DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL
Essentiality of Portal Vein Receptors in Hypoglycemic
Counterregulation: Direct Proof Via Denervation in
Male Canines
Viorica Ionut, Ana Valeria B. Castro, Orison O. Woolcott, Darko Stefanovski,
Malini S. Iyer, Josiane L. Broussard, Hasmik Mkrtchyan, Miguel Burch,
Ram Elazary, Erlinda Kirkman, and Richard N. Bergman
Diabetes and Obesity Research Institute (V.I., A.V.B.C., O.O.W., D.S., M.S.I., J.L.B., H.M., R.N.B.) and
Department of Surgery (M.B., R.E.), Cedars Sinai Medical Center, Los Angeles, California 90048; and
Department of Animal Resources (E.K.), University of Southern California, Los Angeles, California 90033
A major issue of in the treatment of diabetes is the risk of hypoglycemia. Hypoglycemia is detected
both centrally and peripherally in the porto-hepatic area. The portal locus for hypoglycemic detection was originally described using the “local irrigation of the liver” approach in a canine model.
Further work using portal vein denervation (DEN) in a rodent model characterized portal hypoglycemic sensing in detail. However, recent controversy about the relevance of rodent findings to
large animals and humans prompted us to investigate the effect of portal DEN on the hypoglycemic
response in the canine, a species with multiple similarities to human glucose homeostasis. Hypoglycemic hyperinsulinemic clamps were performed in male canines, before (PRE) and after (POST)
portal vein DEN or sham surgery (CON, control). Insulin (30 pmol/kg䡠min) and glucose (variable)
were infused to slowly decrease systemic glycemia to 50 mg/dL over 160 minutes. The average
plasma glucose during clamp steady state was: 2.9 ⫾ 0.1 mmol DEN-PRE, 2.9 ⫾ 0.2 mmol DEN-POST,
2.9 ⫾ 0.1 mmol CON-PRE, and 2.8 ⫾ 0.0 mmol CON-POST. There were no significant differences in
plasma insulin between DEN and CON, PRE and POST experiments. The epinephrine response to
hypoglycemia was reduced by 62% in DEN but not in CON. Steady-state cortisol was 46% lower
after DEN but not after CON. Our study shows, in a large animal model, that surgical disconnection
of the portal vein from the afferent pathway of the hypoglycemic counterregulatory circuitry
results in a substantial suppression of the epinephrine response and a significant impact on cortisol
response. These findings directly demonstrate an essential role for the portal vein in sensing
hypoglycemia and relating glycemic information to the central nervous system. (Endocrinology
155: 1247–1254, 2014)
H
yperinsulinemic hypoglycemia is a serious complication in the management of type 1 and type 2 diabetes.
Tight glycemic control, as recommended to reduce vascular complications of diabetes, increases the risk of hypoglycemia, which in turn has negative implications on the
mortality, morbidity, and quality of life of diabetic patients (1). A decrease in blood glucose below the physiological range results in a sequence of counterregulatory
responses, initiated by activation of glucose sensors
throughout the body (2, 3). A large amount of research has
been devoted to understanding where hypoglycemia is
sensed, what the contribution of these sensors to overall
hypoglycemic response is and under what circumstances
they are activated. It is now known that areas in the brain
(hypothalamus and hindbrain) and outside the brain, such
as the carotid bodies and the hepato-portal area, are involved in glucose sensing (3). It is difficult to study brain
and hepato-portal sensing in humans, so very often animal
models were used to better understand the pathophysiology of hypoglycemic counterregulation.
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received August 23, 2013. Accepted December 4, 2013.
First Published Online January 15, 2014
Abbreviations: AUC, area under the curve; CNS, central nervous system; DEN, denervation;
Ginf, glucose infusion rate; HPLC, high performance liquid chromatography; PBST, PBS
containing 0.05% Tween 20.
doi: 10.1210/en.2013-1794
Endocrinology, April 2014, 155(4):1247–1254
endo.endojournals.org
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Ionut et al
Portal Vein DEN and Hypoglycemic Counterregulation
The role of hepatic portal vein sensing in hypoglycemic
counterregulation was originally described by Donovan et
al (4) in a large animal model using the “local irrigation”
technique. They showed that inducing systemic hypoglycemia by insulin infusion, but maintaining the porto-hepatic area at euglycemia, results in a blunting of catecholamine response. However, much of the subsequent work
on portal vein hypoglycemic sensing was done in rodent
models. Donovan and coworkers (5– 8), in a series of elegant studies, characterized the portal vein-mediated
counterregulatory response to hypoglycemia and provided direct evidence of portal vein sensors to hypoglycemia via portal vein denervation (DEN) in rats. In contrast
to the rodent work, Jackson et al (9) reported in the dog
that afferent signaling from the liver and hepato-portal
region is not required for the normal counterregulatory
response to insulin-induced hypoglycemia. Recently, the
relevance of rodent portal hypoglycemic sensing to humans has also been questioned. Smith et al (10) showed
that administration of oral glucose during a hypoglycemic
clamp reduced the early epinephrine response to hypoglycemia. In contrast, Rossetti et al (11) found that in nondiabetic patients, hyperinsulinemic hypoglycemic counterregulation was not affected by normalization of portal
glucose via an oral glucose load, whereas Heptulla et al
(12) found that oral glucose augmented the counterregulatory hormone response during insulin-induced hypoglycemia. Thus, it remains controversial whether the role of
portal sensing of hypoglycemia plays an essential role in
hypoglycemic counterregulation in larger animals or in
man. Therefore, the aim of the current study was to assess
the importance of portal vein hypoglycemic sensing to the
counterregulatory response in a canine model via portal
DEN and to investigate whether hypoglycemic counterregulation mechanisms other than the catecholamine response are affected by this intervention.
Materials and Methods
Animals
Experiments were performed in male mongrel dogs (1 y old;
body weight 27.7 ⫾ 0.7 kg), in the conscious relaxed state (n ⫽
17), housed under controlled kennel conditions in the Cedars
Sinai Medical Center vivarium, and fed once daily with a standard diet (27% protein, 32% fat, and 41% carbohydrate; Labdiet; PMI Nutrition International). The animals 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 Cedars Sinai Medical Center Institutional
Animal Care and Use Committee.
Endocrinology, April 2014, 155(4):1247–1254
Experimental design
Experiments were all performed in the morning, after 12–16
hours of fasting. In each animal, the metabolic assessment was performed before and at least 1 week after portal DEN or sham surgery.
At the end of the experimental period, the animals were euthanized,
and portal veins were harvested for assessment of catecholamine
content by high performance liquid chromatography (HPLC) and
of tyrosine hydroxylase by immunohistochemistry.
Surgical procedures
Portal vein DEN and sham DEN were performed under general anesthesia induced with propofol and maintained with isoflurane or sevoflurane. For portal vein DEN (n ⫽ 11), after a
midline incision, the hepatic portal vein was exposed. A myelinspecific dye (toluidine blue 1%) was used to visualize nerve bundles along the portal vein, hepatic artery, common bile duct, and
liver hilum. Only the visible nerves along the portal vein were cut,
and the vein was painted with a solution of phenol-alcohol (10%
phenol in alcohol vol/vol). Special care was taken to preserve the
nerves running along the hepatic artery and its branches, the
hepatic hilum nerves where nerve fibers enter the liver, the hepatic branch of the vagus nerve, and innervation of the common
bile duct. Specifically, the portal vein was denervated distally
(1–2 cm) from the liver hilum. It has been shown that, in rat, the
portal vein glucose sensor is situated 1–3 cm from the liver hilum
(5, 13), so this distance is likely to be even longer in larger mammals. The abdomen was then sutured closed. For sham surgery
(n ⫽ 6), after a midline incision, the hepatic portal vein was
exposed and painted with saline. The abdomen was then sutured
closed.
Hypoglycemic clamps
Intracatheters were acutely placed in the cephalic or saphenous vein for infusion of insulin and glucose and for blood sampling. After basal sampling (⫺20 – 0 min) at t ⫽ 0, insulin was
infused at 30 pmol/kg䡠min to slowly decrease glycemia to 3
mmol/L. Glucose (50% dextrose, 454 mg/mL; B Braun) was
infused at a variable rate. The glucose infusion rate (Ginf) was
adjusted every 10 minutes to achieve approximately 0.55
mmol/L reductions in blood glucose over 40-minute periods.
Thus peripheral glucose decreased stepwise to a nadir that was
reached between 120 and 160 minutes (steady state). Blood samples were taken every 10 minutes for measurements of glucose,
insulin, lactate, and cortisol and every 30 minutes for glucagon.
Samples for measurement of catecholamines were taken at basal
and at steady state. Data for cortisol and for catecholamines were
not available for one hypoglycemic clamp.
Blood samples and assay measurements
Samples for determination of glucose, insulin, and cortisol
were collected into tubes coated with LiF and heparin containing
EDTA. Samples for determination of glucagon were collect in
tubes coated with lithium fluoride and containing EDTA and
Trasylol. Samples for determination of catecholamines were collected in tubes containing EDTA/sodium metabisulfite and flash
frozen after plasma separation. Plasma glucose concentrations
were determined using a YSI 2300 autoanalyzer (Yellow Springs
Instruments). Insulin was measured using a human ELISA kit
(Millipore) adapted in our laboratory for dog plasma. Plasma
glucagon was measured using RIA (Millipore). Cortisol was
doi: 10.1210/en.2013-1794
measured using RIA (Siemens Healthcare Diagnostics). Plasma
catecholamines were measured using ELISA (Rocky Mountain
Diagnostics).
endo.endojournals.org
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area of signal (area of signal above the signal threshold), and
total area analyzed was calculated by the software.
Statistical analysis
Portal vein catecholamine content by HPLC
Catecholamine content of organs and tissues has been used in
various studies as an indicator of sympathetic DEN (9, 14, 15).
We assessed DEN of the portal vein via measuring catecholamine
content of portal vein by HPLC (n ⫽ 3 denervated, 2 nondenervated animals). Tissue catecholamines were isolated by binding to activated alumina and eluted with perchloric acid (1:1 or
1:4 wt/vol) and centrifuged for 15 minutes at 4°C (14 000 rpm);
the supernatant was filtered and the filtrate directly injected to
the HPLC (pg/g).
Tyrosine hydroxylase immunohistochemistry
Tyrosine hydroxylase immunohistochemistry was performed
in portal veins from 7 animals (4 denervated and 3 nondenervated). Briefly, the portal tissue was washed with 0.015M PBS
and dehydrated with a series of increasing concentrations of ethanol. After dehydration, the tissue was incubated in xylene (2 ⫻
60 min) and then infiltrated with paraffin, a paraplast-embedding media (Sigma Chemical), at 60°C overnight and then embedded with fresh paraffin. Each pad was sliced across its extent
at 5 ␮m using a rotary microtome (American Optical Instrument). Sections were placed in a tissue-floating water bath
(37°C) immediately after being sliced and were mounted on glass
slides. Slides were left on a slide-warming table (37°C) to air-dry
overnight. Slides were placed in a series of xylene to remove
paraffin, then a series of ethanol. After a wash in tap water, the
slides were placed in 3% hydrogen peroxide/methanol solution
for 10 minutes. After a wash in distilled water, the slides were
incubated for 25 minutes in citrate buffer (pH 6) at 95°C using
a vegetable steamer. The slides were brought to room temperature, rinsed in PBS containing 0.05% Tween 20 (PBST), and
incubated with antityrosine hydroxylase (AB152; Millipore) at
the dilution of 1:100 at 4°C overnight. The slides were rinsed
with PBST and incubated with Dako EnVision⫹ System-HRP
Labelled Polymer Anti-Rabbit (K4003; Dako) at room temperature for 30 minutes. After a rinse with PBST, the slides were
incubated with 3,3⬘-diaminobenzidine for visualization. Subsequently, the slides were washed in tap water, counterstained with
Harris’ hematoxylin, dehydrated in ethanol, and mounted with
media.
Results are presented as means ⫾ SE unless indicated otherwise. Paired t tests with Bonferroni correction were used for
time-point comparisons before and after DEN or sham. Repeated measurements ANOVA was used for timeline comparisons. Area under the curve (AUC) 0 –180 was calculated using
the trapezoid rule. All differences were considered statistically
significant when P ⬍ .05.
Results
Verification of hepatic portal vein DEN
Hepatic portal DEN was measured by tyrosine hydroxylase immunohistochemistry and by portal vein catecholamine content in denervated vs nondenervated animals.
Tyrosine hydroxylase staining, an indicator of DEN,
showed a 79% decrease in denervated animals compared
with nondenervated (Figure 1B). Portal vein catecholamine was performed in a subset of samples (2 nondenervated and 3 denervated) and showed a 47% content
reduction in denervated animals.
Counterregulatory response to hypoglycemia:
hypoglycemic clamps
To test the counterregulatory response to hypoglycemia, hypoglycemic clamps were performed in denervated
and sham, before and after surgery.
Glucose, insulin, lactate, and Ginf
There was no significant difference in baseline glucose
or insulin between the denervated group and the control
group or within groups before and after DEN or sham
(Figure 2).
Insulin infusion during the hypoglycemic clamp resulted in an increase in plasma insulin (to ⬃3000pM) and
a slow decrease in plasma glucose, to a nadir of approximately 3 mmol/L, in the denervated and in the control
Calculations
Tyrosine hydroxylase quantification scanning and analyses
were performed through the Translational Pathology Core Laboratory, Department of Pathology and Laboratory Medicine,
David Geffen School of Medicine at University of California Los
Angeles. Images were acquired and analyzed using Ariol SL-50
(Applied Imaging Corp). The Ariol scanner is based on an Olympus BX61 microscope with motorized stage and autofocus
capabilities, equipped with a black and white video camera
(Jai CVM2CL). Slides were scanned at ⫻20 objective magnification with the 4⬘,6-diamidino-2-phenylindole and fluorescein
isothiocyanate filters. Optimal exposure times were determined before automated scanning. After scanning, threshold
levels of the individual signals were optimized before final
analysis. Analytical readouts of user-defined areas included:
Figure 1. Hepatic portal vein total catecholamine content by HPLC (A)
and tyrosine hydroxylase immunohistochemistry percentage staining
(B) in denervated (䡺) or nondenervated (f) animals.
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Portal Vein DEN and Hypoglycemic Counterregulation
Endocrinology, April 2014, 155(4):1247–1254
Figure 2. Plasma levels of glucose (A), insulin (B), and Ginf (C) before (open symbols) and after (closed symbols) DEN (left panel) or sham (right
panel).
group. There was no difference in glycemia at steady state
(120 –160 min) before and after DEN (2.9 ⫾ 0.1 mmol/L
vs 2.9 ⫾ 0.2 mmol/L, P ⫽ .78) or before and after sham
(2.9 ⫾ 0.1 mmol/L vs 2.8 ⫾ 0.0 mmol/L, P ⫽ .27) (Figure
2A). There was no significant difference in plasma insulin
before and after DEN (AUC0 –180: before 444 888 ⫾ 35
077 pmol/L䡠min, AUC after 460 561 ⫾ 27 370 pmol/
L䡠min; P ⫽ .46), before and after sham DEN (AUC before
500 983 ⫾ 42 224 pmol/L䡠min, AUC after 502 889 ⫾ 25
854 pmol/L䡠min; P ⫽ .94), or between the portally denervated group and the sham DEN group (ANOVA, P ⫽ .63)
(Figure 2B).
Ginf necessary to maintain hypoglycemia tended to be
higher after DEN, without reaching significance (AUC before 2170 ⫾ 101 mg/kg, AUC after 2425 ⫾ 217 mg/kg;
P ⫽ .12), but it was not significantly higher in sham (AUC
before 2261 ⫾ 272 mg/kg, AUC after 2122 ⫾ 208 mg/kg;
P ⫽ .9) (Figure 2C). Plasma lactate concentration was not
different between groups or before and after DEN. AUC0 –
180, DEN: before 1455 ⫾ 108 mg/dL䡠min, after 1356 ⫾ 90
mg/dL䡠min, P ⫽ .25; sham: before 1349 ⫾ 139 mg/dL䡠min,
after 1266 ⫾ 129 mg/dL䡠min, P ⫽ .34.
Plasma catecholamines
Before DEN, hypoglycemia resulted in significant increases in plasma epinephrine and norepinephrine. Epinephrine concentration doubled at steady state (120 –160
min), from 528 ⫾ 66 basal to 1084 ⫾ 153 pmol/L, P ⫽
.001, in the denervated group, and from 444 ⫾ 143 to
1075 ⫾ 433 pmol/L, P ⬍ .05, in the sham group; norepi-
doi: 10.1210/en.2013-1794
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are essential for a full hypothalamo-pituitary axis access
response to hypoglycemia.
Figure 3. Plasma epinephrine at basal (0) and steady state (120 –160)
before (open bar) and after (closed bar) DEN (A) or sham (B).
nephrine rose from 1997 ⫾ 327 to 3184 ⫾ 567 pmol/L,
P ⬍ .01, in the denervated group, and from 804 ⫾ 163
to1840 ⫾ 645 pmol/L, P ⬍ .05, in the sham group. After
surgery, the epinephrine response to hypoglycemia was
dramatically reduced in portally denervated animals
(531 ⫾ 76 to 679 ⫾ 75 pmol/L, P ⫽ .19) but not reduced
in sham (488 ⫾ 156 to 1147 ⫾ 375 pmol/L, P ⬍ .05)
(Figure 3). Interestingly, we did not measure a decrease in
norepinephrine response to hypoglycemia after DEN
(1812 ⫾ 304 to 3226 ⫾ 531 pmol/L, P ⬍ .01) (Figure 4).
Thus, intact portal vein nerves are essential for a normal
epinephrine response to hypoglycemia in the conscious
dog.
Cortisol
There was no significant difference in fasting cortisol
before and after DEN (before 2.9 ⫾ 0.7 ng/L, after 2.5 ⫾
0.6 ng/L; P ⫽ .66) or before and after sham (before 3.3 ⫾
0.8 ng/L, after 3.0 ⫾ 0.8 ng/L; P ⫽ .44). However, at
steady state during the hypoglycemic clamp, cortisol was
45% lower after DEN (4.0 ⫾ 0.5 ng/L before vs 2.2 ⫾ 0.3
ng/L after DEN, P ⬍ .001). In contrast, there was no difference between cortisol before (3.5 ⫾ 0.9 ng/L) and after
(3.6 ⫾ 0.7 ng/L, P ⫽ .28) sham (Figure 5). It appears that,
like the sympathetic response, intact portal vein receptors
Figure 4. Plasma norepinephrine at basal (0) and steady state (120 –
160) before (open bar) and after (closed bar) DEN (A) or sham (B).
Glucagon
Glucagon did not significantly increase from basal during the hypoglycemic clamps in denervated (DEN: before
41 ⫾ 9 to 34 ⫾ 6 ng/L, P ⫽ .12; after 44 ⫾ 6 to 42 ⫾ 7 ng/L,
P ⫽ .16) or sham-operated animals (before 46 ⫾ 4 to 43 ⫾
3 ng/L, P ⫽ .58; after 47 ⫾ 7 to 38 ⫾ 5 ng/L, P ⫽ .09).
There was no difference in glucagon before and after DEN,
or before and after sham.
Discussion
Our study brings direct proof, via portal denvervation, of
the essential role of the hepatic portal vein in hypoglycemic
counterregulation in a nonrodent model, the canine. Previous studies in the canine that explored the portal involvement in hyperinsulinemic hypoglycemia used the local irrigation technique, in which hypoglycemia is induced
peripherally via an insulin infusion, and concomitantly,
glucose is infused into the portal vein to expose the portohepatic area to euglycemia (16 –18). This technique does
not discriminate between liver and portal vein sensing and
does not rule out the former as the exclusive site of portohepatic hypoglycemia sensing. To the contrary, our findings, in which surgical disconnection of the portal vein
from the afferent pathway of the hypoglycemic counterregulatory circuitry resulted in a very substantial suppression of the epinephrine response, demonstrates an essential role for the portal vein in sensing hypoglycemia (when
hypoglycemia is slowly induced) and relating glycemic information to the central nervous system (CNS).
The essentiality of receptors in the portal vein for the
sympathoadrenal response to hypoglycemia has been controversial in all but rodent models. In dogs, Jackson et al
(9, 19) found that hepatic and portal vein denervation
(alone or combined with vagal cooling) in dogs had no
effect on the counteregulatory response to insulin-induced
hypoglycemia. In a study in nondiabetic humans, Rossetti
et al (11) showed that hyperinsulinemic hypoglycemic
counterregulation was not affected by administration of
an oral glucose load. Moreover, other findings from rodents involving hepato-portal area sensing and its role in
glycemic control were not translatable to humans. Infusion of glucose into the portal vein at rates equal to endogenous glucose production resulted in peripheral hypoglycemia in mice but not in dogs (20) or humans (21).
These contrasting results suggest that there might be fundamental differences in hepato-portal sensing and glucoregulation between rodents and large mammals or hu-
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Portal Vein DEN and Hypoglycemic Counterregulation
Endocrinology, April 2014, 155(4):1247–1254
counterregulation. In our study, we
created a slow pattern of glucose
drop, similar to that employed by Saberi et al (8) (120 min to reach 2.5–3
mmol/L). Under these conditions, we
indeed obtained a significant 73%
decrease in the epinephrine response,
supporting the role of portal sensors
as primary responsible for initiation
of a counterregulatory epinephrine
response.
We did not find a significant decrease in the norepinephrine response to hypoglycemia. Unlike the
epinephrine response, which is reliably decreased when portal sensing
of hypoglycemia is eliminated, a significant suppression of norepinephrine is an inconsistent finding (17,
23). The lack of a detectable norepinephrine decrease after denervation
in our study could suggest a dissociated catecholamine response, depending on the source: adrenal medulla vs nonadrenal (such as muscle
sympathetic nerve activity). Indeed,
Figure 5. Plasma concentration of cortisol before and after DEN (A) or sham (C). Cortisol AUC
it has been shown that type 1 diabetic
before and after DEN (B) or sham (D).
subjects have diminished muscle
mans. It is known that such interspecies differences exist sympathetic nerve activity but normal responses to hypowith respect to glucose homeostasis. For example, a pro- glycemia, indicating that epinephrine and norepinephrine
longed fast impairs insulin-stimulated glucose use in hu- might represent independently mediated responses (24).
mans but enhances it in normal mice (22). These glucoThe pattern of the counterregulatory response obhomeostatic differences could extend to hypoglycemic served in the current study does not respect the traditional
sensing and relay of signals to and from the CNS. To re- hierarchy of hypoglycemia response. Under physiological
spond to this criticism, Saberi et al (8) suggested that dif- conditions, the first step is an inhibition of insulin release
ferences in methodology might be responsible for the dif- and an increase in glucagon secretion, followed by changes
ferent results. One of these differences is the rate of in epinephrine and norepinephrine, and if the hypoglyceglycemic fall. Indeed, when hypoglycemia was induced mia persists, cortisol and GH (3). In our study, glucagon
slowly (80 min to reach 2.5 mmol/L), the catecholamine was not changed from basal and was not different with or
response was blunted in the capsaicin-denervated animals, without denervartion. This finding is similar to other studwhereas when hypoglycemia was induced rapidly (20 min ies, in which a hyperinsulinemic hypoglycemic clamp in
to reach 2.5 mmol/L), there was no impact on counter- denervated or sham-operated animals failed to elicit a gluregulation. They proposed that the relative contribution cagon response. Jackson et al (9) found no significant difof the brain vs the hepato-portal sensors in hypoglycemia ferences in glucagon levels during the clamp in liver-devaries with the rate of glycemic fall: when the fall in glucose nervated animals; Donovan et al (17) found a small
is slow, such as most clinical situations, the porto-hepatic increase in glucagon only during the final part of the clamp
area is essential, whereas during more extreme situations, (240 –260 min). In both studies, the plasma insulin levels
when glucose levels drop rapidly and dramatically, CNS were significantly lower than ours (1300 –1800 pmol/L vs
sensing is activated (8). In the canine studies of Jackson et 3000 pmol/L). As suggested by earlier studies (17), it is
al (9, 19), the rate of glycemic drop was rapid (30 min to very likely that the presence of hyperinsulinemia inhibited
reach 2.5 mmol/L), which could explain why they did not glucagon release and that this signal to the ␣-cells domifind a role for hepato-portal denervation in hypoglycemic nates over the hypoglycemia signals. Davis et al (25)
doi: 10.1210/en.2013-1794
showed that exposure to hyperinsulinemia in the presence
of euglycemia for 30 minutes before hypoglycemia completely eliminated the subsequent glucagon response. In
addition, the study of Donovan et al (17), in which changes
in glucagon were found, used the local irrigation technique
instead of portal/liver denervation. It is not known
whether an intact vs denervated liver/portal vein plays a
role in glucagon regulation.
An interesting finding in our study is represented by the
cortisol changes in the denervated group. Basal cortisol
was significantly suppressed in response to glucose infusion, in the euglycemic period. With hypoglycemia, cortisol started a slow rise, and by the end of the clamp, it
reached baseline or slightly higher levels. However, in the
denervated group, the cortisol stayed at lower levels significantly longer, suggesting that denervation affected the
glycemia-dependent cortisol release. Thus, it is possible
that the portal signal-mediated loop involves in the efferent part not only the medullary adrenal gland but also the
cortex and possibly the hypothalamo-pituitary axis. Preliminary studies in which ACTH was measured support
the involvement of the hypothalamo-pituitary axis in the
counterregulatory response mediated by portal vein receptors (Ionut, V., unpublished observation).
Based on the lack of a full epinephrine counterregulatory response to hypoglycemia after denervation, we
would have expected the Ginf in these experiments to be
significantly higher, in order to supply glucose for maintaining the same glycemic levels as before denervation. In
this context, the fact that the Ginf is not significantly
higher is intriguing. However, a lack of change in Ginf was
found in other studies investigating the counterregulatory
response to hypoglycemia as well. Although Saberi et al (8)
found in denervated rats a significant increase in Ginf during a hypoglycemic clamp, the same group did not report
a significant increase in Ginf (P ⫽ .2) in a similar study in
rats (7) or in a study using canines (17). It is possible that,
under our experimental circumstances, the epinephrine
contribution to the overall counterregulatory response is
rather small, precluding a significant effect on Ginf. Another possibility is that we did not have enough power to
detect significance, because the study was powered for the
catecholamine response. Indeed, the Ginf tended to be
higher after denervation (P ⫽ .12) without reaching significance (in contrast, in sham tends to be lower).
Previous studies have not elucidated whether portal
vein receptors are essential for full counterregulatory response to hypoglycemia in large animals or in man. This
study shows clearly the critical role played by receptors in
the portal vein per se in hypoglycemic counterregulation.
Whether this putative mechanism is dysfunctional in states
of metabolic disease remains to be elucidated.
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Acknowledgments
We thank Dr Igor Rebrin at the University of Southern California
for measuring catecholamines by HPLC and Dr Clara Magyar at
UCLA for her assistance with quantification of tyrosine hydroxylase staining.
Address all correspondence and requests for reprints to:
Viorica Ionut, MD, PhD, Diabetes and Obesity Research
Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, THAL 104, Los Angeles, CA 90048. E-mail:
[email protected].
This work was supported by National Institutes of Health
(NIH)/National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK) Grants R37DK027619 and 5R01DK029867
(to R.N.B.).
Disclosure Summary: The authors have nothing to disclose.
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