Z. Q. Shi

Importance of peripheral insulin levels for insulin-induced suppression of glucose production in depancreatized dogs.

It is generally believed that glucose production (GP) cannot be adequately suppressed in insulin-treated diabetes because the portal-peripheral insulin gradient is absent. To determine whether suppression of GP in diabetes depends on portal insulin levels, we performed 3-h glucose and specific activity clamps in moderately hyperglycemic (10 mM) depancreatized dogs, using three protocols: (a) 54 pmol.kg-1 bolus + 5.4 pmol.kg-1.min-1 portal insulin infusion (n = 7; peripheral insulin = 170 +/- 51 pM); (b) an equimolar peripheral infusion (n = 7; peripheral insulin = 294 +/- 28 pM, P < 0.001); and (c) a half-dose peripheral infusion (n = 7), which gave comparable (157 +/- 13 pM) insulinemia to that seen in protocol 1. Glucose production, use (GU) and cycling (GC) were measured using HPLC-purified 6-[3H]- and 2-[3H]glucose. Consistent with the higher peripheral insulinemia, peripheral infusion was more effective than equimolar portal infusion in increasing GU. Unexpectedly, it was also more potent in suppressing GP (73 +/- 7 vs. 55 +/- 7% suppression between 120 and 180 min, P < 0.001). At matched peripheral insulinemia (protocols 2 and 3), not only stimulation of GU, but also suppression of GP was the same (55 +/- 7 vs. 63 +/- 4%). In the diabetic dogs at 10 mM glucose, GC was threefold higher than normal but failed to decrease with insulin infusion by either route. Glycerol, alanine, FFA, and glucagon levels decreased proportionally to peripheral insulinemia. However, the decrease in glucagon was not significantly greater in protocol 2 than in 1 or 3. When we combined all protocols, we found a correlation between the decrements in glycerol and FFAs and the decrease in GP (r = 0.6, P < 0.01). In conclusion, when suprabasal insulin levels in the physiological postprandial range are provided to moderately hyperglycemic depancreatized dogs, suppression of GP appears to be more dependent on peripheral than portal insulin concentrations and may be mainly mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and by the suppressive effect of insulin on glucagon secretion. These results suggest that a portal-peripheral insulin gradient might not be necessary to effectively suppress postprandial GP in insulin-treated diabetics.

Effect of Diabetes on Glucoregulation: From glucose transporters to glucose metabolism in vivo

Peripheral resistance to insulin is a prominent feature of both insulin-dependent and non-insulin-dependent diabetes. Skeletal muscle is the primary site responsible for decreased insulin-induced glucose utilization in diabetic subjects. Glucose transport is the rate-limiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. The transport of glucose across the muscle cell plasma membrane is mediated by glucose transporter proteins, and two isoforms (GLUT1 and GLUT4) are expressed in muscle. Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT4 transporter (but not GLUT1) from an intracellular pool to the plasma membrane. In skeletal muscles of streptozocin-induced diabetic rats, there is a decreased GLUT4 protein content in intracellular and plasma membranes. In these rats, insulin induced the mobilization of GLUT4 from the internal pool, but the incorporation of the transporter protein into the plasma membrane is diminished. Conversely, the content of the GLUT1 transporter increases in the plasma membrane of these diabetic rats. Normalization of glycemia with phlorizin fully restores the amount of GLUT1 and GLUT4 proteins to normal levels in the plasma membrane without altering insulin levels. This suggests that glycemia regulates the number of glucose transporters at the cell surface, GLUT1 varying directly and GLUT4 inversely, to glycemia. The regulatory role of glycemia also can be seen in diabetic dogs in vivo, where correction of hyperglycemia with phlorizin restores, at least in part, the defective metabolic clearance rate of glucose seen in these animals. In addition to acutely stimulating glucose transport in muscle, insulin controls exercise- and possibly stress-mediated glucose uptake in vivo, by preventing hyperglycemia and by restraining the effects of catecholamines on lipolysis and/or muscle glycogenolysis. Finally, we postulated a neural pathway that requires the permissive effect of insulin to increase glucose uptake by the muscle. Thus, insulin, glucose, and neural pathways regulate muscle glucose utilization in vivo and are, therefore, important determinants of glucoregulation in diabetes.

A moderate decline in specific activity does not lead to an underestimation of hepatic glucose production during a glucose clamp

We have previously shown that modeling errors lead to underestimation of hepatic glucose production (HGP) during glucose clamps when specific activity (SA) declines markedly. We wished to assess whether the failure to keep SA constant substantially affects calculation of HGP during insulin infusion when glucose requirements to maintain the glucose clamp are moderate. Therefore, 150-minute hyperinsulinemic (5.4 pmol - kg (-1) - min (-1) clamps were performed in depancreatized dogs that were maintained hyperglycemic (approximately 10 mmol/L with either (l) unlabeled glucose infusate (COLD Ginf, n = 5) or (2) labeled glucose infusate (HOT Ginf, n = 6) containing high-performance liquid chromatography (HPLC purified [6-3H]glucose. Insulinemia and glucagonemia were similar between the two groups. Additionally, glucose infusion rates were equivalent with COLD and HOT Ginf, indicating comparable insulin effects on overall glucose metabolism. The SA decreased a maximum of 32% with COLD Ginf, but remained constant with HOT Ginf. HGP was suppressed equally with COLD or HOT Ginf treatments at each time point during the clamp (mean suppression during last hour of clamp, 69% +/- 4% and 69% +/- 5%, P = NS, COLD and HOT Ginf, respectively). We conclude that when glucose requirements are moderate and SA changes slowly, as in the diabetic dog, it is not necessary to keep SA perfectly constant to avoid significant modeling errors when calculating HPG during hyperinsulinemic clamps.

Quantitative Measurement of Islet Glucagon Response to Hypoglycemia by Confocal Fluorescence Imaging in Diabetic Rats

AbstractWe have shown that the glucagon irresponsiveness to hypoglycemia in diabetic rats is markedly improved by correction of hyperglycemia independent of insulin. In contrast, normalization of glycemia by insulin did not improve this response. To find out whether these glucagon responses reflect changes in islet glucagon, we directly quantified glucagon area and content in each pancreatic islet by using fluorescent immunostaining and computerized image analysis with confocal laser scanning microscopy (CLSM). The pancreases were analyzed in four groups of rats.1.Normal controls (NC,n=4), streptozotocin (65 mg/kg) diabetic rats.2.Diabetic untreated (DU,n=4).3.Diabetic Phlorizin-treated, (0.4 g/kg), twice daily for 4 d (DP,n=4).4.Diabetic insulin-treated, using sustained release (2–3 U/d) insulin implant for 5 d (DI,n=4). Basal plasma glucose was 7.4 ± 0.3 mM in NC, increased to 14.5±2.2 mM in DU, which was normalized in DP (5.5 ±0.5) and DI (6.7±0.8). Acute hypoglycemia (H) was induced by iv insulin injection. The rats were sacrificed 2 h after insulin injection and the pancreas was removed. By imaging with CLSM, we quantified:1.Percent of glucagon containing A-cell area/islet area,2.Fluorescence intensity per islet area, which indicated glucagon content in the islet.3.Fluorescence intensity per glucagon area indicating glucagon concentration in A-cells. In NC, glucagon containing A cell area was 21±2% of the islet area, and glucagon intensity and concentration was 11±1 U and 36±3.0 U, respectively, in basal (O) state and did not change in (H). In DU, glucagon area increased 183%. (O) and 166% (H), and islet glucagon intensity increased by 235% (O) (p<0.05), but decreased to 135% in H. Glucagon area in DP and DI did not differ significantly from DU. However, hypoglycemia in DP increased glucagon intensity in islet further to 306% of normal control (p<0.05), suggesting marked increase in glucagon content indicating increased synthesis. In contrast, DI compared to DP showed a decrease in glucagon intensity in islet (46±3, DP to 22±2 DI;p<0.05) in (H) state. Glucagon concentration followed the same pattern as its intensity. Conclusion:1.Increase in islet glucagon content in diabetic rats was associated with increase in glucagon containing A-cell area per islet.2.Phlorizin-induced insulin independent correction of hyperglycemia increased glucagon content per islet in hypoglycemic state. This, in part, probably contributed to improved glucagon response to hy poglycemia observed earlier3.Normalization of glycemia with insulin reduced glucagon content of each islet during hypoglycemia. This may explain, in part, unresponsiveness of glucagon to hypoglycemia often observed in insulin-dependent diabetes mellitus (IDDM) with intensive insulin therapy.

Effect of Stress on Glucoregulation in Physiology and Diabetes

To examine the glucoregulatory responses to stress and their impact on diabetes, we used the following models of stress: A) Hypoglycemia; B) Epinephrine infusion; C) intracerebroventricular (ICV) injection of carbachol, an analog of acetylcholine. A) Hypoglycemia induces release of all counterregulatory hormones. During acute hypoglycemia, glucose production increases initially mainly due to glucagon release but eventually also due to a very large increment in catecholamines. In newborn dogs, neither epinephrine nor glucagon respond to a decrease in plasma glucose. This lack of a safeguard against hypoglycemia may indicate that the brain in pups is less dependent on a normal supply of glucose as a fuel, than in adult dogs. Counterregulation is enhanced when the effects of endogenous opiates are blocked by naloxone, indicating that endogenous opiates play a regulatory role during hypoglycemia. However, beta-endorphins which can be released with epinephrine during various stress situations, potentiate the peripheral effect of epinephrine. Glucoregulatory responses, even to slight changes in plasma glucose, are greatly enhanced during glucocorticoid treatment. This apparently reflects the greater sensitivity of the liver to glucagon. In diabetic dogs, similar to human diabetics, the glucagon response is abolished and the response of the catecholamines is partially decreased. On the basis of histological studies, we proposed that the deficient glucagon response in diabetes could be related to an increase in the somatostatin-glucagon ratio in the diabetic pancreas. This ratio is further augmented when normoglycemia is maintained with insulin. In response to a decrease in plasma glucose, there is a biphasic increment in glucose production in normal dogs, which is missing in diabetes. When normoglycemia is restored in diabetic dogs with phlorizin treatment, the second but not the first increment in glucose production is restored. We postulated, therefore, that the toxic effect of hyperglycemia, in addition to the lack of glucagon response, is the main reason why in diabetes, glucose production cannot respond promptly to a decrease in plasma glucose. The low rate of metabolic clearance of glucose seen in diabetes in the post-absorptive state, also reflects, at least in part, the toxic effect of glucose, because with acute normalization of glucose with phlorizin, metabolic glucose clearance substantially improves. Hyperglycemia is the main reason for the decreased number of glucose transporters in diabetic muscle. B) Epinephrine infusion in normal dogs mimics some effects of stress, in that it increases glucose production, inhibits metabolic glucose clearance and increases lipolysis. These metabolic effects of epinephrine are independent of glucagon release.(ABSTRACT TRUNCATED AT 400 WORDS)

Importance of substrate changes in the decrease of hepatic glucose cycling during insulin infusion and declining glycemia in the depancreatized dog

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G⇆G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6–9 h at a starting dose of 45–150 pmol-kg−1-min−1 to normalize plasma glucose from 23.9 ± 1.4 to 5.0 ± 0.4 mmol/1 and gradually decreased to and maintained at a basal rate (1.7 ± 1.0 pmol · kg−1 · min−1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 ± 2.7 and normalized at 1.3 ± 0.6 μmol · kg−1 · min−1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 ± 3.1 to 11.6 ± 1.2) than did glucose production (from 25.9 ± 1.9 to 10.3 ± 1.0 μmol · kg−1 · min−1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 ± 24 vs. 71 ± 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 ± 0.8 to 19.6 ± 2.6 U/g) and glucokinase (from 1.1 ± 0.2 to 1.0 ± 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content. We therefore conclude that in the depancreatized dog, 1) acute insulin infusion with a concurrent correction of hyperglycemia can markedly reduce and normalize GC, and 2) the acute reduction in GC is primarily mediated by reduced substrate fluxes through glucokinase and G6Pase and does not require stable changes in maximal activities of the two enzymes. However, in vivo allosteric changes in enzyme activities (particularly G6Pase), immeasurable in vitro, cannot be excluded. In addition to enzyme measurements, the importance of tracer methods that allow for in vivo measurements of fluxes through glucokinase and G6Pase is emphasized.