Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada M5G 2C4
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 · kg1 · 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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)
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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%].
|
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).
|
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 · kg1 · 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).
|
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) atDextrose 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 · kg1 · 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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ader, M.,
and
R. N. Bergman.
Peripheral effects of insulin dominate suppression of fasting hepatic glucose production.
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E1020-E1032,
1990
2.
Boden, G.,
X. Chen,
R. A. Desantis,
and
Z. Kendrick.
Effects of insulin on fatty acid reesterification in healthy subjects.
Diabetes
42:
1588-1593,
1993[Abstract].
3.
Bradley, D. C.,
G. M. Steil,
and
R. N. Bergman.
Quantitation of measurement error with Optimal Segments: basis for adaptive time course smoothing.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E902-E911,
1993
4.
Campra, J. L.,
and
T. B. Reynolds.
The hepatic circulation.
In: The Liver. Biology and Pathobiology, edited by I. M. Arias,
H. Popper,
D. Schachter,
and D. A. Shafritz. New York: Raven, 1982, p. 627-645.
5.
De Feo, P.,
G. Perriello,
S. De Cosmo,
M. M. Ventura,
P. J. Campbell,
P. Brunetti,
J. E. Gerich,
and
G. B. Bolli.
Comparison of glucose counterregulation during short-term and prolonged hypoglycemia in normal humans.
Diabetes
35:
563-569,
1986[Abstract].
6.
Faber, O. K., C. Binder, J. Markussen, L. G. Heding, V. K. Naithani, H. Kuzuya, P. Blix, D. L. Horwitz, and A. H. Rubenstein. Characterization of seven
C-peptide antisera. Diabetes 27, Suppl. 1: 170-177, 1978.
7.
Fanelli, C.,
S. Calderone,
L. Epifano,
A. De Vincenzo,
F. Modarelli,
S. Pampanelli,
G. Perriello,
P. De Feo,
P. Brunetti,
J. E. Gerich,
and
G. B. Bolli.
Demonstration of a critical role for free fatty acids in mediating counterregulatory stimulation of gluconeogenesis and suppression of glucose utilization in humans.
J. Clin. Invest.
92:
1617-1622,
1993[Medline].
8.
Finegood, D. T.,
and
R. N. Bergman.
Optimal segments: a method for smoothing tracer data to calculate metabolic fluxes.
Am. J. Physiol.
244 (Endocrinol. Metab. 7):
E472-E479,
1983
9.
Finegood, D. T.,
R. N. Bergman,
and
M. Vranic.
Estimation of endogenous glucose production during hyperinsulinemic-euglycemic glucose clamps. Comparison of unlabeled and labeled exogenous glucose infusates.
Diabetes
36:
914-924,
1987[Abstract].
10.
Finegood, D. T.,
R. N. Bergman,
and
M. Vranic.
Modeling error and apparent isotope discrimination confound estimation of endogenous glucose production during euglycemic glucose clamps.
Diabetes
37:
1025-1034,
1988[Abstract].
11.
Garner, C. W.
Boronic acid inhibitors of porcine pancreatic lipase.
J. Biol. Chem.
255:
5064-5068,
1980
12.
Giacca, A.,
S. J. Fisher,
R. H. McCall,
Z. Q. Shi,
and
M. Vranic.
Direct and indirect effects of insulin in suppressing glucose production in depancreatised dogs: role of glucagon.
Endocrinology
138:
999-1007,
1997
13.
Giacca, A.,
S. J. Fisher,
Z. Q. Shi,
R. Gupta,
H. L. Lickley,
and
M. Vranic.
Importance of peripheral insulin levels for insulin-induced suppression of glucose production in depancreatized dogs.
J. Clin. Invest.
90:
1769-1777,
1992[Medline].
14.
Lewis, G. F.,
G. Steiner,
K. S. Polonsky,
B. Weller,
and
B. Zinman.
A new method for comparing portal and peripheral venous insulin delivery in humans: tolbutamide versus insulin infusion.
J. Clin. Endocrinol. Metab.
79:
66-70,
1994[Abstract].
15.
Lewis, G. F.,
M. Vranic,
and
A. Giacca.
Glucagon enhances the direct suppressive effect of insulin on hepatic glucose production in humans.
Am. J. Physiol.
272 (Endocrinol. Metab. 35):
E371-E378,
1997
16.
Lewis, G. F.,
M. Vranic,
P. Harley,
and
A. Giacca.
Fatty acids mediate the acute extrahepatic effects of insulin on hepatic glucose production in humans.
Diabetes
46:
1111-1119,
1997[Abstract].
17.
Lewis, G. F.,
B. Zinman,
Y. Groenewoud,
M. Vranic,
and
A. Giacca.
Hepatic glucose production is regulated both by direct hepatic and extrahepatic effects of insulin in humans.
Diabetes
45:
454-462,
1996[Abstract].
18.
Lewis, G. F.,
B. Zinman,
K. D. Uffelman,
L. Szeto,
B. Weller,
and
G. Steiner.
VLDL production is decreased to a similar extent by acute portal vs. peripheral venous insulin.
Am. J. Physiol.
267 (Endocrinol. Metab. 30):
E566-E572,
1994
19.
Mittelman, S.,
Y. Y. Fu,
K. Rebrin,
G. Steil,
and
R. N. Bergman.
Indirect effect of insulin to suppress endogenous glucose production is dominant, even with hyperglucagonemia.
J. Clin. Invest.
100:
3121-3130,
1997
20.
Prager, R.,
P. Wallace,
and
J. M. Olefsky.
Direct and indirect effects of insulin to inhibit hepatic glucose output in obese subjects.
Diabetes
36:
607-611,
1987[Abstract].
21.
Randle, P. J.,
P. B. Garland,
C. N. Hales,
and
E. A. Newsholme.
The glucose-fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.
Lancet
1:
785-789,
1963.
22.
Rebrin, K.,
G. M. Steil,
L. Getty,
and
R. N. Bergman.
Free fatty acid as a link in the regulation of hepatic glucose output by peripheral insulin.
Diabetes
44:
1038-1045,
1995[Abstract].
23.
Rebrin, K.,
G. M. Steil,
S. D. Mittelman,
and
R. N. Bergman.
Causal linkage between insulin suppression of lipolysis and suppression of liver glucose output in dogs.
J. Clin. Invest.
98:
741-749,
1996
24.
Saloranta, C.,
M. R. Taskinen,
E. Widen,
M. Harkonen,
A. Melander,
and
L. Groop.
Metabolic consequences of sustained suppression of free fatty acids by acipimox in patients with NIDDM.
Diabetes
42:
1559-1566,
1993[Abstract].
25.
Schwenk, W. F.,
P. C. Butler,
M. W. Haymond,
and
R. A. Rizza.
Underestimation of glucose turnover corrected with high-performance liquid chromatography purification of [6-3H]glucose.
Am. J. Physiol.
258 (Endocrinol. Metab. 21):
E228-E233,
1990
26.
Shi, Z. Q.,
D. Wasserman,
and
M. Vranic.
Metabolic implications of exercise and physical fitness in physiology and diabetes.
In: Ellenberg and Rifkin Diabetes Mellitus, edited by D. Porte,
and R. Sherwin. Norwolk, CT: Appleton and Lange, 1997, p. 653-687.
27.
Sindelar, D. K.,
J. H. Balcom,
C. A. Chu,
D. W. Neal,
and
A. D. Cherrington.
A comparison of the effects of selective increases in peripheral or portal insulin on hepatic glucose production in the conscious dog.
Diabetes
45:
1594-1604,
1996[Abstract].
28.
Sindelar, D. K.,
C. Chu,
M. Rohlie,
D. W. Neal,
L. L. Swift,
and
A. D. Cherrington.
The role of fatty acids in mediating the effects of peripheral insulin on hepatic glucose production in the conscious dog.
Diabetes
46:
187-196,
1997[Abstract].
29.
Stumvoll, M.,
C. Meyer,
A. Mitrakou,
V. Nadkarni,
and
J. E. Gerich.
Renal glucose production and utilization: new aspects in humans.
Diabetologia
40:
749-757,
1997[Medline].
30.
Van Cauter, E.,
F. Mestrez,
J. Sturis,
and
K. S. Polonsky.
Estimation of insulin secretion rates from C-peptide levels. Comparison of individual and standard kinetic parameters for C-peptide clearance.
Diabetes
41:
368-377,
1992[Abstract].