Department of Physiology, Medicine, and Surgery, University of Toronto, Toronto, Ontario, Canada M5S 1A8; and Department of Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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To determine whether the predominant effect of
insulin in suppressing tracer-determined glucose production
(Ra) is hepatic or peripheral,
we infused insulin peripherally (PER) and portally (POR) at both low
(0.75 pmol · kg1 · min
1)
and high physiological rates (2.7 pmol · kg
1 · min
1)
during euglycemic clamps in normal dogs. We also infused insulin peripherally at one-half these rates (1/2 PER) to match the peripheral insulin levels in POR and thus obtain a selective POR vs. 1/2 PER
difference in hepatic insulin levels. At the high-rate insulin infusion, peripheral insulin levels were greatest with PER (PER = 212 ± 10 pM, n = 5; POR = 119 ± 5 pM, n = 6; 1/2 PER = 122 ± 5 pM, n = 6). Calculated hepatic
insulin levels were greatest with POR (POR = 227 ± 13 pM, PER = 206 ± 19 pM, 1/2 PER = 123 ± 8 pM). High-dose PER yielded a greater
suppression of Ra than POR (79 ± 18 vs. 56 ± 6%, P < .001). Ra was only suppressed by 45 ± 6% with 1/2 PER (P < 0.01 vs. POR on 6 paired experiments). Free fatty acid (FFA) was
suppressed by 57 ± 8% with PER and only by 33 ± 5 and 37 ± 2% with POR and 1/2 PER, respectively. The low-dose PER and POR
yielded an equal Ra suppression
(PER = 46 ± 9%, POR = 43 ± 4%). Only 1/2 PER was associated
with a lower suppression of Ra (36 ± 8, P < 0.05 vs. POR). FFA
showed similar suppression in all three groups (~25%). Using both
insulin infusion rates, the percent
Ra suppression per unit difference
in peripheral insulin was approximately twofold greater than that per
unit difference in hepatic insulin. These results suggest that, during
euglycemic clamps without somatostatin in normal dogs,
Ra suppression is mediated by both
peripheral and hepatic effects of insulin and that peripheral insulin,
at least at high physiological infusion rates, is more potent than
hepatic insulin in suppressing Ra.
portal-peripheral insulin gradient; glucose turnover; gluconeogenic precursors; free fatty acids; glucagon
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INTRODUCTION |
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IN NORMAL PHYSIOLOGY, insulin has a strong inhibitory effect on tracer-determined glucose production (rate of glucose appearance = Ra), but the extent to which this represents a direct action of hepatic sinusoidal insulin (i.e., inhibition of glycogenolysis and/or gluconeogenesis via insulin's interactions with its hepatocyte receptor) or action of peripheral insulin via its effects on extrahepatic tissues is still unclear. The peripheral action of insulin on Ra has been recently linked to insulin-induced inhibition of lipolysis (16, 24, 28).
In obese humans, Prager et al. (22) infused insulin peripherally and, by suppressing endogenous insulin secretion, increased peripheral but not estimated portal insulin levels. They found that Ra was significantly suppressed (82%), which is consistent with Ra inhibition by peripheral insulin. Ader and Bergman (1), using improved glucose tracer techniques in normal dogs, directly demonstrated that Ra suppression was mediated by peripheral insulin. They could not detect any hepatic action of insulin on Ra. Sindelar et al. (27) showed that a selective increase in peripheral insulin levels suppressed Ra, and a selective increase in portal insulin levels also suppressed Ra in normal dogs. Thus, although all of the recent studies show that Ra is inhibited by peripheral insulin, there is still disagreement as to whether Ra can also be inhibited by hepatic insulin. We have shown that insulin-induced inhibition of Ra is proportional to peripheral insulin levels in depancreatized dogs (12) and to both hepatic and peripheral insulin levels in normal humans (17). The difference between dogs and humans might relate to the diabetic state, to species specificity, or to the experimental model used to induce portal insulin secretion in humans (tolbutamide infusion). Because the dog is generally considered a good model for the study of glucose metabolism and because we have shown that, in type 1 diabetic individuals, tolbutamide does not affect glucose turnover independent of its insulin-releasing action (17), we hypothesized that similar results would be obtained in normal dogs and humans, and therefore the differences between our results in dogs (12) and humans (17) could be related to the diabetic state of our dogs. To test this hypothesis, we investigated the mechanism of Ra inhibition by insulin in normal dogs. In contrast to previous studies (1, 27), we did not use somatostatin to inhibit endogenous insulin secretion for two reasons: first, although under most conditions somatostatin has no significant metabolic effects (6), it may independently affect glucose turnover and lipolysis (3, 13) under some conditions and thus could potentially alter the balance between insulin's hepatic and peripheral effects; second, we wished to make our results comparable with those obtained in depancreatized dogs and in humans, where somatostatin was not used. However, we determined dog C-peptide levels to obtain a measure of endogenous insulin secretion and its suppression by insulin. The experimental design consisted of the following protocols: 1) portal insulin infusion (POR), 2) equidose peripheral insulin infusion (PER), and 3) one-half dose peripheral insulin infusion (1/2 PER). The peripheral insulin levels were higher with PER than with POR, whereas the calculated hepatic insulin levels were lower. Thus a greater Ra suppression with PER than with POR would indicate that Ra is suppressed by a peripheral effect of insulin. POR and 1/2 PER only differed in the hepatic insulin levels, which were greater with POR, whereas the peripheral insulin levels were matched by experimental design. Thus a greater Ra suppression with POR than with 1/2 PER would indicate that Ra is suppressed by a direct hepatic effect of insulin. Our aim was to determine whether peripheral or hepatic insulin levels or both suppress Ra in normal dogs and to quantify their effects. Because the relative contribution of peripheral vs. hepatic effects of insulin may depend on the insulin dose, we studied two insulin doses, a high dose resulting in postprandial insulin levels and a low dose resulting in insulin concentrations only slightly above the fasting range.
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METHODS |
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Experimental animals and preparation. The studies were performed on seven postabsorptive, normal dogs of either sex. Mongrel dogs weighing 22-34 kg and of at least 1 yr of age underwent vessel cannulation performed under general anesthesia induced with sodium thiopental and maintained with halothane and nitrous oxide and assisted ventilation. A Silastic cannula (0.04-in. internal diameter; Baxter Healthcare, McGaw Park, IL) was inserted into the portal vein through a branch of the splenic vein and advanced until the tip was ~1.0 cm beyond the point of confluence of the splenic vein with the portal vein, i.e., ~5 cm from the branching point of the portal vein into its left and right bifurcations to the liver. The portal cannula served for infusion. We did not have sampling catheters in the portal vein, as we chose not to use the triple-catheter technique (20) for these studies. This was due to the high failure rate of the catheters with this technique, which makes it difficult to carry out at least three paired experiments in the same dog. Three additional Silastic cannulas served for peripheral infusion (one 0.04-in. internal diameter and two 0.03-in. internal diameter catheters). The three cannulas were inserted into the jugular vein and advanced into the superior vena cava. In addition, a Silastic cannula (0.04-in. internal diameter) was inserted into a carotid artery and advanced into the aortic arch. The arterial cannula served for sampling, and the jugular and portal cannulas served for infusions. The cannulas were tunneled subcutaneously and exteriorized at the back of the neck. They were filled with heparin (1,000 U/ml, Hepalean; Organon Teknika, Toronto, Canada) and were regularly flushed (every 4-5 days) with saline to maintain patency. Circulating heparin activates lipoprotein lipase, which results in release of free fatty acid (FFA) and glycerol. When using heparin as an anticoagulant, we do observe an increase in basal FFA vs. noncatheterized dogs (FFA = 572 ± 62 µmol/l, n = 8) despite our efforts to avoid systemic heparinization during catheter flushing. We have tried using citrate as an alternative anticoagulant; however, we rapidly abandoned the latter method because the failure rate of the citrate-filled catheters is close to 100%. When we tried using less heparin, the failure rate of the catheters increased to the point of making it difficult to perform repeated experiments in the same dog.
The dogs received 400 g of dry chow and 330 g of canned meat one time per day. The dog food was supplemented with folic acid and iron. Body weight, body temperature, hematocrit, stools, and food intake were monitored regularly. Only animals that were healthy according to these parameters were allowed to undergo experiments. The hematocrit of our dogs at the time of experiments was 45 ± 0.7% (mean ± SE). The experiments were performed after an 18-h overnight fast. All procedures were in accordance with the Canadian Council of Animal Care Standards and were approved by the Animal Care Committee of the University of Toronto.
Experimental design. At each insulin dose (high or low), three paired experiments were performed in random order. Of the seven dogs, four underwent six experiments (3 high-dose experiments + 3 low-dose experiments), one dog underwent five experiments (2 high-dose + 3 low-dose experiments), one dog underwent three high-dose experiments, and another dog underwent two low-dose experiments. Failure to complete the series of experiments was due to catheter problems. Therefore, we had three paired experiments in five dogs, plus two paired experiments in another dog at each insulin dose. At both high and low insulin doses, the missing experiment was the full-dose peripheral infusion (PER).
At the onset of the experiment (time = 160 min), a primed
[5.55 × 107
disintegrations/min (dpm)] infusion of high-performance liquid chromatography (HPLC)-purified
[6-3H]glucose (New
England Nuclear, Boston, MA) was initiated (5.55 × 105 dpm/min). HPLC
purification of tracers removes contaminants that have
been shown to induce errors in the determinations of
Ra (26). The tracer infusion was
continued throughout the experiment. From
40 to 0 min, basal
samples were taken every 10 min. At 0 min, insulin was infused at both
low or high physiological rates according to the following three
treatments: 1) PER,
2) POR, or
3) 1/2 PER, for 180 min. The 1/2 PER
treatment was designed to match peripheral insulin levels achieved with
POR and thus obtain a selective difference in hepatic insulin levels.
The insulin dose for the high-rate insulin infusion study was 27 pmol/kg (small prime) plus 2.7 pmol · kg
1 · min
1
for PER and POR or half-dose peripheral insulin infusion of 13.5 pmol/kg plus 1.35 pmol · kg
1 · min
1
for 1/2 PER. The insulin dose for the low-rate insulin infusion study
was 7.5 pmol/kg plus 0.75 pmol · kg
1 · min
1
for PER and POR or half-dose peripheral insulin infusion of 3.75 pmol/kg + 0.375 pmol · kg
1 · min
1
for 1/2 PER. All insulin infusates were prepared in saline containing ~4% (vol/vol) of the dog's own plasma.
During insulin infusion, plasma glucose was clamped at euglycemia with a variable exogenous dextrose infusion (20% dextrose for the high dose and 10% dextrose for the low dose), which was adjusted according to plasma glucose concentrations determined every 5 min. The glucose infusion was spiked with [6-3H]glucose tracer according to Finegood et al. (8) to prevent the decline in the glucose specific activity during the glucose clamp and thus minimize errors that are associated with the use of a one-compartment, fixed-pool volume model method for calculations of Ra (8). The amount of tracer in the glucose infusate was based on estimates of suppression of Ra and glucose requirements. The following equation by Finegood et al. (8), modified as in Giacca et al. (12) to account for partial suppression of Ra, was used to calculate the specific activity of the glucose infusate
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The SAGlcinf
for the study with basal insulin infusion was calculated on
Glcinf,
Ra b, and F estimates of
6.67 µmol · kg1 · min
1, 12.2 µmol · kg
1 · min
1,
and 0.4, respectively. The Glcinf
and F values were estimated to be 11.1 µmol · kg
1 · min
1
and 0.5 for POR and 1/2 PER groups, respectively, and 22.2 µmol · kg
1 · min
1
and 0.6 for the PER group, respectively, in the study with high-rate insulin infusion. The initial estimates, which were based on previous studies (8, 12), were updated according to the results in this study.
Arterial samples were taken every 10 min for 40 min in the basal period
and then every 10 min in the 1st and 3rd h and every 15 min in the 2nd
h of the hyperinsulinemic clamp. The blood samples for
[6-3H]glucose (4.0 ml)
and insulin analysis (2.0 ml) were collected in tubes containing sodium
fluoride (Fisher, Lawn Park, NJ) and dried heparin (50 U/ml of blood).
The samples for glucagon, FFA, and C-peptide analysis (2.5 ml) were
collected in tubes containing EDTA (24 mg/dl; Sigma, St. Louis, MO) and
Trasylol (2,000 kallikrein inhibitory units; Bayer, Etobicoke, Canada;
1:1 ratio, 0.1 ml/ml of blood). Blood samples for alanine, glycerol,
and lactate (1.5 ml) were collected in tubes containing an equal volume
of 10% perchloric acid (BDH, Toronto, Canada). A total blood volume of <150 ml was withdrawn per experiment. Within 1 h after collection, the blood samples were centrifuged at 800 g at 4°C. The supernatant was
stored at
20°C for later analysis.
Laboratory methods. Plasma glucose concentrations were measured by the glucose oxidase method on a glucose analyzer (Glucose Analyzer II; Beckman Instruments, Fullerton, CA). The plasma glucose determinations were performed during the experiments. The other determinations were performed within 3 mo of sample collection. The radioimmunoassays for plasma insulin and glucagon have been previously described (12). Plasma C-peptide levels were determined by a nonequilibrium double-antibody radioimmunoassay procedure (21). The FFA levels were determined with the fluorometric method of Miles et al. (18). The plasma concentrations of the gluconeogenic precursors lactate, alanine, and glycerol were measured by enzymatic fluorometric methods as previously described (12).
For the determination of [6-3H]glucose specific activity, plasma was deproteinized in equal volumes of 5% (wt/vol) zinc sulfate and 0.3 N barium hydroxide (BDH), which had been titrated and adjusted for strength. The supernatant was run through columns containing anion and cation ion-exchange resins (AG 50 W-X8, AG 2-X8; Bio-Rad Laboratories, Richmond, CA) to remove labeled three-carbon metabolites formed from labeled glucose. Ion-exchange resins remove charged metabolites such as lactate, pyruvate, and alanine. Glycerol is not retained by the resins; however, it has been shown that, when using 14C tracers, which, unlike tritiated tracers, do not result in labeled water, 99.1% of the radioactivity in the column eluate could be ascribed to glucose, as it was recovered in the potassium gluconate derivative (15). An aliquot of the eluate was evaporated to dryness to eliminate tritiated water. After addition of water and liquid scintillation solution (ReadySafe; Beckman, Fullerton, CA), the radioactivity from [6-3H]glucose was measured in a beta scintillation counter (Canberra Packard, Meriden, CT). Aliquots of the infused glucose tracer and of the labeled glucose infusate were diluted in nonradioactive plasma of the same dog and assayed together with the plasma samples.
Calculations. Ra was calculated as the endogenous rate of appearance measured with [6-3H]glucose. A modified one-compartmental model of Steele (8) was used for the calculations of Ra and glucose utilization (Rd). This model accounted for the exogenously infused mixture of labeled and unlabeled glucose. Data were smoothed with the optimal segments routine using the optimal error algorithm (4). When using radiolabeled glucose infusates, the monocompartmental assumption becomes minor because the nonsteady part of Steele's equation is close to zero.
The portal insulin levels were calculated using Fick's principle of dye dilution modified by Ader and Bergman (1)
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Statistical analysis. The data are expressed as means ± SE. The calculations were performed on the last 90 min of the study. One-way or two-way analysis of variance (ANOVA) for repeated measures followed by Tukey's t-test was carried out to determine differences between experimental groups. When the experiments were paired (POR, n = 6, vs. 1/2 PER, n = 6, at both high- and low-rate insulin infusions), two-way ANOVA was used; when experiments were not paired (PER, n = 5, vs. POR, n = 6, or 1/2 PER, n = 6, at both high- and low-rate insulin infusions), one-way ANOVA was used. For each insulin infusion rate, we also repeated the statistical analysis of the comparison between PER vs. POR or 1/2 PER after excluding the dog that failed to complete the PER experiments. Because the groups were now paired, we could use two-way ANOVA on the data from five dogs. The results did not change, i.e., 1) at the high-rate insulin infusion, Ra suppression with PER was greater than that with POR or 1/2 PER, as expected, and 2) at the low-rate insulin infusion, Ra suppression with PER was greater than that with 1/2 PER but remained nonsignificantly different from that obtained with POR. If, for each infusion rate, the comparison between POR and 1/2 PER was also performed on only five dogs (those that had all 3 experiments), Ra suppression with POR remained significantly greater than that with 1/2 PER at the low-rate insulin infusion; however, the difference failed to reach significance at the high-rate infusion (P = 0.08). Data were also analyzed within each group for differences between the experimental periods. Correlations were assessed with linear regression analysis. Calculations were performed with SAS software (SAS, Cary, NC).
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RESULTS |
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High-rate insulin infusion. The following results (means ± SE) are based on a n = 6 for POR and 1/2 PER and n = 5 for PER. The peripheral, portal, and hepatic insulin levels and the C-peptide levels (absolute values) in the basal state and during the clamp are shown in Table 1. PER induced a rise in peripheral insulin levels that was greater (P < 0.001) than that with POR or 1/2 PER, as expected (Fig. 1, top left). As per experimental design, there was no difference in the increase in peripheral insulin levels induced by POR or 1/2 PER. The C-peptide levels allowed us to estimate a 40-100% range of suppression of endogenous insulin secretion, since the decrease from basal was >40% and the clamp values were not significantly different from the lower detection limit of our assay. The increase in the estimated hepatic insulin levels (Fig. 1, top right) was greatest with POR (P < 0.05 vs. PER), intermediate with PER (P < 0.001 vs. 1/2 PER), and lowest with 1/2 PER. The results shown in the graph are based on 100% suppression of basal endogenous insulin secretion (which explains why hepatic insulin levels increased minimally with 1/2 PER). However, also assuming only a 40% suppression of basal endogenous insulin secretion, the increase in hepatic insulin levels was greatest with POR, intermediate with PER (P < 0.05 vs. POR), and least with 1/2 PER (P < 0.001 vs. POR).
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Figure 1, bottom left, shows that the plasma glucose levels were maintained constant at euglycemia in all treatments. The amount of glucose required to maintain euglycemia is shown in Fig. 1, bottom right. The greatest requirements of glucose were observed with PER, whereas the requirements with POR and 1/2 PER were similar.
Specific activity of
[6-3H]glucose
decreased in all treatments (Table 2);
however, the decrease was <25% of the basal levels. The basal rate
of glucose turnover (= Ra = Rd) was similar between treatments (13.0 ± 0.4, 13.4 ± 0.5, and 12.2 ± 0.6 µmol · kg1 · min
1
with PER, POR, and 1/2 PER, respectively). Tracer-determined Rd in the last 90 min of the clamp
was 42.7 ± 2.2, 21.5 ± 0.6, and 19.9 ± 1.1 µmol · kg
1 · min
1
in the PER, POR, and 1/2 PER treatments, respectively. As expected, PER
increased Rd more than POR or 1/2
PER (Fig. 2,
top). In the clamp period,
Ra was suppressed to 2.7 ± 0.7, 6.3 ± 0.4, and 6.9 ± 0.4 µmol · kg
1 · min
1
(90- to 180-min values) with the PER, POR, and 1/2 PER treatments, respectively. Figure 2, bottom, shows
the percent suppression of Ra. PER
suppressed Ra more than POR or 1/2
PER (PER 79 ± 18%, P < 0.001, vs. POR 56 ± 6% or 1/2 PER 45 ± 6%). POR suppressed Ra slightly more than 1/2 PER
(P < 0.05, ANOVA for repeated
measures).
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With regard to the possible mediators of insulin's peripheral effect on Ra, the PER, POR, and 1/2 PER treatments suppressed glucagon levels by a similar extent (~15%; Fig. 3, top) from basal levels of 172 ± 25, 150 ± 24, and 158 ± 25 pmol/l, respectively; however, FFA levels declined more (P < 0.001) with PER (57 ± 8%) than POR (33 ± 5%) or 1/2 PER (37 ± 2%) from basal values of 1,165 ± 133, 1,253 ± 82, and 1,164 ± 55 µmol/l, respectively (Fig. 3, bottom). Of the gluconeogenic precursors that could also mediate part of the peripheral effect of insulin on Ra, only glycerol decreased to a significantly greater extent with PER (Table 3). Lactate increased with PER and did not change significantly with POR or 1/2 PER (Table 3). Alanine did not change significantly from basal with either treatment (Table 3).
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Low-rate insulin infusion. The following results are based on an n = 6 for POR and 1/2 PER and n = 5 for PER. The peripheral, portal, and hepatic insulin levels and the C-peptide levels (absolute values) in the basal state and during the clamp are shown in Table 4. As expected, PER resulted in a greater increase in peripheral insulin levels (P < 0.05) than POR and 1/2 PER, and the latter two treatments yielded similar increases (Fig. 4, top left). The calculation of the hepatic sinusoidal insulin levels was based on a 0% suppression of endogenous insulin secretion, since the C-peptide levels did not decrease significantly from basal in either treatment. As expected, the increase in the calculated hepatic insulin levels was greatest (P < 0.01) with POR, intermediate with PER, and lowest (P < 0.01) with 1/2 PER (Fig. 4, top right). The glucose levels were maintained at constant euglycemia in all treatments (Fig. 4, bottom left). Figure 4, bottom right, shows the glucose infusion rates necessary to maintain euglycemia. PER required the greatest glucose infusion rate, whereas POR and 1/2 PER required similar amounts.
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Table 5 shows that plasma glucose specific
activity was kept constant. Basal glucose turnover rate (=
Ra = Rd) was similar between
treatments (11.5 ± 0.6, 11.9 ± 0.5, and 12.8 ± 0.6 µmol · kg1 · min
1
with PER, POR, and 1/2 PER, respectively).
Rd increased minimally to 15.5 ± 0.7, 12.9 ± 0.3, and 14.0 ± 0.4 µmol · kg
1 · min
1,
respectively, in the last 90 min of the clamp. As expected, the PER
treatment increased Rd more than
POR or 1/2 PER (Fig. 5,
top).
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The Ra values in the clamp period
(last 90 min) were suppressed to 6.1 ± 0.4, 6.9 ± 0.3, and 7.8 ± 0.3 µmol · kg1 · min
1
with the PER, POR, and 1/2 PER treatments, respectively. The percent
suppression of Ra is shown in Fig
5, bottom.
Ra suppression was similar with
PER (46 ± 9%) and POR (43 ± 4%) and significantly less with
1/2 PER (36 ± 8%, P < 0.01 vs.
POR, ANOVA for repeated measures).
Glucagon levels declined by a similar extent (~15%; Fig. 6, top) from basal levels of 173 ± 29, 134 ± 16, and 172 ± 21 pmol/l in PER, POR, and 1/2 PER groups, respectively. The FFA levels declined by 29 ± 10, 26 ± 4, and 21 ± 6% from a basal level of 1,145 ± 72, 1,260 ± 43, and 1,013 ± 49 µmol/l in the PER, POR, and 1/2 PER groups, respectively (Fig. 6, bottom). Unlike the high-rate insulin infusion, there were no significant differences between treatments. Alanine and lactate did not change from basal levels, and glycerol levels decreased minimally but not significantly (Table 6).
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DISCUSSION |
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In normal dogs at high physiological insulin concentrations, PER suppressed tracer-determined Ra more than equidose POR or 1/2 PER. Because peripheral insulin levels were greater in PER, whereas hepatic insulin levels were lower in PER vs. POR, this result is consistent with a peripheral effect of insulin in suppressing Ra. However, in the normal dogs at both low and high physiological insulin concentrations, POR suppressed Ra more than 1/2 PER despite peripheral insulin levels being matched in both studies. Because hepatic insulin levels were greater with POR than with 1/2 PER, this result is consistent with a direct hepatic effect of insulin in suppressing Ra. The novelty of the present study is the combination of the following two findings, both obtained for the first time during euglycemic clamps without using somatostatin: 1) in normal dogs, both hepatic and peripheral effects of insulin contribute to Ra suppression, and 2) the contribution of the peripheral effect predominates (we have addressed the second point in the end of the DISCUSSION).
In the low-rate insulin infusion study, plasma glucose specific activity remained constant. In the high-rate insulin infusion study, specific activity declined by ~25% with PER, more than with POR or 1/2 PER; therefore, we cannot exclude that some underestimation of Ra occurred. However, we (9) and others (14) have found that, provided the decline in specific activity is slow and <30% of the basal levels, the underestimation is negligible. In addition, we have repeated our calculation on the steady-state specific activities of the last hour of the clamp using the steady-state formula. The resulting values for Ra suppression were similar to those reported here (high rate: PER 79 ± 15%, POR 60 ± 9%, 1/2 PER 50 ± 5%, PER vs. POR P < 0.02, POR vs. 1/2 PER P < 0.01 using two-way ANOVA for repeated measures), and our conclusions remained unchanged.
At both low and high insulin concentrations, Rd was proportional to the peripheral insulin levels, as expected. Similar to Rd, the glucose infusion rate required to maintain euglycemia was greatest with PER but similar with POR and 1/2 PER despite Ra suppression being greater with POR than with 1/2 PER. This discrepancy is likely to be explained by the fact that small changes in Ra represent a sizable percentage of basal Ra, whereas they represent only a small fraction of the glucose requirements during a clamp.
At both insulin infusion rates, POR suppressed Ra more than 1/2 PER (high rate, P < 0.01, low rate, P < 0.05, two-way ANOVA for repeated measures). Because the peripheral insulin levels were similar between POR and 1/2 PER, the difference in Ra suppression should be ascribed to the difference in the hepatic insulin levels and is therefore consistent with a direct hepatic effect of insulin on Ra. With high-rate insulin infusion, Ra suppression was greater with PER than with POR. With low-rate insulin infusion, Ra suppression was not significantly different between PER and POR; however, because estimated hepatic insulin levels were greater with POR than with PER, one would expect a greater Ra suppression with POR. Therefore, even a similar degree of suppression by the two treatments implies that insulin suppressed Ra by a peripheral effect. Because Ra suppression was greater with PER than with POR at the high but not the low insulin infusion rate, our data suggest that the relative contribution of the peripheral effect is greater at higher insulin concentrations.
Insulin-induced inhibition of FFA levels has been found to play a dominant role in the peripheral effect of insulin on Ra (16, 24, 28). The present study is consistent with these results, as FFA levels decreased more with PER than with POR or 1/2 PER at the high-rate insulin infusion where Ra suppression was greater with PER. If the percent suppression of Ra was correlated with that of FFA as previously reported (23), the steady-state values were significantly correlated at the high-rate insulin infusion (r = 0.69, P < 0.01, n = 17 experiments) and almost significantly correlated at the low-rate insulin infusion (r = 0.48, P = 0.051, n = 17). The time course of Ra suppression was significantly correlated with that of FFA suppression at both the high-rate insulin infusion [r = 0.73, P < 0.001, n = 21 (7 time points × 3 groups)] and the low-rate infusion (r = 0.82, P < 0.001, n = 21). It might be argued that, at the high-rate infusion, the insulin-induced decrease in FFA levels did not appear to precede Ra suppression. This suggests, in our view, that insulin's suppression of Ra is partly independent of FFA [early Ra suppression may be mediated by insulin's hepatic effect (27)]. However, the difference in FFA between high-dose PER and POR did precede the difference in Ra, suggesting that FFA suppression is linked to the peripheral effect of insulin on Ra. In keeping with this observation, decreased FFA levels obtained with antilipolytic agents, such as acipimox, have been shown to enhance Ra suppression during hyperinsulinemic clamps (10, 25).
The actions of peripheral insulin in suppressing Ra may include decreased availability of gluconeogenic precursors, i.e., alanine and glycerol, due to insulin's suppression of proteolysis and lipolysis. In depancreatized dogs (12), we observed slight differences in alanine and glycerol levels between treatments; however, we calculated that these differences could have only minimally contributed to the effect of peripheral insulin on Ra. In the present study, only glycerol was significantly different between treatments at the high insulin infusion rate.
In depancreatized dogs, we have shown that glucagon may be an important mediator of the peripheral effect of exogenous insulin on Ra (11). In the present study, steady-state Ra suppression was correlated with steady-state glucagon suppression at both insulin infusion rates (high rate: r = 0.57, P < 0.05, n = 17; low rate: r = 0.63, P < 0.01, n = 17) and the time course of Ra suppression was correlated with that of glucagon suppression at the low insulin infusion rate (r = 0.75, P < 0.001, n = 21). However, some degree of correlations between variables that are both affected by insulin is to be expected. Figures 3 and 6 show that there was no difference in glucagon levels between any of the three treatments at either insulin infusion rate, indicating that suppression of glucagon may not play a major role in the peripheral effect of insulin in normal dogs. The difference between normal and diabetic dogs might relate to the presence of residual B cell insulin secretion directly inhibiting A cell glucagon secretion in normal dogs, whereas, in the absence of endogenous insulin secretion in depancreatized dogs, insulin-induced inhibition of glucagon is only proportional to the peripheral insulin levels.
It has recently been reported that, in dogs (5) and humans (30), the kidney may contribute to >20% of glucose Ra. It is therefore possible that a substantial part of the peripheral effect of insulin is due to insulin's direct effect in suppressing renal Ra. Our study, which was aimed at investigating the hepatic and peripheral effects of insulin in normal, conscious dogs, does not allow us to quantify the contribution of renal Ra to insulin's peripheral effect. This important issue can only be clarified if further studies in nephrectomized, anesthetized dogs (2) are carried out.
It is also possible that other mechanisms, which remain hypothetical, account for at least part of insulin's peripheral effect. One possibility is that hepatocytes could be more sensitive to the insulin concentrations reaching the liver with the hepatic artery than those reaching the organ through the portal vein. Also, insulin in the systemic circulation could inhibit Ra by activating peripheral or central neural pathways, thereby influencing the activity of hepatic nerves.
The results of this protocol are in accordance with those of Ader and Bergman (1) during euglycemic clamps in somatostatin-infused normal dogs in that suppression of Ra was mainly proportional to peripheral insulin levels. Although in Ader and Bergman's study Ra suppression was unaffected by hepatic insulin, the same authors found a small hepatic effect of insulin at low insulin doses in a subsequent study (23). Similar to our results, Sindelar et al. (27) also found both hepatic and peripheral effects of insulin on Ra in normal dogs during euglycemic clamps using somatostatin.
Our results in normal dogs are in excellent agreement with our study in
humans, where with the use of a high-rate tolbutamide-induced insulin
secretion (0.88 mU · kg1 · min
1),
both hepatic and peripheral effects of insulin in suppressing Ra were found (17). At the
high-rate insulin infusion in dogs, the percent difference in
suppression of Ra per unit
difference in the rise in hepatic insulin levels (POR vs. 1/2 PER) was
11/104 = 0.11%/pM. Taking into account that the rise in
hepatic insulin levels was 24 pM greater in POR than in PER, the
percent difference in suppression of
Ra per unit difference in the rise
in peripheral insulin levels (PER vs. POR) was [(0.11 × 24) + 23]/90 = 0.28%/pM. At the low-rate insulin infusion in dogs,
the percent difference in suppression of
Ra per unit difference in the rise
in hepatic insulin levels (POR vs. 1/2 PER) was 7/32 = 0.22%/pM.
Taking into account that the rise in hepatic insulin levels was 15 pM
greater in POR than in PER, the percent difference in suppression of
Ra per unit difference in the rise
in peripheral insulin levels (PER vs. POR) was [(0.22 × 15) + 3]/15 = 0.42%/pM. Thus the potency of peripheral insulin per
unit concentration was 0.28/0.11 = 2.6 greater than that of hepatic
insulin at the high-rate insulin infusion and 0.42/0.22 = 1.9 greater than that of hepatic insulin at the low-rate insulin infusion.
These data suggest that the peripheral effect of insulin prevails over
the hepatic effect at both low and high insulin concentrations and its
relative contribution to suppression of
Ra may increase with the insulin
dose. If the same calculations are performed with our data in humans
(17), the results show that peripheral insulin levels were
approximately threefold more potent than hepatic levels in suppressing
Ra.
These results are affected by the assumption used in calculating the hepatic insulin levels. If our calculated portal insulin levels are compared with those determined directly by Sindelar et al. (27), it would appear that we might have underestimated the differences in portal insulin levels (and therefore hepatic insulin levels) between POR and PER or 1/2 PER. If the difference in hepatic insulin levels was underestimated, we could have overestimated the effect of hepatic insulin. This would strengthen our conclusion about the predominance of the peripheral effect of insulin vs. the hepatic effect.
In contrast, Sindelar et al. have reported that a selective rise of 96 pmol in either portal or peripheral insulin levels resulted in similar suppression of Ra, consistent with equal potency of insulin delivered by either route per unit insulin concentration. The discrepancy between our results and theirs may relate to the use of the pancreatic clamp technique (somatostatin infusion + glucagon replacement) in their study. As previously discussed, we cannot exclude that somatostatin might alter the balance between insulin's hepatic and peripheral effects. In addition, we (11) and other authors (19) have found that glucagon (which was allowed to decrease in our study but is kept constant during pancreatic clamps) potentiates the direct effect of insulin. However, it is unlikely that the pancreatic clamp technique is the main reason for the quantitative discrepancy between Sindelar's study and ours, since, in Ader and Bergman's (1) study, pancreatic clamps were used, but Ra suppression was even more dependent on peripheral insulin than in our study. Alternatively, the discrepancy between Sindelar's data and ours may be due to the fact that, in our experiments, similar to those of Ader and Bergman's, hepatic and peripheral insulin levels rise simultaneously, whereas, in Sindelar's protocol, a selective rise in either portal or peripheral insulin levels was obtained. The latter authors have recently shown that the effects of portal and peripheral insulin are synergistic on hepatic glucose uptake and are additive on net hepatic glucose balance but not on tracer-determined Ra (29). Thus it is possible that, when both levels rise, as occurs physiologically, the effect of peripheral insulin on Ra predominates, thus masking insulin's hepatic effect.
We have previously reported that, in depancreatized diabetic dogs, we could only detect a peripheral effect of insulin on Ra (12). Thus it appears that insulin's hepatic effect may be abolished or masked by the peripheral effect under diabetic conditions.
In conclusion, at both low and high physiological insulin levels in normal dogs, insulin can suppress Ra by both peripheral and hepatic effects. Quantitatively, the peripheral effect predominates, being at least two times as potent as the hepatic effect.
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ACKNOWLEDGEMENTS |
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We are grateful to L. Lam, C. Chisholm, W. Pugh, and P. Rue for excellent technical assistance and to Dr. M. Vranic and Dr. G.F. Lewis for helpful criticism of the manuscript.
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FOOTNOTES |
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This work was supported by the Juvenile Diabetes Foundation (grant no. 193135 to A. Giacca). Financial contributions for personnel and manuscript editing were also provided by the Medical Research Council of Canada and by the Canadian Diabetes Association. A. Giacca was supported by a Career Development Award from the Juvenile Diabetes Foundation.
Address for reprint requests: A. Giacca, Dept. of Physiology, Medical Sciences Bldg., Rm. 3363, 8 King's College Circle, Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8.
Received 7 February 1997; accepted in final form 29 October 1997.
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