1 Department of Molecular Physiology and Biophysics, and 2 Department of Preventive Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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Our aim was to
determine whether complete hepatic denervation would affect the
hormonal response to insulin-induced hypoglycemia in dogs. Two weeks
before study, dogs underwent either hepatic denervation (DN) or sham
denervation (CONT). In addition, all dogs had hollow steel coils placed
around their vagus nerves. The CONT dogs were used for a single study
in which their coils were perfused with 37°C ethanol. The DN dogs
were used for two studies in a random manner, one in which their coils
were perfused with 20°C ethanol (DN + COOL) and one in which
they were perfused with 37°C ethanol (DN). Insulin was infused to
create hypoglycemia (51 ± 3 mg/dl). In response to hypoglycemia
in CONT, glucagon, cortisol, epinephrine, norepinephrine, pancreatic
polypeptide, glycerol, and hepatic glucose production increased
significantly. DN alone had no inhibitory effect on any hormonal or
metabolic counterregulatory response to hypoglycemia. Likewise, DN in
combination with vagal cooling also had no inhibitory effect on any
counterregulatory response except to reduce the arterial plasma
pancreatic polypeptide response. These data suggest that afferent
signaling from the liver is not required for the normal
counterregulatory response to insulin-induced hypoglycemia.
hepatic glucose production; liver nerves; parasympathetic blockade; vagus nerve
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INTRODUCTION |
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LOW BLOOD GLUCOSE results in counterregulatory hormone responses (glucagon, cortisol, epinephrine, norepinephrine, and growth hormone) that increase glucose production and decrease glucose utilization. Although the effects of these hormones have been well characterized, the signal initiating their release remains controversial.
Glucosensors for hypoglycemia have been proposed to lie both within the hepatoportal region and the central nervous system (CNS). Their function is to sense low blood glucose and to signal the brain to coordinate a counterregulatory response. Frizzell et al. (11) brought about cerebral euglycemia during generalized hypoglycemia and almost totally eliminated the counterregulatory response. From these data, they suggested that the brain contained the glucose-sensing sites. In contrast, Donovan et al. (7) conducted a study in which they maintained hepatic euglycemia during generalized hypoglycemia. They observed a 40% decrement in the sympathoadrenal response to hypoglycemia and concluded that the liver contained glucosensors and that they play the dominant role in initiating the counterregulatory response.
It has been suggested that glucose sensors in the hepatoportal region are linked to the brain via afferent fibers traveling within the vagus nerves (1, 23). In a previous study, we blocked vagal afferent signaling during hyperinsulinemic hypoglycemia in conscious dogs (14). We showed that, under those conditions, functioning vagus nerves were not necessary for a complete counterregulatory hormone response to moderate hypoglycemia. It remains possible, however, that hepatic glucosensors communicate with the brain through afferent fibers traveling via nonparasympathetic nerves. In support of this possibility, Lamarche et al. (15), using the anesthetized dog, suggested that liver denervation resulted in a diminution in the normal response of plasma norepinephrine and epinephrine to hypoglycemia. Furthermore, in a recent paper, Hevener et al. (13) suggested that chemical denervation of the hepatic portal vein decreased the sympathoadrenal response to sustained systemic hypoglycemia in rats. The question thus arises as to whether, in the conscious dog, hepatic denervation would result in a diminution of the counterregulatory response to insulin-induced hypoglycemia.
To clearly address the question of afferent neural signaling in the counterregulatory response to hypoglycemia, we conducted the present study using conscious dogs that had undergone hepatic denervation. In addition, we used a vagal cooling technique to ensure the elimination of both parasympathetic and sympathetic signaling, because it is not possible to confirm the former on biopsy. With this model, we were able to completely interrupt neural communication between the hepatoportal region and the brain. The aim of our study, therefore, was to determine whether chronic hepatic denervation alone or in combination with vagal cooling would reduce the counterregulatory response to insulin-induced hypoglycemia in the conscious dog.
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MATERIALS AND METHODS |
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Animal care. Experiments were conducted on 10 conscious mongrel dogs (24.8 ± 1.5 kg) of either sex. The animals were fed once daily with a meat (KalKan; Vernon, CA) and chow (Purina Lab Canine Diet No. 5006, St. Louis, MO) diet (34% protein, 46% carbohydrate, 14.5% fat, and 5.5% fiber based on dry weight). Before study, the dogs were deprived of food for 18 h. The animals were housed in a facility that met the standards of the American Association for the Accreditation of Laboratory Animal Care International, and the protocols were approved by the Vanderbilt University Medical School Animal Care Committee.
Surgical procedures. Two weeks before the initial experiment, the dogs were injected intravenously with a short-acting general anesthetic (sodium pentothal, 15 mg/kg), after which they were intubated and placed on an inhalation anesthetic (1% isoflurane) for the entire surgical procedure. All dogs underwent a laparotomy. Five dogs underwent complete surgical denervation of the liver (DN). Nerves were stripped along the common hepatic artery and its branches from the celiac ganglion to the liver. Nerves were also stripped beginning at the entry of the splenic vein into the portal vein and continuing rostrally to the liver. The other five dogs underwent a sham procedure (CONT), in which the structures along the hepatic nerves were manipulated but innervation was not interrupted. The success of the surgical denervation procedure was verified by measuring liver norepinephrine levels after each study. The liver norepinephrine concentration in DN dogs was 2.5 ± 1% of that in normal innervated liver (2). Stainless steel cooling coils, with Silastic extension tubing attached, were placed around the vagus nerves in all dogs as described previously (14). The effectiveness of cooling (COOL) to block parasympathetic signaling was verified by measuring the heart rate and arterial plasma pancreatic polypeptide concentrations (both of which are under vagal control).
For the placement of a femoral artery catheter, a 1-cm incision was made parallel to the vessel in the left inguinal area. The artery was isolated by blunt dissection and ligated distally. A Silastic catheter (ID 0.04 in., OD 0.085 in.) was inserted through a small hole in the artery, and its tip was positioned in the iliac artery. The catheter was secured and filled with heparin (1,000 U/ml; Abbott, North Chicago, IL) and its free end was knotted and buried in a subcutaneous pocket to allow complete closure of the incision. Penicillin G (100,000 U) was administered intramuscularly on one occasion preoperatively, and ampicillin (1,000 mg orally) was given postoperatively. Each dog was used for an experiment only if it had a leukocyte count below 18,000/mm3, a hematocrit >35%, a good appetite, normal stools, and normal serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, and bilirubin. On the day of the study, the femoral artery catheter and the Silastic tubes connected to the cooling coils were exteriorized from their subcutaneous pockets under local anesthesia (2% lidocaine; Astra Pharmaceutical Products, Worcester, MA). The contents of the femoral catheter were aspirated, and heparinized saline (1 U/ml) was slowly infused through it during the study. The ends of the Silastic tubes connected to the vagal cooling coils were joined to inflowing lines (ID 0.125 in., OD 0.25 in.) from the cooling bath and to outflowing lines linking them to the collection reservoir. Angiocaths (18 gauge; Becton-Dickinson, Sandy, UT) were inserted percutaneously into the left cephalic vein for [3-3H]glucose infusion, the left saphenous vein for insulin infusion, and the right cephalic vein for peripheral glucose infusion (20% dextrose), as needed. After coil preparation, the dog was allowed to stand calmly in a Pavlov harness for 30 min before the start of the experiment. Each DN dog was reanesthetized (sodium pentothal, 15 mg/kg) after the experiment, and its femoral artery catheter was filled with heparin. The free ends of the arterial and cooling coil catheters were knotted and placed into a new subcutaneous pocket. The incisions were closed, and antibiotics were administered as described earlier. The animals were then studied 7-10 days later in a random manner. Three days before each experiment, the leukocyte count and hematocrit of the animal were again measured. Only dogs which met the study criteria described earlier were reused.Experimental design.
Eighteen-hour-fasted dogs underwent an experiment consisting of a
100-min tracer equilibration period (140 to
40 min), a 40-min
control period (
40 to 0 min) and a 90-min experimental period
(0-90 min). A priming dose (33 µCi) of
[3-3H]glucose (Du Pont-NEN, Boston, MA) was administered
at
140 min, followed by a continuous infusion of 0.29 µCi/min of
[3-3H]glucose. This tracer infusion was adjusted as
needed in each of the three groups (see below) to clamp the glucose
specific activity (SA) at a constant value approximately equal to the
basal SA (11,945 ± 1,673 to 9,742 ± 1,234 dpm/mg in CONT,
11,671 ± 1,415 to 9,989 ± 1,045 dpm/mg in DN, and
12,157 ± 1,256 to 10,227 ± 680 dpm/mg in DN + COOL).
At time 0, a peripheral porcine insulin infusion (5.0 mU · kg
1 · min
1; Eli Lilly,
Indianapolis, IN) was started and continued for the entirety of the
experiment in all protocols. To maintain a hypoglycemic clamp (
50
mg/dl), glucose (20% dextrose; Baxter, Deerfield, IL) was infused
through a leg vein as needed. During CONT and DN experiments, the coils
were perfused for the entire experimental period (90 min) with 37°C
fluid concurrent with the insulin infusion. In contrast, in the DN + COOL group, a perfusion of
20°C fluid was begun 5 min before the
initiation of insulin infusion (time 0) and continued
throughout the 90-min experimental period. The order of the DN and
DN + COOL protocols was randomly determined. Arterial blood was
sampled every 10 min during the basal period and every 15 min
thereafter. The collection and processing of blood samples have been
described previously (24). Approximately 8% of the dog's
total blood volume was removed during each study.
Hormone and metabolic assays.
Plasma glucose levels were assayed using the glucose-oxidase method
with a Beckman glucose analyzer. Small blood samples were taken every 5 min to measure the glucose concentration so that exogenous glucose
could be administered as needed to maintain the hypoglycemic clamp.
Plasma insulin and glucagon were measured by a double-antibody
radioimmunoassay described previously (20) with interassay
coefficients of variation (CV) of 7 and 5%, respectively. Plasma
samples used for glucagon determination contained 100,000 kallikrein
inhibitor units of aprotinin (Trasylol; Miles, Kankakee, IL) added at
collection. Catecholamines were assayed by high-performance liquid
chromatography, as previously described (18). The
interassay CV for epinephrine and norepinephrine were 7 and 5%,
respectively. The samples for catecholamine analysis contained 60 µl
of glutathione/EGTA added at collection. Liver tissue norepinephrine
levels were assayed as described previously (18). Plasma
cortisol was assayed with the Clinical Assays Gamma Coat RIA kit with
an interassay CV of 6% (9). Pancreatic polypeptide was
assayed by use of the method described by Hagopian et al.
(12) with an interassay CV of 8%. Whole blood levels of
lactate, glycerol, alanine, and -hydroxybutyrate were determined
from perchloric acid-treated (3%) samples according to the method of
Lloyd et al. (16), as adapted to the Monarch 2000 centrifugal analyzer (Lexington, MA). Plasma nonesterified fatty acids
(NEFA) were measured using a Wako FFA C test kit (Wako Chemicals,
Richmond, VA), also applied to the Monarch 2000 centrifugal analyzer.
Plasma [3-3H]glucose was determined by liquid
scintillation counting after Somogyi-Nelson deproteinization and a
3H2O exclusion procedure (24).
Endogenous glucose production. The total rate of glucose production was determined by means of a primed tracer infusion. The data were calculated according to the method of Wall et al. (26), as simplified by DeBodo et al. (5), and also according to a two-compartment model described by Mari (17), using parameters for the dog as determined by Dobbins et al. (6). The results obtained with the two methods were not significantly different, and we therefore chose to display the data from the two-compartment model in the figures. To obtain endogenous glucose production, the amount of glucose infused during the hypoglycemic clamp was subtracted from total glucose production.
Statistical analysis. Data are expressed as means ± SE. Area under the curve measurements (AUC) for the counterregulatory hormones (glucagon, cortisol, epinephrine, and norepinephrine) are represented as the mean difference between the last 60 min of the experimental period (i.e., when stable hypoglycemia was present) and the basal period ± SE. Statistical comparisons among and between groups were made using analysis of variance with repeated measures (25). Post hoc analysis was performed using universal F tests. Significance was presumed at P < 0.05.
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RESULTS |
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Insulin and glucose.
With peripheral insulin infusion, plasma insulin levels rose similarly
in all groups from basal levels (CONT, 13 ± 4; DN, 10 ± 1;
DN + COOL, 12 ± 2 µU/ml) to average values during the last
hour of 297 ± 42, 296 ± 50, and 338 ± 39 µU/ml in CONT, DN, and DN + COOL, respectively (Fig.
1). The average basal glucose levels were 112 ± 2, 108 ± 2, and 107 ± 4 mg/dl in
the three protocols (CONT, DN, and DN + COOL, respectively; Fig.
1). Upon insulin infusion, the glucose level quickly declined to a
hypoglycemic plateau (51 ± 3, 50 ± 2, and 48 ± 2 mg/dl during the final hour in CONT, DN, and DN + COOL,
respectively). There were no significant differences among the glucose
levels in the three protocols at any time.
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Heart rate and pancreatic polypeptide.
The heart rate (Fig. 2) increased
significantly from basal in both CONT and DN in the hypoglycemic period
(averaging 113 ± 10 and 108 ± 11 beats/min, respectively,
during the last hour). The addition of vagal cooling resulted in a
marked increase in heart rate to 211 ± 16 beats/min within 15 min. Pancreatic polypeptide levels increased significantly in the CONT
and DN groups to 941 ± 304 and 871 ± 277 pg/ml,
respectively, and then fell slightly as hypoglycemia continued (Fig.
2). In the DN + COOL group, there was a slight decrease from basal
during the experimental period, attesting to the effectiveness of
cooling in preventing parasympathetic input to the pancreas.
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Glucagon and cortisol.
Glucagon peaked by 30 or 45 min of hypoglycemia (101 ± 18, 120 ± 41, and 97 ± 19 pg/ml in CONT, DN, and DN + COOL
groups, respectively) and then began to decline as the hypoglycemia
continued (Fig. 3). There were no
significant differences in glucagon levels among groups. Similarly, AUC
measurements showed no significant differences in arterial plasma
glucagon levels (1,087 ± 457, 1,483 ± 327, and 2,329 ± 523 pg/ml for CONT, DN, and DN + COOL, respectively), although
there was a tendency for the glucagon change to be greater in the
presence of combined denervation and cooling than in the other two
groups. Clearly, hepatic denervation (DN) did not significantly reduce
the glucagon response to hypoglycemia.
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Epinephrine and norepinephrine.
The plasma epinephrine level (Fig. 4)
increased markedly during the hyperinsulinemic hypoglycemic period to
averages of 1,492 ± 263, 1,778 ± 449, and 1,945 ± 839 pg/ml for the final 30 min of the CONT, DN, and DN + COOL
protocols, respectively. The AUCs were 63,228 ± 10,825, 68,520 ± 12,136, and 89,480 ± 27,862 pg/ml for each
protocol, respectively. The levels were not significantly different
among protocols, indicating that neither denervation nor denervation
plus vagal cooling significantly altered the adrenal response to
hypoglycemia. Similarly, plasma norepinephrine levels (Fig. 4) rose to
averages of 400 ± 34, 494 ± 79, and 514 ± 165 pg/ml
for the final 30 min in CONT, DN, and DN + COOL protocols, respectively. The AUCs were, respectively, 14,039 ± 2,508, 13,049 ± 666, and 15,457 ± 5,445 pg/ml in each protocol.
Neither denervation nor denervation plus vagal cooling significantly
reduced the sympathetic nervous system's response to hypoglycemia.
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Blood glycerol and endogenous glucose production.
Arterial blood glycerol levels (Fig.
5) began to rise after the first 15 min
of the hypoglycemic period in all groups, reaching a similar plateau in
the last 30 min of the study (194 ± 19, 229 ± 61, and
198 ± 53 µmol/l in CONT, DN, and DN + COOL, respectively). The lipolytic response to hypoglycemia was unaffected by hepatic denervation or vagal cooling. Figure 5 shows that endogenous glucose production began to rise by 30 min of hypoglycemia and averaged 4.7 ± 0.7, 5.7 ± 0.5, and 5.8 ± 0.6 mg · kg1 · min
1 in CONT,
DN, and DN + COOL, respectively, during the last hour of the
study. Glucose production was not significantly different from the
control response when the liver was denervated or when denervation was
combined with vagal blockade.
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Substrates.
Plasma free fatty acids, blood alanine, lactate, and
-hydroxybutyrate are shown in Table 1.
ANOVA failed to show any period or treatment effects for free fatty
acids or
-hydroxybutyrate. There was a period effect for alanine
that failed to reach significance when post hoc analysis was applied.
There was a significant increase in blood lactate levels during the
hypoglycemic period in the DN and DN + COOL groups
(P < 0.05).
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DISCUSSION |
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It has been suggested that the counterregulatory hormone responses to hypoglycemia are, at least in part, initiated by hepatic glucoreceptors that send afferent messages to the CNS (7, 8). We have previously shown that the vagus nerves are not involved in the transmission of this signal (14). In the present study, we attempted to define the role of afferent nerves, not traveling along the parasympathetic system, in transmitting signals from the liver to the brain in response to hypoglycemia. This is particularly important in light of recent suggestions that the afferent signals may not travel along the vagal trunk. The present results demonstrate that afferent nerves originating in the hepatoportal region, regardless of their path to the brain, are not necessary for a complete counterregulatory response to the insulin-induced hypoglycemic challenge we presented.
To examine the effects of afferent nerve transmission from the liver to the CNS, we chronically denervated the liver. This surgical technique has been utilized previously in our laboratory (2, 19), and as before, its effectiveness was determined by measuring liver norepinephrine concentrations from each of the seven liver lobes and comparing them with levels in normal dog liver. In the present study, the norepinephrine content of the liver decreased to 2.5 ± 1.0% of normal concentration, thus indicating complete denervation. Because we could not directly measure liver acetylcholine, we further ensured blockade of afferent signals by combining our previously described vagal cooling technique (14) with liver denervation. The completeness of parasympathetic blockade was confirmed by measuring plasma pancreatic polypeptide levels and heart rate. The former failed to rise in response to hypoglycemia, whereas the heart rate increased twofold.
A similar hypoglycemic nadir (50 mg/dl) was achieved in all three
protocols. Likewise, hyperinsulinemia (
310 µU/ml) was similar in
all three groups. Hepatic denervation (DN) caused no impairment in the
hypoglycemia-induced elevations in plasma norepinephrine, epinephrine,
cortisol, or glucagon. This is most easily demonstrated by our
observation that the increase in AUC for each of the above hormones
was, if anything, slightly greater in the DN group than in the CONT
group. Despite the lack of evidence of a decreased response, it is
possible that we could have missed a small change in the
counterregulatory hormone response due to the variation in the data.
With regard to norepinephrine, we had excellent power and could have
detected a change of 75 pg/ml with a power of 0.95. With epinephrine,
the variation was greater, thereby lessening our precision;
nevertheless, we could have detected a change in epinephrine of 800 pg/ml with a power of 0.80.
Because there was a possibility of residual parasympathetic activity at
the liver (upon biopsy, we could not rule out the presence of
parasympathetic nerve endings), we utilized our vagal cooling technique
in addition to hepatic denervation (DN + COOL) to ensure complete
neural isolation of the liver. Even in the absence of both sympathetic
and parasympathetic signaling, there was still no impairment in any
counterregulatory response to hypoglycemia. The addition of vagal nerve
cooling to liver denervation (DN + COOL) caused a significant rise
(5 µg/dl) in cortisol levels at the initial 15-min time point that
was not evident in the CONT group. As a result, the AUC for the plasma
cortisol level was significantly greater with the DN + COOL
treatment than in DN. We noted such a change previously on two
occasions (14, 27), and it probably represents a response
to a mild stress created by the cooling per se. In a previous study
(27), we showed that under euglycemic conditions with
insulin and glucagon clamped at basal values, vagal cooling resulted in
a
3 µg/dl increase in the plasma cortisol level that was evident
for the 90 min of the experiment. When one calculates the AUC for
cortisol in the present study (DN + COOL) and subtracts the part
probably attributable to the cooling stress (assuming it was sustained
for 90 min) there was no difference in the cortisol response to
hypoglycemia in the control and DN + COOL protocols.
In the present study, we also observed a small initial rise in plasma glucagon in response to cooling that was not evident in the control protocol. Glucagon secretion, therefore, may also have been stimulated by the cooling stress. It should be noted, however, that this phenomenon was not observed in our earlier euglycemic experiments because we used somatostatin to clamp plasma glucagon (27). Because we do not know the time course of any potential effect of cooling on glucagon secretion, it is difficult to interpret the glucagon data for the DN + COOL protocol. It should be remembered, however, that liver denervation had no inhibitory effect on hypoglycemia-induced glucagon secretion in the present study and that in none of the studies by Donovan and colleagues (7, 8, 13) was there any evidence for an alteration in the response of glucagon to hypoglycemia. Interestingly, as we showed previously and as is evident here, neither epinephrine nor norepinephrine appeared to show any increase in response to the stress of vagal cooling per se.
We also assessed the effect of hepatic denervation on the metabolic responses to insulin-induced hypoglycemia. Not only was there no diminution in the counterregulatory hormone responses to low plasma glucose when the liver was denervated, there was also no decrease in the increment in glucose production. Removal of both parasympathetic and sympathetic nerves to the liver had no apparent effect on its ability to produce glucose in response to hypoglycemia. Similarly, the lipolytic response to hypoglycemia, as reflected in the plasma glycerol level, was also unaffected by hepatic denervation with or without the addition of vagal cooling.
In a study by Connolly et al. (3), dogs were
adrenalectomized and given somatostatin to completely block the
hormonal response to insulin-induced hypoglycemia (36 mg/dl). When
the dogs were given glucose via carotid and vertebral catheters to
maintain cerebral euglycemia, there was little or no blunting of the
hypoglycemia-induced increase in glucose production. It was concluded,
therefore, that the liver either senses the hypoglycemia and directly
responds to it or it transmits a signal to the brain with a subsequent relay back to the liver (despite euglycemia in the brain). Because we
know that 50% of the increase in hepatic glucose production during
insulin-induced hypoglycemia is dependent on hormonal response (10), and because in the present study we found that
neither the counterregulatory hormone response nor the increase in
hepatic glucose production changed when the liver was denervated, it
would seem that neural input to the liver is of little significance to
the hepatic response. This would suggest that, in the study of Connolly
et al., the liver increased glucose production by responding directly
to the low glucose concentration and not by responding reflexly to
neural input from the CNS.
Although we have demonstrated that there appears to be no transmission
of signals from the liver to the brain via sympathetic or
parasympathetic nerves, it must be noted that our finding may be
specific to our experimental conditions, in which hypoglycemia (50 mg/dl) was induced by a high dose of insulin (5.0 mU · kg1 · min
1
administered via a peripheral leg vein). It is possible that the liver
plays a role in initiating counterregulation at different levels of
hypoglycemia or hyperinsulinemia. For example, in the study of Donovan
et al. (7), a peripheral insulin infusion of 3-3.5
mU · kg
1 · min
1 was used to
create a milder hypoglycemia (
58 mg/dl). When liver euglycemia
(
95 mg/dl) was maintained by portal glucose infusion, they observed
a decrement of
40% in the sympathoadrenal response to hypoglycemia.
The discrepancy between our findings and theirs may relate to the
different levels of insulin and hypoglycemia used, although this seems
unlikely given the relatively small differences in the experimental
conditions that were used. A more plausible explanation is that, in the
study of Donovan et al., a feeding signal was created by the
establishment of a negative arterioportal glucose gradient
(22). This has been shown to be associated with a decrease
in afferent nerve firing to the adrenal glands (21), thus
potentially explaining the decrease in the sympathoadrenal response
seen by Donovan et al. Because the portal glucose level does not exceed
the arterial glucose level in most hypoglycemic settings, this
mechanism would not normally play a part in the defense against low
blood glucose. In a recent paper, Hevener et al. (13)
diminished the magnitude of the sympathoadrenal response to
insulin-induced hypoglycemia in rats by chemically denervating the
portal vein. They therefore concluded that attributing the diminished
sympathoadrenal response to a negative arterioportal glucose difference
is not valid. The difficulty with their experimental design is that, by
chronically denervating the portal vein, the input from the afferent
fibers to the brain is eliminated, thereby simulating a high glucose level in the portal vein (an increase in the portal vein glucose concentration decreases afferent vagal firing rates). Their results are, therefore, not surprising and can be interpreted as supporting the
concept of a feeding signal reducing the response to hypoglycemia.
Hevener et al. (13) suggested that we might not have completely stripped the nerves of the portal vein for a sufficient distance to completely remove all the hepatoportal sensors. This seems unlikely, given the nature of the surgical procedure, and because in a previous publication (2) we abolished the increase in net hepatic glucose uptake induced by the portal signal by using the same denervation procedure. This suggests that our denervation technique is sufficient to eliminate glucose sensors within the hepatoportal region. It is possible, on the other hand, that, after chronic liver denervation, the organism develops an adaptive mechanism to react to hypoglycemia, thus explaining why we did not see any differences among the groups in the present study.
It is also possible that, in the presence of very high insulin levels,
the brain can integrate neural signals differently. It is known that
the brain is responsive to the insulin level in plasma per se, but the
significance of this finding is unknown. Davis et al. (4)
showed that, with a selective physiological increase in the plasma
insulin level in the carotid and vertebral arteries (200 µU/ml),
the counterregulatory response to hypoglycemia (
57 mg/dl) was
amplified relative to that seen with the same glycemia but less
insulin. The additional increase in the plasma levels of epinephrine,
norepinephrine, and cortisol led to an augmented increase in hepatic
glucose production. It is possible, therefore, that, in the present
study, by choosing a high rate of insulin infusion we have created a
scenario in which the brain is dominant. Nevertheless, this study has
demonstrated that, in the conscious dog, afferent hepatic nerves were
not essential for eliciting a normal counterregulatory hormone response
to hypoglycemia in the presence of high (310 µU/ml) arterial insulin
levels. It remains to be seen whether, under different hypoglycemic or
insulin conditions, a role for liver glucose sensors emerges.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent technical assistance of Jon Hastings. The help of Dr. Mary Courtney Moore was greatly appreciated during the preparation of the manuscript.
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FOOTNOTES |
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The work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants 2RO1 DK-18243 and 5PO60 DK-20593 and by Juvenile Diabetes Foundation International Grant no. 397008.
Address for reprint requests and other correspondence: S. Cardin, Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School of Medicine, 702 Light Hall, Nashville, TN 37232-0615. (SYLVAIN.CARDIN{at}McMail.Vanderbilt.EDU).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 June 2000; accepted in final form 19 July 2000.
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