., Kuhn, C.M., Anthony, D.C., Branum, G.D.,
Epstein, R., and W.C. Meyers. 1990. Evaluation of selective liver denervation methods.
Am. J. Physiol. 259(22):G781-G785.
49. Dagogo-Jack, S.E., Craft, S., and P.E. Cryer. 1993. Hypoglycemia-associated autonomic
failure in insulin-dependent diabetes mellitus. J Clin Invest 91:819-828.
50. Dagogo-Jack, S.E., Rattarasam, C., and P.E. Cryer. 1994. Reversal of hypoglycemia
unawareness but not defective glucose counterregulation, in IDDM. Diabetes 43:1426-
1434.
51. Davis, M.R., Mellman, M., and H. Shamoon. 1992. Further defects in counterregulatory
responses induced by recurrent hypoglycemia in IDDM. Diabetes 41:1335-1340.
52. Davis, S.N., Dobbins, R., Tarumi, C., Colburn, C., Neal, D., and A.D. Cherrington. 1992.
Effects of differing insulin levels on response to equivalent hypoglycemia in conscious
dogs. Am. J. Physiol. 263(26):E688-E695.
53. Davis, M., and H. Shamoon. 1991. Counterregulatory adaptation to recurrent
hypoglycemia in normal humans J Clin. Endocrinol. Metab. 73:995-1001.
54. Davis, S., Shavers, C., Neal, D., Allen, E., and P. Williams. 1995. Hepatic hypoglycemia
is unable to initiate counterregulation in conscious dogs. Diabetes (Suppl.) 44:3A:1.
55. The DCCT Research Group. 1991. Epidemiology of severe hypoglycemia in the Diabetes
Control and Complications Trial. 1991. Am. J. Med. 90:450-459.
56. The DCCT Research Group. 1993. The effect of intensive treatment of diabetes on the
development and progression of long-term complications in insulin dependent diabetes
mellitus. N. Engl. J. Med. 329:977-986.
57. The DCCT Research Group. 1997. Hypoglycemia in the Diabetes Control and
Complications Trial. Diabetes 46:271-286.
58. DeFeo, P., Perriello. G., Torlone, E., Fanelli, C., Ventura, M.M., Santeusanio, F., Brunetti,
P., and G.B. Bolli. 1991. Contribution of adrenergic mechanisms to glucose
counterregulation in humans. Am. J. Physiol. 261:E725-E736.
93
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
59. DeFronzo, R.A., Tobin, J., and R. Andrea. 1979. Glucose clamp technique: a method for
quantifying insulin secretion and resistance. Am. J. Physiol. 237(3):E214-E223.
60. DiRocco, R.J., and H. Grill. 1979. Hie fbrebrain is not essential for sympathoadrenal
hyperglycemic response to glucoprivadon. Science 204:112-114.
61. Donovan, C.M., Halter, J.B., and R.N. Bergman. 1991. Importance of hepatic
glucoreceptors in sympathoadrenal response to hypoglycemia. Diabetes. 40:155-158.
62. Donovan, C.M., Hamilton-Wessler, M., Halter, J.B., and R.N. Bergman. 1994. Primacy of
liver glucosensors in the sympathetic response to progressive hypoglycemia. Proc. Natl.
Acad. Sci. 91:2863-2867.
63. During, M., Leone, P., Davis, K., Kerr, D., and R. Sherwin. 1995. Glucose modulates rat
substantia nigra GABA releases in vivo via ATP-sensitive potassium channels. J. Clin.
Invest. 95:2403-2408.
64. Esler, M., Jennings, G., Lambert, G., Meredith, I., Home, M., and G. Eisenhofer. 1990.
Overflow of catecholamine neurotransmitters to the circulation: source, fate, and functions.
Phys.Rev. 70(4):963-985.
65. Evans, M.I., Halter, J.B., and D. Porte. 1978. Comparison of double and single enzymatic
derivative methods for measuring catecholamines in human plasma. Clin. Chem. 24:567-
570.
66. Exton, J.H., Robinson, G., Sutherland, E., and C. Park. 1971. Studies on the role of
adenosine 3 ’,5’-monophosphate in the hepatic actions of glucagon and catecholamines. J.
Biol. Chem. 246(20):6166-6177.
67. Faloona, G.R., and R.H. Unger. 1965. In: Methods of Hormone Radioimmunoassays.
Jaffe, B.M., and H.R. Berman, eds. Academic, New York. pp. 1375-1384.
68. Fanelli, C.G., DeFeo, P., Porcellati, F., Perriello, G., Torlone, E., Santeusanio, F.,
Brunetti, P., and G. Bolli. 1992. Adrenergic mechanisms contribute to hypoglycemic
glucose counterregulation in man by stimulating lipolysis. J Clin. Invest. 89:2005-2013.
69. Figlewicz, D.P., Brot, M.D., McCall, A.L., and P. Szot. 1996. Diabetes causes
differential changes in CNS noradrenergic and dopaminergic neurons in the rat: a molecular
study. Brain Research 736:54-60.
70. Finegood, D.T., Bergman, R.N., and M. Vranic. 1987. Estimation of endogenous glucose
production during hyperinsulinemic-euglycemic glucose clamps: comparison of unlabeled
and labeled exogenous glucose infiisates. Diabetes 36:914-924.
94
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
71. Forssmann, W.G., and S. Ito. 1977. Hepatocyte innervation in primates. J. Cell Biol.
74:299-313.
72. Frizzell, R.T., Hendrick, G.K., Biggers, D.W., Lacy, D.B., Donahue, D.P., Green, D.R.,
Carr R.K., Williams, P.E., Stevenson, R.W., and A.D. Cherrington. 1988. Role of
gluconeogenesis in sustaining glucose production during hypoglycemia caused by
continuous insulin infusion in conscious dogs. Diabetes 37:749-759.
73. Frizzel, R.T., Hendrick, G.K., Brown, L.L., Lacy, D.B., Donahue, E.P., Carr, R.K.,
Williams, P.E., Parlow, A.F., Stevenson, R.W., and A.D. Cherrington. 1988. Stimulation
of glucose production through hormone secretion and other mechanisms during insulin-
induced hypoglycemia. Diabetes 37:1531-1541.
74. Frizzel, R.T., Jones, E.M., Davis, S.N., Biggers, D.W., Myers, S.R., Connolly, C.C., Neal,
D.W., Jaspan, J.B., and A.D. Cherrington. 1993. Counterregulation during hypoglycemia
is directed by widespread brain regions. Diabetes 42:1253-1261.
75. Frohman, L.A., and L. Bemardis. 1971. Effect of hypothalamic stimulation on plasma
glucose, insulin, and glucagon levels. Am. J. Physiol. 221(6): 1596-1603.
76. Fukuda, M., Tanaka, A., Tahara, Y., Ekegama, H., Yamamoto, Y., Kumarhara, Y., and K.
Shima. 1988. Correlation between minimal secretory capacity of pancreatic p-cells and
stability of diabetic control. Diabetes 37:81-88.
77. Garber, A.J., Cryer, P.E., Santiago, J.V., Haymond, M.W., Pagliara, A.S., and D.M.
Kipins. 1976. The role of adrenergic mechanisms in the substrate and hormonal response
to insulin-induced hypoglycemia in man. J. Clin. Invest. 58:7-15.
78. Gauthier, C., Vranic, M., and G. Hetenyi. 1980. Importance of glucagon in regulatory
rather than emergency responses to hypoglycemia. Am. J. Physiol. 238(1):E131-140.
79. Gerbitz, K.D., Gempel, K., and D. Brdiczka. 1996. Mitochondria and diabetes: genetic,
biochemical, and clinical implications of the cellular energy circuit. Diabetes 45:113-126.
80. Gerich, J., Cryer, P., and R. Rizza. 1980. Hormonal mechanisms in acute glucose
counterregulation: the relative roles of glucagon, epinephrine, norepinephrine, growth
hormone, and cotisol. Metabolism 29:1164-1175.
81. Gerich, J., Davis, J., Lorenzi, M., Rizza, R., Bohannon, N., Karam, J., Lewis, S., Kaplan,
R., Schultz, T., and P. Cryer. 1979. Hormonal mechanisms of recovery from insulin-
induced hypoglycemia in man. Am. J. Physiol. 236(4):E380-E385.
82. Gerich, J., Langlois, M., Noacco, C., Karam, J., and P. Forsham. 1973. Lack of glucagon
response to hypoglycemia in diabetes: evidence of an intrinsic pancreatic a-cell defect.
Science 182:171-173.
95
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
83. Gisel, E., and D. Innes. 1979. Glycemic responses induced by hypothalamic stimulation.
Neuroendocrinology 28:21-216.
84. Goldstein, R.E., Reed, G.W., Wasserman, D.H., Williams, P.E., Brooks Lacy, D.,
Buckspan, R., Abumrad, N.N., and A. Cherrington. 1992. The effects of acute elevations
in plasma cortisol levels on alanine metabolism in the conscious dog. Metabolism
41(12): 1295-1303.
85. Gray, D.E., Lickley, H., and M. Vranic. 1980. Physiologic effects of epinephrine on
glucose turnover and plasma free fatty acid concentrations mediated independently of
glucagon. Diabetes 29:600-608.
86. Hamilton-Wessler, M., Bergman, R.N., Halter, J.B., and C.M. Donovan. 1994. The role
of integrated sympathetic response induced by deep hypoglycemia in dogs. Diabetes
43:1052-1060.
87. Havel, P.J., Flatness, D.E., Halter, J.B., Best, J.D., Veith, R.C., and G.J. Taborsky. 1987.
Halothane anesthesia does not suppress sympathetic activation produced by
neuroglucopenia. Am. J. Physiol. 252(15):E667-E672.
88. Hay, M., and D.L. Kunze. 1994. Calcium-activated potassium channels in rat visceral
sensory afferents. Brain Research 639:33-336.
89. Haylett, D.G., and D.H., Jenldnson. 1972. Effects of noradrenaline on potassium efflux,
membrane potential and electrolyte levels in tissue slices prepared from guinea-pig liver. J.
Physiol. 225:721-750.
90. Heller, S.R., and P.E. Cryer. Reduced neuroendocrine and symptomatic responses to
subsequent hypoglycemia after 1 episode of hypoglycemia in nondiabetic humans.
Diabetes 41:1597-1602.
91. Hepburn, D.A., Patrick, A.W., Eadington, D.W., Ewing, D.J., and B.M. Frier. 1990.
Unawareness of hypoglycemia in insulin-treated diabetic patients: prevalence and
relationship to autonomic neuropathy. Diabetic Med. 7:711-717.
92. Herbert, V., Law, K., Gottlieb, C., and S. Bleicher. 1965. Coated charcoal immunoassay
of insulin. J. Clin. Endocrinol. Metab. 25:1375-1384.
93. Hermann, G.E., Kohlerman, N.J., and R.C. Rogers. 1983. Hepatic-vagal and gustatory
afferent interactions in the brainstem of the rat. J. Auton. Nev. Syst. 9:477-495.
94. Hevener, A.L., Bergman, RN., and C.M. Donovan. 1997. Novel glucosensor for
hypoglycemic detection localized to the portal vein. Diabetes 46:1521-1525.
96
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
95. Hilsted, J., Madsbad, S., Krarup, T., Sestoft, L., Christenson, M., Tronier, B., and H.
Gaibo. 1981. Hormonal, metabolic, and cardiovascular responses to hypoglycemia in
diabetic autonomic neuropathy. Diabetes 30:626-633.
96. Himsworth, R.L. 1970. Hypothalmic control of adrenaline secretion in response to
insufficient glucose. J. Physiol. 206:411-417
97. Hirsh, I.B., Boyle, P.J., Craft, S., and P.E. Cryer. 1991. Higher glycemic thresholds for
symptoms during p-adrenergic blockade in IDDM. Diabetes 40:1177-1186.
98. Hirsch, B.R., and H. Shamoon. 1987. Defective epinephrine and growth hormone
responses in type I diabetes are stimulus specific. Diabetes 36:20-26.
99. Hoelstke, R.D., Boden, G., Shulman, C., and O. Owen. 1982. Reduced epinephrine
secretion and hypoglycemia unawareness in diabetic autonomic neuropathy. Ann. Intern.
Med. 96:459-462.
100. Hutson, N.J., Brumley, F., Assimacopoulos, F., Herper, S., and J. Exton. 1976. Studies
on the a-adrenergic activation of hepatic glucose output: studies on the a-adrenergic
activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in
isolated rat liver parenchymal cells. J. Biol. Chem. 251(17):5200-5208.
101. Ishise, S., Pegram, B.L., Yammamoto, J., Kitamura, Y., and E.D. Frolich. 1980.
Reference sample microsphere method: cardiac output and blood flows in conscious rat.
Am. J. Physiol. 239:H443-H449.
102. Jackson, P.A., Cardin, S., Neal, D., and A. Cherrington. 1997. The effect of hepatic
denervation on the counter-regulatory response to insulin induced hypoglycemia in
conscious dogs. Diabetes (Suppl.) 46:68A:266.
103. Kennedy, E.D., Rizzuto, R., Theler, J.M., Pralong, W.F., Bastianutto, C., Pozzan, T.,
and C.B. Wollheim. Glucose stimulated insulin secretion correlates with changes in
mitochondrial and cytosolic Ca2 + in aequorin-expressing INS-1 cells. J. Clin. Invest. 98:
2524-2538.
104. Kinsley, BT., Widom, B., Utzschneider, K., and D.C. Simonson. 1994. Stimulus
specificity of defects in counterregulatory hormone secretion in insulin-dependent
diabetes mellitus: effect of glycemic control. J. Clin.. Endo. and Metab. 79(5): 1383-
1389.
105. Krishtal, O.A. 1994. Modulation of excitatory synaptic transmission by adenosine:
possibility of interaction with Ca-delivering machinery. Neurophysiology 26(l):26-28.
97
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
106. Kummer, W., and W.L. 1989. Vagal paraganglia of the rat. J. Electron. Microsc.
Techn. 12:343-355.
107. Lamarche, L., Yamaguchi, N., and F. Peronnet. 1996. Hepatic denervation reduces
adrenal catecholamine secretion during insulin induced hypoglycemia. Am. J. Physiol.
268(37): R50-R57.
108. lamarche, L., Yamaguchi, N., and F. Peronnet. 1996. Selective hypoglycemia in the
liver induces adienolmedullary counterregulatory response. Am. J. Physiol.
270(39):R1307-R1316.
109. Lamarche, L., Yamaguchi, N., Peronnet, F., and F. Giutard. 1992. Evidence against a
humoral control mechanism in adrenal catecholamine secretion during insulin-induced
hypoglycemia. Am. J. Physiol. 262(3 l):R659-665.
110. Lautt, W.W. 1980. Hepatic nerves: a review of their functions and effects. Can. J.
Physiol. Pharmacol. 58(2): 105-123.
111. Lautt W.W. 1983. Afferent and efferent neural roles in liver function Progress in
Neurobiology. 21:323-348.
112. Lautt, W.W. 1984. The effect of intraportal and intravenous glucose tolerance test on
insulin and glucagon levels in conscious cats with normal and chronically phenol-
denervated livers. J. Auton. Nerv. Syst. 10:135-143.
113. Lautt, W.W, and A.M Carroll. 1984. Evaluation of topical phenol as a means of
producing autonomic denervation of the liver. Can. J. Physiol. Pharmacol. 62:849-853.
114. Lecavalier, L., Bolli, G., Cryer, P., and J. Gerich. 1989. Contributions of
gluconeogenesis and glycogenolysis during glucose counterregulation in normal man.
Am. J. Phys. 256(19):E844-E851.
115. Lee, K.C., and RE. Miller. 1985. The hepatic vagus nerve and the neural regulation of
insulin secretion. Endocrinology 117(l):307-314.
116. Liu, D., Moberg, E., Kollind, M., Lins, P-E, and U. Adamson. 1991. A high
concentration of circulating insulin suppresses the glucagon response to hypoglycemia in
normal man. J. Clin. Endocrinol. Metab. 73:1123-1128.
117. Louis-Sylvestre, J. 1983. Validation of tests of completeness of vagotomy in rats. J.
Auton. Nerv. Syst. 9:301-304.
98
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
118. Maggs, D.G., Jacob, R., Francis, R., Caprio, S., Tamborlane, W.V, and R.S. Sherwin.
1997. Counterregulation in peripheral tissues: effect of systemic hypoglycemia on levels
of substrates and catecholamines in human skeletal muscle and adipose tissue. Diabetes
46:70-76.
119. Maran, A., Cranston, I., Lomas, J., and S.A. Amiel. 1994. Protection by lactate of
cerebral function during hypoglycemia. The Lancet 343:16-20.
120. Matchinsky, F.M. 1990. Glucokinase as glucose sensor and metabolic signal generator
in pancreatic P-cells and hepatocytes. Diabetes 39:647-652.
121. Matschinsky, F.M. and I.R. Sweet. 1996. Annotated questions and answers about
glucose metabolism and insulin secretion of P-cells. Diabetes Reviews 4(2): 130-144.
122. Mellman, M.J., Davis, M.R., and H. Shamoon. 1992. Effect of physiological
hyperinsulinemia on counterregulatory hormone responses during hypoglycemia in
humans. J. Clin. Endocrinol. Metab. 75:1293-1297.
123. Miles, D.G., Yamatani, K., Lavina, H., Lickley, H., and M. Vranic. 1991. Mechanism
of glucoregulatory responses to stress and their deficiency in diabetes. Proc. Natl. Acad.
Sci. 88:1296-1300.
124. Mitrakou, A., Ryan, C., Veneman, T., Mokan, M., Jenssen, T., Kiss, I., Durrant, J.,
Cryer, P., and J. Gerich. 1991. Hierarchy of glycemic thresholds for counterregulatory
hormone secretion, symptoms, and cerebral dysfunction. Am. J. Physiol. 260(23):E67-
74.
125. Morgan, A.J., and R. Jacob. 1996. Ca2 + influx does more than provide releasable Ca2 +
to maintain repetitive spiking in human umbilical vein endothelial cells. Biochem. J.
320:505-517.
126. Morishita, R., Nakamura, S., Nakamura, Y., Aoki, M., Moriguchi, A., Kida, I.,
Matsumoto, K., Nakamura, T., Higaki, J., and T. Ogihara. 1997. Potential role of an
endothelium-specific growth factor, hepatocyte growth factor, on endothelial damage in
diabetes. Diabetes 46:138-142.
127. Niijima A. 1969. Afferent impulse discharges from glucoreceptors in the liver of the
guinea pig. Ann. N.Y. Acad. Sci. 157:690-700.
128. Niijima A. 1980. Glucose sensitive afferent nerve fibers in the liver and regulation of
blood glucose. Brain Res. Bull. 5:175-179.
129. Niijima A. 1981. Visceral afferents and metabolic functions. Diabetologia 20:325-330.
99
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
130. Niijima, A. 1983. Electrophysiological study on nervous pathway from splanchnic nerve
to vagus nerve in rat. Am. J. Physiol. 244:R888-R890.
131. Niijima A. 198S. Blood glucose levels modulate efferent activity in the vagal supply to
the rat liver. J. Physiol. 364:105-112.
132. Niijima A. 1989. Nervous regulation of metabolism. Prog. Neurobiol. 33:135-147.
133. Novin, D., Rogers, R.C., and G. Hermann. 1981. Visceral afferent and efferent
connections in the brain. Diabetologica 20:331-336.
134. Oomura, Y., and H. Yoshimatsu. 1984. Neural network of glucose monitoring system.
J. Auton. Nerv. Syst. 10:359-372.
135. Popp, D.A., Shah, S., and P. Cryer. 1982. Role of epinephrine-mediated P-adrenergic
mechanisms in hypoglycemic glucose counterregulation and post-hypoglycemic
hyperglycemia in insulin-dependent diabetes mellitus. J. Clin. Invest. 69:315-326.
136. Porte, D., 1967. A receptor mechanism for the inhibition of insulin release by
epinephrine in man. J. Clin. Invest. 46(l):86-94.
137. Powell, A.M., Sherwin, R.S., and G.I. Shulman. 1993. Impaired hormonal response to
hypoglycemia in spontaneously diabetic and recurrently hypoglycemic rats. J. Clin.
Invest. 92:2667-2674.
138. Powley, T.L. and Berthoud, H.R. 1991. A fluorescent labeling strategy for staining the
enteric nervous system. J. Neuroscience Methods 36:9-15.
139. Prechtl, J.C., and T.L. Powley. 1987. A light and electron microscopic examination of
the vagus hepatic branch of the rat. Anat. Embryol. 176:115-126.
140. Prechtl, J.C., and T.L. Powley. 1990. The fiber composition of the abdominal vagus of
the rat. Anat. Embryol. 181:101-115.
141. Pueler, J.D., and G.A. Johnson. 1977. Simultaneous single isotope derivative
radioenzymatic assay for plasma norepinephrine, epinephrine, and dopamine. Life
Sciences 21:625-636.
142. Rattarasarn, C., Dagogo-Jack, S.E., Zachwieja, J.J., and P.E. Cryer. 1994.
Hypoglycemia-induced autonomic failure in IDDM is specific for the stimulus of
hypoglycemia and is not attributable to prior autonomic activation per se. Diabetes
43:809-818.
100
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
143. Robertson, R.P., and D. Porte Jr. 1973. The glucose receptor A defective mechanism in
diabetes mellitus distinct from the beta adrenergic receptor. J. Clin. Invest. 52:870-876.
144. Reitman, S., and S. Frankel. 1957. A colorimetric method for the determination of
serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Pathol. 28:56-
63.
145. Ritter, R.C., Slusser, P., and S. Stone. 1981. Glucoreceptors controlling feeding and
blood glucose: Location in the hindbrain. Science 213:451-453.
146. Rizza, R., Haymon, M., Cryer, P., and J. Gerich. 1979. Differential effects of
epinephrine on glucose production and disposal in man. Am. J. Physiol. 237(4):E356-
362.
147. Rogers, R.C., and G.E. Hermann. 1983. Central connections of the hepatic branch of
the vagus nerve: a horseradish peroxidase histochemical study. J. Auton. Nerv. Syst.
7:165-174.
148. Russek, M. 1963. Participation of hepatic glucoreceptors in the control of intake of
food. Nature 197:79-90.
149. Sacca, L., Sherwin, R _, Hendler, R., and P. Felig. 1979. Influence of continuous
physiologic hyperinsulinemia on glucose kinetics and counterregulatory hormones in
normal and diabetic humans. J. Clin. Invest. 63:849-857.
150. Santiago, J.V., White, N., Skor, D., Levandoski, L., Bier, D., and P. Cryer. 1984.
Defective glucose counterregulation limits intensive therapy in diabetes mellitus. Am. J.
Physiol. 247(10):E215-220.
151. Sawchenko, P.E., and M.I. Friedman. 1979. Sensory functions of the liver-a review.
Am. J. Physiol. 236:R5-R20.
152. Schmitt M. 1973. Influence of hepatic portal receptors on hypothalamic feeding and
satiety centers. Am. J. Physiol. 225:1089-1095.
153. Schwartz, N.S., Clutter, W., Shah, S., and P. Cryer. 1987. Glycemic thresholds for
activation of glucose counterregulatory systems are higher than thresholds for symptoms.
J. Clin. Invest. 79:777-781.
154. Shamoon, H., Hendler, R., and R. Sherwin. 1980. Altered responsiveness to cotisol,
epinephrine, and glucagon in insulin-infused juvenile-onset diabetics: A mechanism for
diabetic instability. Diabetes 29:284-291.
101
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
155. Shimazu, T. 1981. Central nervous system regulation of liver and adipose tissue
metabolism. Diabetologica 20:343-356.
156. Shimazu, T., Fukuda, A., and T. Ban. 1966. Reciprocal influences of the ventromedial
and lateral hypothalamic nuclei on blood glucose level and liver glycogen. Nature
(Lond), 210:1178-1179.
157. Shimazu, T., and S. Ogasawara. 1975. Effect of hypothalaminc stimulation on
gluconeogenesis and glycolysis in rat liver. Am. J. Physiol. 228(6):1787-1793.
158. Shimizu, N., Oomura, Y., Novin, D., Grijala, C.V., and P.H. Cooper. 1983. Functional
correlations between lateral hypothalmic glucose-sensitive neurons and hepatic portal
glucose-sensitive units in rat. Brain Research 265:49-54.
159. Skaaring, P., and F. Bierring. 1976. On the intrinsic innervation of normal rat liver.
Histochemical and scanning electron microscopic studies. Cell Tissue Res. 171:141-
155.
160. Steele, R., Wall, DeBodo, R., and N. Altszuler. 1956. Measurement of size and
turnover rate of body glucose pool by the isotope dilution method. Am. J. Physiol.
187:15-24.
161. Suda, S., Shinohara, M., Lucignani, G., Kennedy, C., and L. Sokoloff. 1990. The
lumped constant of deoxyglucose method in hypoglycemia: Effects of moderate
hypoglycemia on local cerebral glucose utilization in the rat. J. Cereb. Blood Flow
Me tab. l0(4):499-509.
162. Sutherland, S. D. 1964. An evaluation of cholinesterase techniques in the study of the
intrinsic innervation of the liver. J. Anat. 98:321-326.
163. Towler, D.A., Havlin, C.E., Craft, S., and P.E. Cryer. 1993. Mechanism of awareness
of hypoglycemia: perception of neurogenic (predominantly cholinergic) rather than
neuroglycopenic symptoms. Diabetes 42:1791-1798.
164. Vranic, M., Gauthier, C., Bilinski, D., Wasserman, D., El Tayeb, K , Hetenyi, G., and H.
Lickley. 1984. Catecholamine responses and their interactions with other
glucoregulatory hormones. Am. J. Physiol. 247:E145-E156.
165. Widom, B., and D.C. Simonson. 1992. Intermittent hypoglycemia impairs glucose
counterregulation. Diabetes 41:1597-1602.
166. Whipple, A.O. 1944. Hyperinsulinism in relation to pancreatic tumor. Surgery 16:289-
298.
102
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
167. Wolfe, R.R. 1992. Calculation of substrate kinetics: single pool model. In: Radioactive
and stable isotope tracers in biomedicine: principles and practice o f kinetic analysis.
Wiley-Liss, New York. pp. 119-124.
168. Wollner. I.S., Knutsen, C.A., Ullrich, K.A., Niederhuber. J.E., Crudup, J.W., Juni, J.E.,
Warber, S.L., Gyves, J., Stetson, P.L., and W.D. Ensminger. 1986. A dog model using
an implanted system for protracted hepatic artery chemotherapy. J. Surg. Res. 41:510-
517.
169. Yamaguchi, N., and L. Lamarche. 1994. Adrenal medullary counterregulatory response
to hypoglycemia in dogs with hepatic cross-perfusion. Can. J. Physiol. Pharamacol.
72:353-360.
170. Yasuhito, S., and H.N. Wagner Jr. 1971. Measurement of the distribution of cardiac
output in unanesthetized rats. J. Appl. Physiol. 30(6):879-884.
171. Yoshida, T., Kemnitz, J.W., and G.A. Bray. 1983. Lateral hypothalamic lesions and
norepinephrine turnover in rats. J. Clin.. Invest. 72:919-927.
172. Yoshimatsu, H., Oomura, Y., Katafuchi, T., and A. Niijima. 1985. Lesions of the
ventromedial hypothalmic nucleus enhance sympathoadrenal system. Brain Res.
339:390-392.
173. Young, J.B., and L. Landsberg. 1980. Impaired suppression of sympathetic activity
during fasting in the gold thioglucose-treated mouse. J. Clin. Invest. 65:1086-1094.
R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Creator Hevener, Andrea Lynn (author)

Core Title Characterization of portohepatic glucosensors in sympathoadrenal counterregulation

Degree Doctor of Philosophy

Degree Program Biophysics

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, animal physiology,biophysics, medical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), [illegible] (committee member), Turcotte, Lorraine (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-347893

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Legacy Identifier 9902809.pdf

Dmrecord 347893

Document Type Dissertation

Rights Hevener, Andrea Lynn

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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