SPECIAL COMMUNICATION
Role of free fatty acids and glucagon in the peripheral effect of insulin on glucose production in humans

Gary F. Lewis, Mladen Vranic, and Adria Giacca

Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5G 2C4

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We have shown previously that the greater suppression of endogenous glucose production (GP) with equimolar peripheral vs. portal insulin cannot be detected or is minimally reversed when the insulin-induced suppression of either free fatty acids (FFA) or glucagon alone is prevented. The present experiments were designed to minimize the insulin suppression of both glucagon and FFA in an attempt to further examine the mechanism of insulin's peripheral effect on GP. In nine healthy men, we investigated the effect of limiting the insulin suppression of both FFA and glucagon by infusing heparin (250 U/h), Intralipid 10% (25 ml/h), and glucagon (0.65 ng · kg-1 · min-1) during 1) portal (n = 9), 2) equimolar peripheral (n = 9), and 3) half-dose peripheral insulin delivery (n = 4) by use of our previously published tolbutamide infusion method, with calculation and matching of insulin secretion rate. GP decreased by 57.2 ± 2.6% with portal, 39.0 ± 4.1% with equimolar peripheral, and 31.5 ± 2.7% with half-dose peripheral insulin delivery (P < 0.001 for portal vs. peripheral and P < 0.001 for portal vs. half-dose peripheral). In contrast, in six control subjects in whom glucagon and FFA were not replaced, GP decreased by 62.6 ± 2.4% with portal (n = 6), 75.7 ± 3.0% with peripheral (n = 6), and 56.3 ± 3.0% with half-dose peripheral (n = 4) insulin delivery (P < 0.01 for portal vs. peripheral and P = not significant for portal vs. half-dose peripheral). In summary, the greater suppression of GP with equimolar peripheral vs. portal insulin is eliminated and markedly reversed if the acute insulin-induced suppression of both plasma FFA and glucagon is minimized. This suggests that the insulin-induced suppression of glucagon and FFA has additive or cooperative effects in mediating the acute extrahepatic effect of insulin on GP.

portal vein; tolbutamide; glucose clamp technique

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

APPROXIMATELY 50% OF INSULIN secreted into the portal vein is extracted on first pass through the liver, with subsequent dilution in the greater systemic circulation, creating a physiological portal-peripheral insulin concentration gradient. The metabolic effects of insulin delivery via the portal vs. peripheral venous circulation have been the subject of intensive investigation in recent years and have great bearing on methods of insulin replacement therapy for individuals with diabetes mellitus. It has recently been shown that ~75% of total endogenous glucose production (GP) in the postabsorptive state is derived from the liver and ~25% from renal gluconeogenesis (29). Insulin exerts multiple metabolic effects in the liver, including the acute suppression of GP. Although one would logically expect insulin to exert these metabolic effects by a direct action on the hepatocyte, we and others have shown in recent years that insulin also acutely suppresses GP indirectly by extrahepatic mechanisms (1, 13, 17, 19, 20, 22, 23, 27, 28).

This extrahepatic effect of insulin on GP has been demonstrated experimentally in a variety of human and animal models. Insulin infused by peripheral vein has a greater suppressive effect on GP than equimolar insulin delivered by the portal vein (13, 17), and a selective increase in peripheral insulin without altering hepatic insulin levels suppresses GP (27). The precise mechanism whereby insulin can indirectly affect GP has been intensively investigated in recent years (12, 15, 16, 19, 22, 23, 28) and is the subject of investigation in the present study. In addition to the likely greater suppression of renal gluconeogenesis by systemic (peripherally administered) insulin vs. portal insulin, two other potentially important mechanisms are 1) insulin's potent antilipolytic effect in peripheral tissues, which reduces the flux of free fatty acids (FFA) to the liver (2), and 2) the insulin-induced suppression of alpha -cell glucagon secretion. Glucagon stimulates GP, and suppression of plasma glucagon levels results in suppression of GP (26). Likewise, FFA stimulate gluconeogenesis, and an acute suppression of FFA has been shown to suppress GP (7, 24). Preventing the acute insulin-induced suppression of either FFA or glucagon has been shown to prevent or diminish the indirect suppressive effect of insulin on GP (12, 15, 16, 23, 28). There have, however, been no previously published studies in humans in which both glucagon and FFA were simultaneously replaced during acute portal and peripheral hyperinsulinemia to investigate how this would impact on insulin's ability to regulate GP.

In the present study we used our previously published method of noninvasively matching the rate of pancreatic insulin secretion with a peripheral venous insulin infusion in healthy nondiabetic individuals (14-18). This is achieved with a programmed intravenous tolbutamide infusion and calculation of the insulin secretion rate from peripheral venous C-peptide levels, followed by a euglycemic hyperinsulinemic clamp in the same individual 4-6 wk later in which the exogenous insulin infusion rate is matched with the calculated rate from the earlier tolbutamide study. Alternatively, insulin can be infused at one-half the calculated delivery rate to match the peripheral insulin levels obtained during portal infusion. In control experiments, we permitted both the glucagon and FFA levels to decline during hyperinsulinemia and did not attempt to replace either glucagon or FFA. In a second group of subjects, we infused glucagon and a combination of heparin (to stimulate lipoprotein lipase) and Intralipid (a synthetic triglyceride emulsion) to limit the insulin-induced decline in glucagon and FFA. The measurement of primary interest was the percent suppression of GP. The present experiments were designed to minimize the insulin suppression of glucagon and FFA to diminish the peripheral effects of insulin.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Subjects

Fifteen healthy nondiabetic men participated in the study, with nine receiving heparin, Intralipid, and glucagon during portal and peripheral insulin delivery as detailed below (FFA-glucagon clamp studies) and with six participating in control experiments in which no heparin, Intralipid, or glucagon was administered during hyperinsulinemia (controls). The mean age of the subjects participating in the FFA-glucagon clamp studies was 24.3 ± 1.2 yr and their body mass index was 26.1 ± 1.1 kg/m2, whereas those of the controls were 26.5 ± 2.4 yr and 25.2 ± 1.3 kg/m2, respectively. No subject had a history of systemic illness, and none was taking any medication at the time of the study. Informed written consent was obtained from all participants in accordance with the guidelines of the Human Subjects Review Committee of The Toronto Hospital, University of Toronto. Permission was granted by the Health Protection Branch, Health and Welfare Canada, for the use of intravenous tolbutamide (IND 034076, to Dr. G. Lewis).

Experimental Protocol

All subjects were studied on two occasions each, 6-8 wk apart. In addition, four subjects in each group underwent a third study on a subsequent occasion. In the first study, hyperinsulinemia was induced by an intravenous tolbutamide infusion; it will be referred to as the portal study. In the second study, hyperinsulinemia was induced by an exogenous insulin infusion; it will be referred to as the full-rate peripheral insulin study. In the third study, exogenous insulin was infused at one-half the rate infused in study 2; it will be referred to as the half-rate peripheral insulin study.

Portal study. Subjects were admitted to the Clinical Investigation Unit of The Toronto Hospital after a 12-h overnight fast and did not eat until completion of the study that afternoon. At ~0800 (-150 min), a primed (3.3 × 106 dpm) continuous infusion (0.33 × 106 dpm/min) of [3-3H]glucose (New England Nuclear, Boston, MA) was started and maintained throughout the study. The tracer had been submitted to the HPLC purification procedure (25). After 110 min, five samples of arterialized venous blood were drawn every 10 min for basal determinations from an intravenous catheter placed in a dorsal hand vein of the opposite arm, which was maintained in a warming device. At 0 min, tolbutamide sodium USP (Upjohn, Kalamazoo, MI; 3 g in 250 ml normal saline) was infused into a peripheral arm vein at a rate of 1 g/h for the 1st hour, 800 mg/h for the 2nd hour, and 600 mg/h for the 3rd hour. This dosage regimen was empirically determined in earlier studies to produce sustained and steady rates of pancreatic insulin secretion in nondiabetic individuals (14). The mean steady rate of pancreatic insulin secretion between 60 and 180 min in response to tolbutamide was used to determine the exogenous insulin infusion rate needed for the second study. Blood samples were drawn for glucose, [3-3H]glucose specific activity, insulin, glucagon, C-peptide, and FFA at baseline and at regular intervals throughout the study. Samples for measurement of FFA were drawn into chilled EDTA tubes on ice containing 0.4 µmol/ml blood of the lipase inhibitor APBA (m-aminophenylboronic acid, Sigma Pharmaceuticals, St. Louis, MO) (11). Plasma glucose levels were measured every 5 min during the tolbutamide infusion. The values were used to adjust the rate of a 20% dextrose infusion to maintain constant euglycemia (glucose 5.0-5.5 mmol/l). An aliquot of [3-3H]glucose was added to the 20% dextrose infusate (0.388 µCi/kg [3-3H]glucose added to a 500-ml dextrose solution) to minimize the decline in glucose specific activity during the clamp ("hot Ginf" method) (9, 10). Potassium chloride was infused at ~10 meq/h in all subjects.

To make these results comparable with those of our previous studies (15, 16), in the FFA-glucagon clamp studies, at -150 min a constant intravenous infusion of glucagon (glucagon for injection, USP, Eli Lilly Canada, Scarborough, ON, Canada) was started at 0.65 ng · kg-1 · min-1 and was maintained throughout the study. Fifteen minutes after the start of the tolbutamide infusion, heparin sodium (Organon Tieknika, Toronto, ON, Canada) and Intralipid 10% solution (Baxter Healthcare, Mississauga, ON, Canada) were infused at 250 U/h and 25 ml/h, respectively, and continued for the duration of the study. Tolbutamide was discontinued after 3 h, and the subjects were permitted to eat, thus ending the active phase of the study. Because tolbutamide has a prolonged action, the rates of the potassium chloride and the 20% dextrose infusions were gradually reduced overnight, maintaining euglycemia at all times according to half-hourly blood glucose measurements drawn through a sampling intravenous catheter. In most cases the dextrose infusion was discontinued within 12 h of the end of tolbutamide infusion.

In the control studies, no heparin, Intralipid, or glucagon was infused, and the FFA and glucagon levels were allowed to decrease during hyperinsulinemia.

Full-rate peripheral insulin study. The study with an exogenous insulin infusion was performed 6-8 wk later by use of crystalline human insulin (Novo Nordisk Canada, Toronto, ON, Canada) infused into a peripheral vein between 0 and 180 min. Plasma glucose levels were maintained in the euglycemic range, as described above. Heparin, Intralipid, and glucagon were infused in an identical fashion to that described above in the FFA-glucagon clamp studies.

The rate of infusion of exogenous insulin was matched in each individual to the calculated mean steady rate of pancreatic insulin secretion between 60 and 180 min of the earlier tolbutamide infusion. Pancreatic insulin secretion had been calculated from peripheral plasma C-peptide levels by deconvolution by means of a two-compartmental mathematical model for C-peptide distribution and metabolism, as previously described (30). (The software program for calculation of insulin secretion was kindly provided by Drs. K. Polonsky and J. Sturis, University of Chicago, Chicago, IL.) The use of standard parameters for C-peptide clearance and distribution has been shown to result in insulin secretion rates that differ in each subject by only 10-12% from those obtained with individual parameters, and there is no systematic over- or underestimation of insulin secretion (30). Over the 1st hour of the infusion (0 to 60 min), the insulin infusion rate was increased in increments of 25% of the calculated maximal rate every 15 min to mimic as closely as possible the gradual increase in insulin secretion seen in the earlier portal insulin study.

Half-rate peripheral insulin study. This study was performed in four individuals in the FFA-glucagon clamp study and four control subjects. The study was identical to the full-rate peripheral insulin study described above, with the exception that insulin was infused at one-half the rate in an attempt to match peripheral venous insulin concentrations.

Calculations

The specific activity (SA) of the infusate was calculated as previously described (17). Briefly, calculations were based on estimation of the parameters of the formula of Finegood et al. (9), modified to allow for incomplete suppression of GP. GP was calculated as the endogenous rate of appearance measured with [3-3H]glucose, and glucose utilization was calculated as the rate of disappearance (Rd) measured with [3-3H]glucose. For glucose turnover calculations, a modified one-compartmental model (9) was used to account for the exogenously infused mixture of labeled and unlabeled glucose. Data were smoothed with the optimal segments routine (8) by use of the optimal error algorithm (3). With the hot Ginf method, the monocompartmental assumption becomes minor, because the non-steady-state part of Steele's equation is close to zero. At euglycemia, Rd corresponded to glucose utilization and plasma clearance rate of glucose (Rd/glycemia) to glucose metabolic clearance rate.

Portal insulin levels were calculated according to the method of De Feo et al. (5)
PI<SUB><IT>t</IT></SUB> = AI<SUB><IT>t</IT></SUB> + [PI<SUB>0</SUB> − AI<SUB>0</SUB>]ISR<SUB><IT>t</IT></SUB>/ISR<SUB>0</SUB>
PIt, AIt, and ISRt are the portal venous insulin concentration, arterial (peripheral) plasma insulin concentration, and insulin secretion rate, respectively, at time t. PI0, AI0, and ISR0 are portal venous insulin concentration, arterial (peripheral) plasma insulin concentration, and insulin secretion rate, respectively, at baseline. ISR0 was taken as the average of the basal insulin secretion rates determined in the three experiments. PI0 was assumed to be 2.4 × AI0 (5). Hepatic insulin levels were calculated assuming a 72% vascularization of the liver by the portal vein and 28% by the hepatic artery (4).

Laboratory Methods

Glucose was assayed enzymatically at the bedside using a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). Insulin was measured by radioimmunoassay with a double-antibody separation method (kit supplied by Pharmacia Diagnostic, Uppsala, Sweden). C-peptide was measured by radioimmunoassay with previously described techniques (6). Glucagon was measured by radioimmunoassay with a double-antibody procedure by use of a kit by Linco (Linco Research, St. Charles, MO). FFA values were measured by a colorimetric method (kit supplied by Wako Industrials, Osaka, Japan). Triglycerides were measured as esterified glycerol with an enzymatic colorimetric kit (no. 450032, Boehringer Mannheim Diagnostica). Free glycerol was eliminated from the sample in a preliminary reaction followed by enzymatic hydrolysis of triglyceride with subsequent determination of the liberated glycerol by colorimetry.

For the determination of [3-3H]glucose SA, plasma was deproteinized with Ba(OH)2 and ZnSO4. An aliquot of the supernatant was then evaporated to dryness to eliminate tritiated water. After addition of water and liquid scintillation solution, the radioactivity from [3-3H]glucose was counted by liquid scintillation spectrometry. An external standard was used for quench corrections. Aliquots of the infused [3-3H]glucose and of the labeled glucose infusate were assayed together with the plasma samples.

Statistical Methods

The data were expressed as means ± SE. Analysis of variance for repeated measurements followed by Tukey's t-test was performed for differences between experimental groups during the basal period and the 90- to 180-min hyperinsulinemic period. Analysis of variance was also performed within each group for differences between the basal and the 90- to 180-min experimental periods. A P value of <0.05 was regarded as significant. Calculations were performed with SAS software (Statistical Analysis System, Cary, NC).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The tolbutamide infusion study will be referred to as the portal study, the exogenous insulin infusion as the full-rate peripheral insulin study, and the half-rate peripheral insulin infusion as the half-rate peripheral insulin infusion study. All mean values reported below for the baseline period are the means for -40 to 0 min, and those for the hyperinsulinemic period are the means for 90-180 min.

Insulin Infusion and Secretion Rates

The calculated insulin secretion rate in response to tolbutamide (in the portal study) reached a plateau in the last 2 h of the tolbutamide infusion (353 ± 38 pmol/min). In the FFA-glucagon clamp studies, the mean tolbutamide-stimulated insulin secretion rate was slightly lower (319 ± 16 pmol/min, P < 0.05) than in the control studies. In both studies the insulin secretion rate in each individual was matched with an equimolar peripheral insulin infusion. In addition, four individuals in each group underwent a third study with a half-rate peripheral insulin infusion. The mean rate for each study is illustrated in Fig. 1.


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Fig. 1.   Insulin infusion and secretion rates. Calculated pancreatic insulin secretion rate in response to tolbutamide in the portal study (bullet ) and exogenous insulin infusion rate in full-rate peripheral study (open circle ) and half-rate peripheral (triangle ) study in the control experiments (A) and free fatty acid (FFA)-glucagon studies (B). Tolbutamide (in portal study) or exogenous insulin (in peripheral studies) was infused from 0 to 180 min. By design, peripheral insulin infusion rate was matched with calculated insulin secretion rate in each individual. In addition, 4 subjects in each group underwent a 3rd study in which exogenous insulin was infused at one-half the rate. In FFA-glucagon clamp studies, mean tolbutamide-stimulated insulin secretion rate was slightly lower (P < 0.05) than that in control studies.

Control Studies

Peripheral venous glucose, insulin, and C-peptide concentrations. Glucose levels (Fig. 2) were clamped at euglycemia throughout the studies in the controls [portal study, mean glucose = 5.04 ± 0.06 mmol/l, coefficient of variation (CV) = 8.9%; full-rate peripheral study, 5.17 ± 0.05 mmol/l, CV = 7.7%; half-rate peripheral study, 5.54 ± 0.06 mmol/l, CV = 6.9%].


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Fig. 2.   Plasma glucose, insulin, FFA, and glucagon. A: data from control experiments; B: data from FFA-glucagon studies. Tolbutamide (in portal study) or exogenous insulin (in peripheral study) was infused from 0 to 180 min.

The peripheral insulin levels (Fig. 2) rose (P < 0.0001) from a basal value of 33.2 ± 1.9 to 141.6 ± 7.8 pmol/l in the portal study, which was less than the rise from 32.4 ± 2.64 to 209.4 ± 13.1 pmol/l in the full-rate peripheral study (P < 0.001). In the half-rate peripheral study, insulin rose from 39.6 ± 5.4 to 153.0 ± 13.2 pmol/l, a level similar to that reached in the portal study but significantly lower than that in the full-rate peripheral study (P < 0.001).

C-peptide levels (not illustrated) rose (P < 0.0001) from 0.38 ± 0.02 to 1.5 ± 0.09 nmol/l with tolbutamide and decreased (P < 0.05) from 0.35 ± 0.06 to 0.22 ± 0.01 nmol/l with the full-rate peripheral insulin infusion and from 0.37 ± 0.04 to 0.28 ± 0.05 nmol/l (P < 0.05) with the half-rate peripheral insulin infusion.

Calculated portal and hepatic insulin levels. Calculated portal insulin levels were similar [P = not significant (NS)] in the basal state in the portal, full-rate peripheral, and half-rate peripheral insulin studies (Table 1). During the last 90 min of the clamp, calculated portal insulin levels were higher (P < 0.001) in the portal insulin study than in the full-rate peripheral study or the half-rate peripheral insulin study (P = NS, half-rate vs. full-rate peripheral study). Calculated hepatic sinusoidal insulin levels were similar (P = NS) in the basal state in the portal, full-rate peripheral, and half-rate peripheral studies. During the last 90 min of the clamp, hepatic sinusoidal insulin levels were higher (P < 0.001) in the portal insulin study than in the full-rate peripheral study or the half-rate peripheral insulin study (P = NS, half-rate vs. full-rate peripheral insulin study).

                              
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Table 1.   Calculated portal vein and hepatic sinusoidal insulin levels

Concentrations of glucagon and FFA. FFA levels (Fig. 2) decreased significantly from baseline (P < 0.001) and were similar (P = NS) in the portal study (baseline 0.48 ± 0.07 mmol/l decreased to 0.11 ± 0.01 mmol/l, P < 0.001), full-rate peripheral study (baseline 0.53 ± 0.06 mmol/l decreased to 0.09 ± 0.01 mmol/l, P < 0.001), and half-rate peripheral study (baseline 0.53 ± 0.06 mmol/l decreased to 0.12 ± 0.07 mmol/l, P < 0.001).

Glucagon levels (Fig. 2) decreased significantly from baseline (P < 0.005) in the portal study (baseline 60.4 ± 4.9 pg/ml decreased to 48.9 ± 2.2 pg/ml, P < 0.001), full-rate peripheral study (baseline 50.4 ± 3.7 pg/ml decreased to 37.0 ± 2.5 pg/ml, P < 0.001), and half-rate peripheral study (baseline 53.5 ± 5.7 pg/ml decreased to 44.0 ± 4.6 pg/ml, P < 0.001). Glucagon levels were slightly higher in the portal vs. the full-rate peripheral insulin study (P < 0.01) throughout.

Dextrose infusion rates, glucose SA, GP, and glucose utilization (Rd). The dextrose infusion rates (Fig. 3) necessary to maintain euglycemia were greater in the full-rate peripheral study (31.1 ± 2.4 µmol · kg-1 · min-1) than in the portal study (21.9 ± 2.0 µmol · kg-1 · min-1, P < 0.001) and the half-rate peripheral study (17.3 ± 2.9 µmol · kg-1 · min-1, P < 0.001). The glucose infusion rate was greater in the portal vs. the half-rate peripheral study (P < 0.001).


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Fig. 3.   [3-3H]glucose specific activity (SA), dextrose infusion rate (Ginf), glucose utilization (Rd), and hepatic glucose production (Ra). A: data from control studies; B: data from FFA-glucagon clamp studies. Tolbutamide (in portal study) or exogenous insulin (in peripheral study) was infused from 0 to 180 min.

Plasma glucose SA (Fig. 3) rose and was higher (P < 0.001) during the last 90 min of the portal study (29.3 ± 4.8%) than in the full-rate peripheral study (13.4 ± 1.7%). In the half-rate peripheral study, plasma SA activity rose by 19.4 ± 13.4% (lower than in the portal study, P < 0.001, but similar to the full-rate peripheral study).

As expected, glucose utilization rose (Fig. 3) proportionally to the peripheral insulin levels. Glucose utilization was greater (P < 0.001) in the full-rate peripheral study (34.2 ± 1.7 µmol · kg-1 · min-1) than in the portal study (26.3 ± 1.7 µmol · kg-1 · min-1) and in the half-rate peripheral study (23.3 ± 2.1 µmol · kg-1 · min-1).

GP (Fig. 3) in the basal period was 11.2 ± 0.6 µmol · kg-1 · min-1 in the portal study, 12.3 ± 0.4 µmol · kg-1 · min-1 in the full-rate peripheral study, and 12.5 ± 0.5 µmol · kg-1 · min-1 in the half-rate peripheral study (no significant difference between studies). The magnitude of suppression of GP (delta GP) in the last 90 min of the clamp was greater in the full-rate peripheral study (75.7 ± 3.0%, delta GP = 9.24 ± 0.36 µmol · kg-1 · min-1) than in the portal study (62.6 ± 2.4%, delta GP = 7.11 ± 0.39 µmol · kg-1 · min-1, P < 0.001) and in the half-rate peripheral study (56.3 ± 3.0%, delta GP = 6.84 ± 0.42 µmol · kg-1 · min-1, P < 0.001). The percent suppression of GP was not significantly different in the half-rate peripheral and portal studies. The early effect (initial 90 min of hyperinsulinemia) of insulin on GP, however, was greater in the portal study than in either full-rate or half-rate peripheral insulin study (P < 0.001).

FFA-Glucagon Studies

Peripheral venous glucose, insulin, and C-peptide concentrations. Glucose levels (Fig. 2) were clamped at euglycemia throughout the studies (portal study, mean glucose = 5.25 ± 0.05 mmol/l, CV = 9.1%; fullrate peripheral study, 5.16 ± 0.04 mmol/l, CV = 7.0%; half-rate peripheral study, 5.51 ± 0.06 mmol/l, CV = 6.7%).

The peripheral insulin levels (Fig. 2) rose (P < 0.0001) from a basal value of 32.4 ± 1.7 to 139.8 ± 12.6 pmol/l in the portal study, which was less than the rise from 34.8 ± 1.7 to 208.8 ± 13.2 pmol/l in the full-rate peripheral study (not shown; P < 0.001). In the half-rate peripheral study, insulin rose from 37.0 ± 6.0 to 150.6 ± 15.0 pmol/l, a level similar to that reached in the portal study but significantly lower than that in the full-rate peripheral study (P < 0.001). There were no differences in insulin levels between control and FFA-glucagon studies.

In the FFA-glucagon clamp studies, the C-peptide levels rose (not shown; P < 0.0001) from 0.41 ± 0.02 to 1.3 ± 0.04 nmol/l with tolbutamide (P < 0.05 vs. controls), decreased with the peripheral insulin infusion (0.30 ± 0.02 to 0.21 ± 0.07 nmol/l, P < 0.01), and decreased from 0.35 ± 0.03 to 0.26 ± 0.04 nmol/l (P < 0.05) with the half-rate peripheral insulin infusion.

Calculated portal and hepatic insulin levels. Calculated portal insulin levels were similar (P = NS) in the basal state in the portal, full-rate peripheral, and half-rate peripheral studies (Table 1). During the last 90 min of the clamp, calculated portal insulin levels were higher (P < 0.001) in the portal insulin study than in the full-rate peripheral study or the half-rate peripheral insulin study (P < 0.05, half-rate vs. full-rate peripheral study). Calculated hepatic sinusoidal insulin levels were similar (P = NS) in the basal state in the portal, full-rate peripheral, and half-rate peripheral studies. During the last 90 min of the clamp, hepatic sinusoidal insulin levels were higher (P < 0.001) in the portal insulin study than in the full-rate peripheral study or the half-rate peripheral insulin study (P < 0.05, half-rate vs. full-rate peripheral insulin study).

Concentrations of glucagon and FFA. FFA levels (Fig. 2) decreased from 0.49 ± 0.04 to 0.32 ± 0.01 mmol/l (P < 0.001) in the portal study, from 0.46 ± 0.04 to 0.33 ± 0.02 mmol/l (P < 0.001) in the full-rate peripheral insulin study, and from 0.52 ± 0.05 to 0.32 ± 0.12 mmol/l (P < 0.001) in the half-rate peripheral insulin study (P = NS for portal vs. peripheral insulin studies; Fig. 2). Levels in the last 90 min of the clamp were about threeefold higher in the FFA-glucagon clamp studies than in the control studies (P < 0.001).

Glucagon levels (Fig. 2) at -150 min (i.e., before the glucagon infusion was begun) were similar in the portal (61.9 ± 4.5 pg/ml), full-rate peripheral (62.8 ± 5.6 pg/ml), and half-rate peripheral (58.5 ± 6.1 pg/ml) studies and were not significantly different from the basal levels in the control studies. During the basal period (between -40 min and 0 min), 110 min after the infusion of glucagon had begun, levels were higher than in the controls during the same time period because of the glucagon infusion (P < 0.001; 77.6 ± 3.5 pg/ml in the portal study, 79.1 ± 4.4 pg/ml in the full-rate peripheral study, and 80.3 ± 5.4 pg/ml in the half-rate peripheral study). Glucagon levels remained constant in the portal study (71.3 ± 2.7 pg/ml) and decreased slightly (P < 0.05; to 68.3 ± 3.0 pg/ml) in the full-rate peripheral insulin study and half-rate peripheral study (69.8 ± 3.9 pg/ml) (P = NS for portal vs. peripheral insulin studies). Glucagon levels in the last 90 min of the clamp were ~60% higher in the FFA-glucagon clamp studies than in the control experiments (P < 0.001).

Dextrose infusion rates, glucose SA, GP, and glucose utilization (Rd). The dextrose infusion rates necessary to maintain euglycemia were greater with the full-rate peripheral insulin infusion (34.1 ± 2.2 µmol · kg-1 · min-1) than in the portal study (22.1 ± 1.5 µmol · kg-1 · min-1, P < 0.001) and half-rate peripheral study (19.9 ± 2.5 µmol · kg-1 · min-1, P < 0.001; Fig. 3). There was no significant difference between the portal and half-rate peripheral insulin studies. In addition, there were no significant differences between the control and the FFA-glucagon clamp studies.

As was the case in the control experiments, plasma glucose SA (Fig. 3) rose and was higher (P < 0.001) during the last 90 min of the portal study (19.1 ± 2.0%) than in the full-rate peripheral study (9.8 ± 1.7%). In the half-rate peripheral study, plasma SA activity rose by 17.3 ± 2.2% (higher than in the full-rate peripheral study, P < 0.001, but not significantly different from the portal study).

As in the control experiments, glucose utilization (Rd) (Fig. 3) rose proportionally to the peripheral insulin levels. Glucose utilization was greater (P < 0.001) in the full-rate peripheral study (43.1 ± 1.7 µmol · kg-1 · min-1, P < 0.001, for control vs. FFA-glucagon clamp studies) than in the portal study (28.0 ± 1.1 µmol · kg-1 · min-1, P = NS for controls vs. FFA-glucagon clamp) and in the half-rate peripheral study (28.0 ± 1.7 µmol · kg-1 · min-1, P = NS for control vs. FFA-glucagon clamp experiments).

GP (Fig. 3) in the basal period was 12.3 ± 0.3 µmol · kg-1 · min-1 in the portal study, 11.7 ± 0.3 µmol · kg-1 · min-1 in the full-rate peripheral study, and 12.8 ± 0.4 µmol · kg-1 · min-1 in the half-rate peripheral study (no significant difference between studies or between control and FFA-glucagon clamp studies). In contrast to the control experiments, in the FFA-glucagon clamp studies there was in fact a greater suppression of GP in the last 90 min of the portal study (57.3 ± 2.6%, delta GP = 6.89 ± 0.34 µmol · kg-1 · min-1) than in the full-rate peripheral study (39.0 ± 4.1%, delta GP = 4.48 ± 0.39 µmol · kg-1 · min-1, P < 0.001) and in the half-rate peripheral study (31.5 ± 2.7%, delta GP = 3.89 ± 0.43 µmol · kg-1 · min-1, P < 0.001). The magnitude of suppression of GP in the full- and half-rate peripheral insulin studies was similar and much less than in the portal study, suggesting that with glucagon and FFA replacement, peripheral effects of insulin are minimized. The percent suppression of GP was greater in the control than in the FFA-glucagon clamp studies for the full-rate peripheral (P < 0.001) and the half-rate peripheral (P < 0.001) but not the portal studies.

A summary of these findings, contrasted with the results of our previous studies when either glucagon alone or FFA alone was clamped, is presented in Fig. 4. As can be seen in Fig. 4, when both glucagon and FFA were allowed to decline during portal and matched peripheral hyperinsulinemia (controls in present study), there was a significantly greater suppression of GP with peripheral insulin administration vs. portal. When the decline in FFA was limited by infusing heparin and Intralipid without glucagon (16), the greater suppressive effect of peripheral insulin was lost. When the decline in glucagon was limited by infusing glucagon (without heparin and Intralipid) (15), there was no longer a greater suppression of GP with peripheral insulin, and there was a slightly greater effect with portal insulin. When the decline in both glucagon and FFA was diminished by infusing a combination of glucagon, heparin, and Intralipid (present glucagon-FFA clamp studies), there was a marked reduction in the effect of peripheral insulin on GP.


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Fig. 4.   Summary slide of change in suppression of glucose production (GP) with peripheral and portal insulin delivery in 4 studies. Heights of bars, difference between %suppression of GP with peripheral insulin - %suppression of GP with equimolar portal insulin (i.e., a positive bar indicates that suppression of GP was greater with peripheral vs. portal insulin, and a negative bar indicates that suppression of GP was greater with portal vs. peripheral insulin) in the following 4 studies (half-rate peripheral insulin studies are not illustrated). 1) Controls: 6 controls, to whom no glucagon, Intralipid, or heparin was given during hyperinsulinemia. 2) Heparin Intralipid: subjects received same dose of heparin and Intralipid administered in glucagon-FFA clamp in present study, but glucagon was not infused (see Ref. 16). 3) Glucagon: subjects received the same dose of glucagon administered in glucagon-FFA clamp in present study, but heparin and Intralipid were not infused (see Ref. 15). 4) Glucagon, Intralipid, Heparin: 9 subjects, who received an infusion of glucagon, heparin, and Intralipid in present study (glucagon-FFA clamp). * P < 0.05, ** P < 0.001, portal vs. peripheral study.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We have shown in the present study that, when the decline in both glucagon and FFA during acute hyperinsulinemia is limited by the infusion of a combination of glucagon, heparin, and Intralipid, the normally greater suppression of GP with peripheral vs. equimolar portal insulin delivery is markedly reversed. Under these conditions, the magnitude of suppression of GP in the full- and half-rate peripheral insulin studies was similar and much less than with the portal study, suggesting that with glucagon and FFA replacement, peripheral effects of insulin are minimized. These data suggest that both glucagon and FFAs play an important role in mediating the indirect effects of insulin on GP, and they demonstrate the importance of the direct hepatic effect of insulin when glucagon and FFA, two putative mediators of the peripheral effects of insulin, are prevented from declining.

In previous studies, in young healthy nondiabetic men of age and weight similar to the participants of the present study, we prevented the decline in FFA (not glucagon) with an identical dose of heparin and Intralipid (16). Under those conditions we noted an equivalent degree of suppression of GP with portal and peripheral insulin delivery, i.e., the previously noted greater effect of peripheral insulin was lost, but there was no greater suppression with equimolar portal insulin. In a second study (15), we limited the decline of glucagon but not of FFA, and we found a slightly greater suppression of GP with portal vs. peripheral insulin delivery. Unfortunately, it is not possible for us to perform more than three experiments in each subject because of limitations on the exposure to radioactivity from radiolabeled glucose tracers. When we compare the difference in suppression of GP between portal and peripheral insulin delivery in different groups of subjects [those of the present study and those of two previously published studies (15, 16), as illustrated in Fig. 4], we see that in the present study the suppression of GP was 18.3% greater with portal than with peripheral insulin when both glucagon and FFA were clamped, whereas it was only 7.4% greater when glucagon alone was clamped and was no different when the FFA decline alone was prevented. Although we cannot draw firm quantitative conclusions from comparing different groups of subjects, these results would suggest that the effect of clamping both glucagon and FFA is at least additive and may be cooperative (synergistic).

It is important to note that, although there was a marked diminution of the suppressive effect of peripherally administered insulin when glucagon and FFA were replaced, there was still an ~40% suppression of GP with the peripheral infusion and an almost 60% suppression in the portal study, suggesting that another mechanism or mechanisms, most likely the direct hepatic effect of insulin, remain very important in regulating GP. It is possible that, had we totally prevented the decline in glucagon and FFA, we might have seen even less suppression of GP with peripheral insulin. However, in previous experiments when we raised FFA levels more than twofold above basal, there remained a significant suppression of GP with peripheral insulin (16). The half-rate peripheral insulin infusion studies, in which peripheral insulin concentrations were matched with those in the portal study, were specifically designed to examine whether the direct hepatic effect of insulin in controlling GP becomes more evident when the decline in FFA and glucagon is limited during hyperinsulinemia. In the control experiments, the magnitude of suppression of GP was slightly but not significantly greater with portal vs. half-rate peripheral insulin, whereas in the glucagon-FFA clamp studies, GP was suppressed to a much greater extent with portal vs. half-rate peripheral insulin. These results indicate, as we have previously shown (17), that insulin suppresses GP not only by an indirect (extrahepatic) effect but also by a direct (hepatic) effect. We should point out, however, that suppression of GP was similar with portal insulin whether FFA and glucagon were replaced (57.2% suppression) or not replaced (62.6% suppressed), despite the fact that part of insulin's effect during portal infusion should also reflect the effect of peripheral signaling. One could interpret this finding as indicating that, under conditions of physiological (portal) insulin secretion, the role of extrahepatic factors cannot be significant. However, it should be considered that different subjects were studied with and without glucagon and FFA replacement and that replacement was not complete. The data could also be explained if normal or increased concentrations of glucagon sensitize the liver to the direct effect of insulin, as has been previously shown (12, 15, 19), resulting in a similar net effect on GP under both conditions.

The mechanism of action of glucagon and FFA may differ, despite a similar net result on GP. In our previous studies we found that glucagon enhanced the direct suppressive effect of insulin on GP (12, 15), in addition to mediating part of the extrahepatic effect of insulin on GP (12, 13). This finding has been confirmed in a recent study by Mittelman et al. (19). Interestingly, this direct potentiating effect of glucagon does not appear to be related to an elevation of basal GP, as evidenced by similar basal GP rates in the FFA-glucagon and control studies. The corollary of this is that, in the absence of glucagon or when glucagon levels are markedly suppressed by insulin, the peripheral effect of elevated insulin dominates the suppression of GP.

Rebrin and co-workers (22, 23) postulated that insulin acts to suppress GP by slowly traversing the capillary endothelium in adipose tissue and inhibiting lipolysis, thus decreasing FFA levels. Decreased FFA presumably act as a signal to the liver to suppress GP by a glucose-fatty acid cycle, as was originally proposed in the theory by Randle et al. (21) in 1963, because reduced fatty acid oxidation results in a compensatory increase in glucose oxidation and consequent reduction of hepatic gluconeogenesis. Furthermore, reduced availability of FFA has been shown to direct glycogenolytic flow from glucose to lacate production (28). In addition, FFA have short-term allosteric effects on glycogenolytic and gluconeogenic enzymes, the importance of which is unclear in vivo, and long-term effects on the gene transcription of these enzymes. The latter effects presumably would require more than 3 h of exposure to elevated FFA. Rebrin and colleagues found a similar time course for insulin action on glucose Rd and GP, supporting the concept that insulin slowly traverses the capillary endothelial barrier of an insulin-sensitive tissue and then activates glucose uptake and modulates a signal that controls GP. The time course of suppression of GP in our control and FFA-glucagon clamp experiments (Fig. 3) would support this concept. In addition, the late suppression of GP with the peripheral insulin infusion reflected the time course of the suppression of FFA. We would suggest that the early suppression of GP mainly reflects the direct hepatic effect of insulin, because in all of our present experiments, the initial GP suppression was more rapid and quantitatively greater with portal vs. peripheral insulin delivery.

Although we did not totally prevent the insulin-induced decline in glucagon or FFA, there was a marked difference in both glucagon and FFA concentrations in the glucagon-FFA clamp studies and the control study, and this difference was sufficient to result in major differences in the effects of portal vs. peripheral insulin delivery on GP. In the control experiments, the insulin-induced suppression of plasma FFA was similar to peripheral and portal insulin delivery, despite significant differences in peripheral insulin levels between studies. This is likely a result of near maximal suppression of tissue lipolysis at these high physiological doses of insulin. Although FFA levels were well matched between portal and peripheral studies, there were unanticipated differences in glucagon levels between portal and peripheral studies. In the control experiments, the glucagon levels were slightly higher throughout the basal and hyperinsulinemic periods in the portal vs. the full-rate peripheral insulin infusion study, whereas in the glucagon-FFA studies, there was a slightly greater suppression of glucagon with peripheral insulin, possibly related to the suppressive effect of the higher peripheral insulin levels on endogenous glucagon secretion. The slightly greater decrease in glucagon levels with peripheral than equidose portal insulin infusion might have arisen from differences in amino acid levels. However, in a previous study (17) we did not find significant differences in alanine levels with portal vs. peripheral insulin delivery. Despite the differences in glucagon levels, there were no significant differences in basal GP, and it is unlikely that these differences could account for the striking differences in suppression of GP that we see in the portal and peripheral studies. It is important to point out that the peripheral glucagon level is a poor indicator of the portal glucagon level. It is therefore difficult, on the basis of measurement of peripheral glucagon concentrations, to estimate glucagon levels in portal blood that may reflect either suppression of pancreatic glucagon secretion or its peripheral replacement.

In conclusion, we have shown that both glucagon and FFA play an important additive or cooperative role in mediating the indirect effect of insulin on GP but that other mechanisms remain important in controlling GP. This presumably indicates that the majority of the residual effect of insulin is hepatic, but it does not exclude the effect of peripheral insulin to suppress renal gluconeogenesis directly or the possibility of other, hitherto unknown, peripheral signals. The early, more rapid effects of portal insulin infusion also indicate that the hepatic insulin effect coexists with a peripheral effect. The variable, relative importance of direct and indirect effects of insulin on GP offers flexibility of metabolic control due to insulin action during different physiological and pathophysiological states.

    ACKNOWLEDGEMENTS

The expert technical assistance of Debbie Bilinski, Loretta Lam, and Linda Szeto is acknowledged with appreciation.

    FOOTNOTES

This study was supported by the Canadian Diabetes Association (a grant in memory of the late George and Vesta Davidge to G. F. Lewis), the Juvenile Diabetes Foundation (Grant 193135 to A. Giacca), and the Medical Research Council of Canada (Grant MT2917 to M. Vranic and A. Giacca). G. F. Lewis is the recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada, and A. Giacca was the recipient of a Career Development Award of the Juvenile Diabetes Foundation.

Address for reprint requests: G. F. Lewis, The Toronto Hospital, General Division, 200 Elizabeth St., Rm. EN 11-229, Toronto, ON, Canada M5G 2C4.

Received 4 October 1997; accepted in final form 27 March 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(1):E177-E186
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