Involvement of the vagus nerves in the regulation of basal hepatic glucose production in conscious dogs

Sylvain Cardin1,2, Konstantin Walmsley3, Doss W. Neal3, Phillip E. Williams3, and Alan D. Cherrington1,3

1 Department of Molecular Physiology and Biophysics, 3 Diabetes Research and Training Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615; and 2 Department of Neuroscience, Pro-Neuron, Gaithersburg, Maryland 20877


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We determined if blocking transmission in the fibers of the vagus nerves would affect basal hepatic glucose metabolism in the 18-h-fasted conscious dog. A pancreatic clamp (somatostatin, basal portal insulin, and glucagon) was employed. A 40-min control period was followed by a 90-min test period. In one group, stainless steel cooling coils (Sham, n = 5) were perfused with a 37°C solution, while in the other (Cool, n = 6), the coils were perfused with -20°C solution. Vagal blockade was verified by heart rate change (80 ± 9 to 84 ± 14 beats/min in Sham; 98 ± 12 to 193 ± 22 beats/min in Cool). The arterial glucose level was kept euglycemic by glucose infusion. No change in tracer-determined glucose production occurred in Sham, whereas in Cool it dropped significantly (2.4 ± 0.4 to 1.9 ± 0.4 mg · kg-1 · min-1). Net hepatic glucose output did not change in Sham but decreased from 1.9 ± 0.3 to 1.3 ± 0.3 mg · kg-1 · min-1 in the Cool group. Hepatic gluconeogenesis did not change in either group. These data suggest that vagal blockade acutely modulates hepatic glucose production by inhibiting glycogenolysis.

vagal cooling; liver nerves; parasympathetic blockade; gluconeogenesis; glycogenolysis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE AUTONOMIC NERVOUS SYSTEM is involved in the regulation of hepatic glucose metabolism. It has been demonstrated, for example, that electrical stimulation of the vagus nerves induces activation of the liver enzyme glycogen synthase, which in turn increases glycogen synthesis and reduces glucose output (19). Conversely, electrical stimulation of the splanchnic nerve induces activation of the liver enzyme glycogen phosphorylase, which in turn increases glycogenolysis and glucose output (20). Taken together, these data have been interpreted to suggest that activation of the parasympathetic nervous system promotes glucose uptake and hepatic glycogen deposition while activation of the sympathetic nervous system promotes glycogenolysis and glucose output.

It is also known that glucosensors within the hepatoportal region have the ability to sense glucose and inform the brain of its concentration via afferent fibers traveling, at least in part, along the hepatic vagus nerves (16). It has been hypothesized that the brain uses this information to minimize fluctuations in the plasma glucose level after feeding (18). The efferent responses of this feedback loop involve the pancreas, adrenal glands, adipocytes, skeletal muscles, and the liver (2). Although it is known that the autonomic nervous system is vital to the regulation of glucose metabolism in times of stress, evidence is accumulating to support a role for it in the postprandial state. Nevertheless, its role in regulating hepatic glucose metabolism after an overnight fast is not clear.

Our aim, therefore, was to cool the vagus nerves, in the presence of a pancreatic clamp, to study the involvement of the parasympathetic nervous system in the control of hepatic glucose output in the 18-h-fasted dog.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care. Experiments were conducted on 11 conscious, mongrel dogs (22 ± 2 kg) of either sex. The animals were fed one time daily with a meat (KalKan, Vernon, CA) and chow (Lab Canine Diet No. 5006; Purina, St. Louis, MO) diet (34% protein, 46% carbohydrate, 14.5% fat, and 5.5% fiber based on dry weight). Before the 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 experiment, the dogs underwent surgery for placement of splenic and jejunal vein infusion catheters, hepatic and portal vein sampling catheters, and a femoral artery sampling catheter, as previous described (4). Each dog was used for an experiment only if it had a leukocyte count <18,000/mm3, a hematocrit >35%, a good appetite, and normal stools. The position of the catheter tips was confirmed upon necropsy at the end of each experiment.

Stainless steel cooling coils, with Silastic extension tubing attached, were placed around the vagus nerves in the neck in all dogs, as described previously (1, 10). The effectiveness of the cooling-induced blockade of parasympathetic signaling was verified by measuring the heart rate (which is under vagal control).

On the day of the study, the abdominal and femoral artery catheters, as well as 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 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 in the left cephalic vein for somatostatin infusion, in the right cephalic vein for peripheral glucose infusion (20% dextrose) as needed, and in the saphenous vein for tracers and indocyanine green infusion. After coil preparation, each dog was allowed to stand calmly in a Pavlov harness for 30 min before the start of the experiment.

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 min to 0 min), and a 90-min experimental period (0-90 min). A priming dose (55 µCi) of [3-3H]glucose (NEN, Boston, MA) was administered at -140 min followed by a continuous infusion of 0.47 µCi/min [3-3H]glucose, 0.42 µCi/min [14C]alanine (NEN), and indocyanine green (0.07 mg/min). At time -130 min, a peripheral somatostatin infusion (0.8 µg · kg-1 · min-1), an intraportal porcine insulin infusion (0.3 mU · kg-1 · min-1; Eli Lilly, Indianapolis, IN), and an intraportal glucagon infusion (0.65 ng · kg-1 · min-1; GlucaGen; Bedford Laboratories, Bedford, OH) were started and continued for the entirety of the experiment in both protocols. Plasma glucose samples were taken every 5 min, and the insulin infusion rate was adjusted as necessary to maintain glucose at basal levels. The last change was made at least 30 min before the start of the control period, and the insulin infusion rates (192 and 214 µU · kg-1 · min-1 in the Sham and Cool groups, respectively) remained unchanged after that. To maintain a euglycemic clamp (approx 100 mg/dl), glucose (20% dextrose; Baxter, Deerfield, IL) was infused through a leg vein as needed during the experimental period. The dogs were randomly assigned to the Sham (n = 5) or Cool (n = 6) groups. During Sham experiments, the coils were perfused for the entire experimental period (90 min) with 37°C fluid. In contrast, in the Cool group, a -20°C fluid was perfused for the entire experimental period (90 min). In a previous experiment, we have shown that perfusing the coils at this temperature maximally blocked the parasympathetic input to the heart and that atropine did not further increase the heart rate, demonstrating the efficacy of the procedure (10). 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 glucose clamp. Arterial, portal, and hepatic 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 (21). 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. Plasma insulin and glucagon were measured using double-antibody RIAs described previously (15) with interassay coefficients of variation (CV) of 7 and 5%, respectively. Plasma samples used for glucagon determination contained 100,000 kallikrein inhibitor units aprotinin (Trasylol; Miles; Kankakee, IL) added at collection. Catecholamines were assayed using HPLC as previously described (13). The interassay CV for epinephrine and norepinephrine were 7 and 5%, respectively. The samples for catecholamine analysis contained 60 µl glutathione/EGTA added at collection. Plasma cortisol was assayed using the Clinical Assays Gamma Coat RIA kit with an interassay CV of 6% (7). Whole blood levels of lactate, glycerol, alanine, and beta -hydroxybutyrate were determined using perchloric acid-treated (3%) blood samples according to the method of Lloyd et al. (11), as adapted to the Monarch 2000 centrifugal analyzer (Lexington, MA). Plasma [3-3H]- and [14C]glucose were determined by liquid scintillation counting after Somogyi-Nelson deproteinization and an 3H2O exclusion procedure (21).

Calculations. 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. (25), as simplified by DeBodo et al. (5), and also according to a two-compartment model described by Mari (12) 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 Figs. 1-5. For calculation of hepatic glucose uptake, the net [3H]glucose uptake was divided by the arterial [3H]glucose specific activity. The net hepatic balances of unlabeled glucose and [3H]glucose were calculated using the formula (H - [0.28 × A + 0.72 × P]) × HF where H, A, and P were the substrate concentrations in hepatic vein, femoral artery, and portal vein blood or plasma, respectively. HF represents the total hepatic blood flow estimated from indocyanine green, and 0.28 and 0.72 are the approximate contributions of the hepatic artery and the portal vein, respectively, to the total hepatic blood flow during a pancreatic clamp. We have shown in a previous study (Cardin and Cherrington, unpublished observation) that vagal cooling did not alter the flow distribution. With this calculation, a positive value represents net production by the liver, whereas a negative value represents net hepatic uptake. Plasma glucose and [3H]glucose values in the calculations were multiplied by 0.73 to convert them to blood glucose values, as validated elsewhere (14). To obtain endogenous glucose production, the amount of glucose infused was subtracted from total glucose production. Gluconeogenesis was assessed by determining gluconeogenic efficiency, as previously described (3). In addition, the total net hepatic uptake of the gluconeogenic precursors was determined and divided by two to convert the rate to glucose equivalents (mg · kg-1 · min-1). This provides an estimate of hepatic gluconeogenic flux to glucose 6-phosphate as previously described (4). Net hepatic glycogenolysis was estimated using the following equation
NHGly = NHGO + NHLO + GO − GNGFlux
where NHGly is net hepatic glycogenolysis, NHGO is net hepatic glucose output, NHLO is net hepatic lactate output, GO is hepatic glucose oxidation, and GNGFlux is gluconeogenic flux to glucose 6-phosphate. GO was assumed to remain unaltered at 0.2 mg · kg-1 · min-1.


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Fig. 1.   Effects of vagal cooling on the plasma glucose level and heart rate in conscious overnight-fasted dogs maintained on a pancreatic clamp. Sham and Cool, stainless steel cooling coils perfused with a 37°C solution or a -20°C solution, respectively. Glucose was infused to maintain euglycemia (Sham n = 5, Cool n = 6 dogs). Data are means ± SE. Heart rate increased significantly (P < 0.05) from 15 min on in the Cool group.



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Fig. 2.   Arterial plasma glucagon and insulin levels resulting from the pancreatic clamp in conscious overnight-fasted dogs subjected to sham or vagal cooling (Sham n = 5, Cool n = 6). Data are means ± SE. Neither parameter changed significantly.



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Fig. 3.   Arterial plasma cortisol levels before and during cooling or sham cooling of the vagus nerves in conscious overnight-fasted dogs maintained on a pancreatic clamp (Sham n = 5, Cool n = 6). Data are means ± SE. The cortisol level rose significantly (P < 0.05) from 30 min on in the Cool protocol.



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Fig. 4.   Arterial epinephrine and norepinephrine levels before and during cooling or sham cooling of the vagus nerves in conscious overnight-fasted dogs maintained on a pancreatic clamp (Sham n = 5, Cool n = 6). Data are means ± SE. Neither parameter changed significantly.



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Fig. 5.   Net hepatic glucose output, hepatic glucose uptake, and the glucose infusion rate before and during cooling or sham cooling of the vagus nerves in conscious overnight-fasted dogs maintained on a pancreatic clamp (Sham n = 5, Cool n = 6). Data are means ± SE. The changes in net hepatic glucose output and glucose infusion in the Cool group were significant (P < 0.05) from 30 min on.

Statistical analysis. Data are expressed as means ± SE. Statistical comparisons among groups and between groups were made using ANOVA with repeated measures. Post hoc analysis was performed using universal F-tests. Significance was presumed at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucose and heart rate. Arterial plasma glucose levels were stable during the experiment (100 ± 5 and 97 ± 5 mg/dl in the Sham and 98 ± 2 and 94 ± 3 mg/dl in the Cool groups for the control and experimental periods, respectively, Fig. 1, top). The heart rate did not increase significantly in the Sham group when the coils were perfused (Fig. 1, bottom). On the other hand, perfusion of the coils with a cold solution resulted in a marked increase (P < 0.05) in the heart rate of 63 ± 13 beats/min within 15 min and 102 ± 22 beats/min by the end of the experimental period (Fig. 1, bottom). This increase attested to the effectiveness of cooling in producing parasympathetic blockade. The hepatic blood flow was similar in the basal period (25.6 ± 3.4 and 25.1 ± 3.2 ml · kg-1 · min-1) and the experimental period (26.9 ± 3.6 and 28.7 ± 3.1 ml · kg-1 · min-1) in both protocols and did not change in response to vagal cooling.

Insulin and glucagon. The average arterial plasma insulin concentrations were 4 ± 1 and 5 ± 1 µU/ml in the basal period and 4 ± 1 and 6 ± 1 µU/ml in the experimental period for the Sham and Cool groups, respectively (Fig. 2, top). Similarly, the average arterial plasma glucagon concentrations were 46 ± 4 and 53 ± 6 pg/ml in the basal period and 44 ± 4 and 51 ± 5 pg/ml in the experimental period for the Sham and Cool groups, respectively (Fig. 2, bottom). In the presence of a pancreatic clamp, vagal cooling had no effect on plasma insulin or glucagon levels.

Cortisol. Basal arterial plasma cortisol levels (Fig. 3) were similar in the two groups (Sham 2.0 ± 0.4 and Cool 2.1 ± 0.4 µg/dl). Perfusing the coils with a 37°C solution did not significantly affect the arterial plasma cortisol level. On the other hand, perfusing the coils with a -20°C solution caused a rapid small increase in the plasma cortisol level of ~3 µg/dl (Fig. 3). The rise was sustained over the 90-min test period.

Epinephrine and norepinephrine. Although the arterial plasma epinephrine levels tended to rise slightly in the Cool group compared with the Sham group, no significant difference between the groups was found at any time point (Sham 56 ± 26 to 73 ± 25 and Cool 99 ± 45 to 145 ± 67 pg/ml for basal and experimental periods, respectively; Fig. 4, top). Similarly, the arterial plasma norepinephrine levels (Fig. 4, bottom) were stable in the Sham (81 ± 11 to 93 ± 20) and in the Cool (84 ± 12 to 76 ± 14 pg/ml) groups.

Gluconeogenic precursors and lipolysis. Arterial blood lactate levels remained at basal values throughout the experiment (Table 1) regardless of treatment. Average basal values for net hepatic lactate output were 8.20 ± 3.06 in the Sham and 8.59 ± 3.26 µmol · kg-1 · min-1 in the Cool groups (Table 2). These values showed a tendency to decrease over time to an average of 5.60 ± 2.40 and 4.78 ± 2.94 µmol · kg-1 · min-1 in Sham and Cool groups, respectively. There were no significant differences between the two groups.

                              
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Table 1.   Arterial blood lactate, alanine, and glycerol in 18-h-fasted dogs maintained on a pancreatic clamp and subjected to cooling or sham cooling of their vagus nerves


                              
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Table 2.   NHLO, NHAU, and NHGlyU in 18-h-fasted dogs maintained on a pancreatic clamp and subjected to cooling or sham cooling of their vagus nerves

Similarly, the arterial blood alanine levels (Table 1) did not change over time. Average basal values for net hepatic alanine uptake were 2.60 ± 0.49 in the Sham and 3.22 ± 0.97 µmol · kg-1 · min-1 in the Cool groups (Table 2). These values did not change (2.99 ± 0.39 and 3.61 ± 0.75 µmol · kg-1 · min-1, respectively) during coil perfusion in either group. Again, there were no significant differences between the two groups.

The arterial blood glycerol levels (Table 1) remained at basal values during the entire protocol. The average net hepatic glycerol uptake did not change significantly in either group (Sham 0.68 ± 0.18 and 0.77 ± 0.21 and Cool 0.94 ± 0.22 and 1.21 ± 0.24 µmol · kg-1 · min-1 for basal and experimental periods, respectively; Table 2) nor were they different in the two groups.

Glucose metabolism. Net hepatic glucose output did not change over time in the Sham group (2.10 ± 0.15 and 2.17 ± 0.25 for the average basal and experimental period values, respectively; Fig. 5, top). Conversely, net hepatic glucose output in the Cool group significantly decreased from an average basal value of 1.95 ± 0.34 to 1.32 ± 0.22 by 30 min and remained significantly reduced for the rest of the perfusion period (Fig. 5, top). The average values for the last 45 min of the experiment were significantly (P < 0.05) lower in the Cool compared with the Sham group (1.51 ± 0.18 and 2.18 ± 0.27 mg · kg-1 · min-1, respectively).

Hepatic glucose uptake determined by the tracer method did not change during the experiment (Sham 0.31 ± 0.13 and 0.30 ± 0.14 and Cool 0.29 ± 0.11 and 0.30 ± 0.12 mg · kg-1 · min-1 for basal and the last 30 min of the experimental period, respectively). This indicates that the drop in net hepatic glucose output was a reflection of decreased hepatic glucose release. Indeed, endogenous glucose production remained unchanged in the Sham group (basal 2.47 ± 0.24 and last 45 min 2.37 ± 0.22 mg · kg-1 · min-1), whereas it decreased significantly in the Cool group (basal 2.43 ± 0.22 and last 45 min 1.93 ± 0.18 mg · kg-1 · min-1). Significant differences were found between the two groups when comparing the average endogenous glucose production for the last 45 min of the experiment. There were no significant differences between the groups when comparing the glucose disposal rate (Table 3). The average glucose infusion (Fig. 5, bottom) rate necessary to maintain euglycemia was significantly lower in the Sham compared with the Cool groups (Sham 0.18 ± 0.08 and Cool 1.03 ± 0.27 mg · kg-1 · min-1). Gluconeogenic efficiency did not change during the experiment (Sham 0.25 ± 0.15 and 0.29 ± 0.12 and Cool 0.37 ± 0.19 and 0.24 ± 0.10 for the basal and the last 30 min of the experimental period, respectively). These results are supported by the estimate of gluconeogenic flux derived using the arteriovenous difference technique (Sham 0.83 ± 0.20 and 1.01 ± 0.10 and Cool 1.00 ± 0.30 and 1.24 ± 0.20 mg · kg-1 · min-1 for the basal and the last 30 min of the experimental period, respectively; data not shown). This indicates that the decrease in hepatic glucose release was attributable to reduced glycogenolysis.

                              
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Table 3.   Glucose utilization in 18-h-fasted dogs maintained on a pancreatic clamp and subjected to cooling or sham cooling of their vagus nerves


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigates the effect of acute blockade of the vagus nerves on hepatic metabolism in the conscious dog. The fact that a maximal increase in heart rate was observed in response to vagal cooling demonstrates the effectiveness of vagal blockade. To prevent changes in pancreatic hormonal and glucose levels that could themselves affect hepatic glucose production, insulin, glucagon, and glucose were fixed at basal levels for the duration of the experiment using a euglycemic pancreatic clamp. Our data indicate that interrupting vagal transmission using the vagal cooling technique decreased net hepatic glucose output resulting from a decrease in glycogenolysis.

The main finding of the present study was that vagal blockade decreased glucose production. This was evident from three independent pieces of data. First, a decrease in net hepatic glucose output was seen by 30 min during vagal blockade, whereas no significant change was seen in net hepatic glucose output in the Sham group. Second, tracer-determined endogenous glucose production also decreased in the Cool group without any change occurring in the Sham group. Third, the rate of glucose infusion required to maintain euglycemia in the Cool group was significantly greater than in the Sham group despite equivalent glucose utilization rates. These three independent measures attest to the veracity of the observation. Because neither gluconeogenic efficiency nor gluconeogenic flux was altered by vagal blockade, it seems likely that a decrease in glycogenolysis was responsible for the decrease in glucose output by the liver.

Our finding of reduced net hepatic glucose output is somewhat surprising in view of the literature that would have predicted a possible increase in net hepatic glucose output when the inhibitory effects of ACh were removed. Indeed an activation of the parasympathetic nervous system promotes glucose uptake by the liver (19, 23). In the absence of parasympathetic input, we would therefore have expected a net increase in hepatic glucose output. It must be remembered, however, that the vagus nerves carry both afferent and efferent fibers. It is possible, therefore, that, by blocking the afferent fibers traveling along the vagus nerves, we reflexively decreased sympathetic inflow to the liver. Thus, by cooling the vagus nerve, we created a situation that reproduced a feeding signal, which has been shown to decrease efferent sympathetic outflow (17). To the extent that basal sympathetic tone is important to glucose production by the liver, this would decrease glycogen phosphorylase activity, glycogenolysis, and eventually glucose production (20). The fact that acutely interrupting vagal transmission decreased hepatic glucose production is in contrast to other studies that have shown that hepatic vagotomy does not change basal hepatic glucose production (24). It should be kept in mind, however, that, when one uses chronic surgical denervation, the organism can adapt to the change, thus potentially obscuring the acute effects of denervation.

The arterial blood glycerol levels remained basal throughout the experiments, and no significant differences were found between the groups. This suggests that lipolysis was not affected by the vagal blockade. Thus one can assume that sympathetic input to the adipocytes did not change. This suggests that, if the decrease in net hepatic glucose output was because of decreased sympathetic input, this was a selective decrease to the liver.

A significant increase in arterial plasma cortisol of congruent 3 µg/dl occurred 15 min after the beginning of the cooling procedure. This increase appears to be a specific response to vagal cooling, since we observed a similar increase in our previous experiments using the vagal cooling technique and since it did not occur in the Sham group. This increase in cortisol cannot, however, explain the fall in glucose production, because if anything a rise in cortisol would be expected to increase glucose production (8, 9). In fact, however, we have previously shown that an acute change of this magnitude would have little effect on glucose production over a 3-h period (9). Likewise, the small nonsignificant rise in epinephrine, even if real, would have done little to alter hepatic glucose production based on our earlier dose-response studies with the catecholamine (22).

In summary, vagal blockade decreased hepatic glucose production without altering hepatic glucose uptake or gluconeogenesis. These data suggest that hepatic vagus nerves play a role in maintaining hepatic glycogenolysis during the basal state in conscious 18-h-fasted dogs. We postulate that they do so by altering the vagosympathetic reflex, to maintain sympathetic input to the liver, which in turn stimulates glucose production to a modest extent.


    ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Jon Hastings.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants 2RO1 DK-18243 and 5PO60 DK-20593 and Juvenile Diabetes Foundation International Grant JDFI no. 397008.

Address for reprint requests and other correspondence: A. D. Cherrington, Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ. School of Medicine, 702 Light Hall, Nashville, TN 37232-0615.

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.

10.1152/ajpendo.00566.2001

Received 26 December 2001; accepted in final form 24 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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