Mayer B. Davidson

The Variability in the Action of Unmodified Insulin is More Dependent on Changes in Tissue Insulin Sensitivity than on Insulin Absorption

Eight normal subjects were studied twice for 360 min after the subcutaneous injection of unmodified insulin (0.15 U kg−1) during euglycaemic clamps. Insulin absorption was assessed by both the area under the insulin—time curve above baseline (AUC) and time course of absorption (time to 25% and 50% of total AUC). Insulin action was measured as the amount of glucose infused. The maximal serum insulin concentration was 0.27 ± 0.02 (± SE) nmol l−1 at 112 ± 10 min. Fifty percent of total glucose infused occurred at 218 ± 7 min. The maximal glucose infusion rate was 5.11 ± 0.70 mg kg−1 min−1 and occurred at 256 ± 12 min. Intrasubject coefficients of variation (CV) for total insulin AUC (11.2%), time to 25% of maximum AUC (12.1%), and time to 50% of maximum AUC (10.2%) were considerably lower than that for total insulin action (22.6%). Total insulin AUC did not correlate with total glucose utilization (r = 0.06, NS). We conclude that when glucose concentrations are maintained by euglycaemic clamps the peak of unmodified insulin action is later and the duration longer than traditionally recognized, insulin AUC does not predict insulin action, and the higher variability of insulin action compared with the indices of absorption suggests that day‐to‐day changes in tissue insulin sensitivity contribute more to the variability in insulin action than changes in absorption.

Effect of tumor necrosis factor-α on basal and insulin-stimulated glucose transport in cultured muscle and fat cells

It has been reported that tumor necrosis factor-alpha (TNF-alpha) inhibits insulin action in adipocytes and plays an important role as mediator of insulin resistance in non-insulin-dependent diabetes. The effect of this cytokine on insulin action in muscle, which is responsible for 80% of the glucose disposal in the body, has not been studied. Therefore, we examined the effect of TNF-alpha on basal and insulin-mediated transport of 2-deoxy[3H]-glucose in L6 rat muscle cells. TNF-alpha treatment for 5 days up to a concentration of 20 ng/mL or 8 days at 10 ng/mL did not inhibit the insulin-stimulated increase in deoxyglucose transport in L6 cells. However, there was a significant increase in basal transport in TNF-alpha- treated cells. Comparative experiments with 3T3-L1 adipocytes showed that in cells cultured with insulin, TNF-alpha decreased basal transport but the insulin-stimulated increase was unaffected. In cells cultured without insulin, basal transport was slightly increased and the insulin-stimulated increase in transport was decreased by approximately 60% but the cell protein was decreased by approximately 60%, suggesting cytotoxicity. Cells cultured without insulin were more sensitive to inhibition of 14C-alanine incorporation into proteins by low concentrations of TNF-alpha compared with cells cultured with insulin. These results suggest that TNF-alpha affects glucose metabolism, causing increased basal uptake in muscle cells and decreased uptake in adipocytes. TNF-alpha appears to affect general cell metabolism, including glucose transport in adipocytes, and not specifically insulin-stimulated glucose transport.

Lack of glucose elevation after simulated tube feeding with a low-carbohydrate, high-fat enteral formula in patients with type I diabetes

PURPOSE Commercially available enteral formulas frequently cause hyperglycemia. This often leads to difficult and complex diabetes management with glucosuria and urinary losses of calories, fluid, and electrolytes. In this study, we compared a new product, EN-8715 (8% soluble fiber, 18% Polycose, 7% fructose, 50% fat, 17% protein), with a standard feeding product, Ensure HN (53% simple carbohydrates, 30% fat, 17% protein), to determine whether the new product would lower the glucose response. PATIENTS AND METHODS Ten subjects (four women and six men) with type I diabetes were evaluated in paired, simulated tube feeding studies. After an overnight fast, a Biostator (artificial endocrine pancreas) was attached to each subject, and a steady-state blood glucose level of 150 mg/dL (8.4 mM) was established. The Biostator was then programmed to deliver a small basal amount of insulin (0.1 mU/kg/minute [718 pmol/kg/minute]), and the patients were given 20 mL of the randomly assigned formula every 15 minutes for 240 minutes (320 mL). Counterregulatory hormone responses to bringing the initial glucose response to the two enteral feeding formulas were measured and compared. RESULTS The glucose response (mg/dL/four hours +/- SEM) essentially did not rise after EN-8715 (-2 +/- 33 mg/dL/four hours [-0.11 +/- 1.83 mM/four hours]) compared with that for Ensure HN (190 +/- 32 mg/dL/four hours [10.64 +/- 1.79 mM/four hours]). Urinary glucose losses (g +/- SEM) were significantly (p = 0.01) less after the new product (1.4 +/- 0.6 g [7.8 +/- 3.3 mmol] versus 5.7 +/- 1.5 g [30.2 +/- 8.3 mmol]). There were no significant differences in counterregulatory hormone responses. Side effects were minimal and product acceptance was similar. CONCLUSION A low-carbohydrate, fiber-containing enteral feeding formula can limit hyperglycemia in patients with type I diabetes.

Inverse relationship of metabolic clearance rate of insulin to body mass index

Twenty-five human subjects, whose body mass indices (BMI) ranged from 18.0 to 34.1 kg/m2, underwent euglycemic clamps at both low (1.5 mU/kg/min) and high (10 mU/kg/min) insulin infusion rates. Mean C-peptide concentrations were less than or equal to 0.5 ng/mL at the end of each two-hour period. The metabolic clearance rates (MCR) of insulin (mL/kg/min) were significantly (P less than .001) inversely correlated with BMI at both the low (r = -.65) and high (r = -.71) insulin infusion rates. The negative inverse correlations with BMI remained significant at both the low (r = -.42, P less than .05) and high (r = -.61, P less than .005) insulin infusion rates if the MCR were expressed as mL/m2/min. There were no significantly correlations (r less than .21) between the MCR at either rates of insulin infusion and age or tracer insulin binding to monocytes. Decreased MCR of insulin may contribute to the hyperinsulinemia seen in obese subjects.

Insulin resistance in rats harboring growth hormone—secreting tumors: Decreased receptor number but increased kinase activity in liver

Growth hormone (GH) is a potent antagonist of insulin action, and this resistance occurs primarily at a post-binding step(s). To elucidate the underlying mechanisms, the effects of chronic GH excess on the structure and function of insulin receptors partially purified from the liver were examined in rats harboring GH-secreting tumors. Insulin resistance was established in this animal model of GH hypersecretion by a hyperinsulinemic euglycemic clamp. Specific binding of 125I-insulin and receptor number were reduced in tumor animals by 40% and 62%, respectively, reflecting downregulation of the insulin receptor by hyperinsulinemia in these animals. Receptors from tumor animals showed a 50% increase in beta-subunit phosphorylation and in the kinase activity toward the synthetic polypeptide Glu4:Tyr1 when measured in vitro in the absence of insulin; however, the incremental stimulation by insulin (170 nmol/L) of the phosphorylation of either the beta-subunit or Glu4:Tyr1 was not different between control and experimental animals. There was no difference between the two groups in Glu4:Tyr1 phosphorylation measured after immunodepletion of receptors by antibodies to the insulin receptor, indicating that the observed alteration in the kinase activity of tumor animals was intrinsic to the insulin receptor. Exposure to chronic GH excess did not alter insulin receptor structure, as evidenced by electrophoretic mobility under reducing and nonreducing conditions. The enhanced basal kinase activity of the receptor from tumor animals may reflect a more highly phosphorylated state of the receptor (and hence elevated enzyme activity) in these animals due to elevated serum insulin levels. These results demonstrate that the hepatic insulin resistance in rats chronically exposed to GH excess is not due to impaired insulin receptor kinase activity.

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexosamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) L-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (L form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosamine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L6 myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L6 myotubes and although the leucine derivative (20 microM) inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L6 myotubes. Thus, this phenomenon is regulated differently in muscle and fat.

Effect of glyburide on glycogen metabolism in cultured rat hepatocytes

Sulfonylurea agents decrease hepatic glucose production and fasting glucose levels in type II diabetic patients without changing fasting insulin concentrations. This raises the possibility that these drugs may act directly on hepatic carbohydrate metabolism. Cultured rat hepatocytes were used to test this hypothesis. To ascertain whether this in vitro system was suitable to demonstrate an effect of sulfonylurea agents (eg, the well-documented insulin-potentiating action), we initially measured the effect of glyburide (2 micrograms/mL) on insulin-stimulated net glucose-14C incorporation into glycogen. Glyburide increased sensitivity to insulin (ie, shifted the dose-response curve to the left) without affecting either responsiveness or insulin binding. Thus, the ED50 was significantly lowered (8.4 v 15.2 ng/mL), whereas the percent increase (181% v 170%) over the basal level, specific tracer insulin binding (5.3% v 5.1% per mg protein), and the Scatchard plots were similar. Since an effect of sulfonylurea agents could be demonstrated in this system, and the glycogen pathways supply 75% of hepatic glucose production after an overnight fast, we next measured the direct effect of glyburide (2 micrograms/mL) on glycogen storage and breakdown. Glycogenolysis was assessed by measuring the breakdown of prelabeled glycogen (from galactose-14C) and glycogen synthesis by the incorporation of glucose-C14 into glycogen. Glyburide significantly inhibited glycogenolysis and stimulated glycogen synthesis. Furthermore, glyburide significantly stimulated glycogen synthase while glycogen phosphorylase was unaffected. In conclusion, glyburide directly inhibited glycogenolysis, stimulated glycogen synthesis and glycogen synthase, and potentiated the action of insulin on glycogen synthesis at a postbinding site in cultured rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Review: Pathogenesis of Type 2 Diabetes Mellitus: An Interpretation of Current Data

The available evidence concerning the pathogenesis of type 2 diabetes mellitus suggests that although insulin antagonism is common to many patient groups (i.e., obese, older), overt NIDDM will only develop in a subset who may have a genetic susceptibility for inadequate beta-cell reserves. Since the role of a receptor defect in causing type 2 diabetes is doubtful, the insulin antagonism in these patients is due to a post-receptor defect.

Effect of sodium butyrate on glucose transport and glucose-phosphorylating enzymes in RIN-m5F cells

Sodium butyrate is widely used to differentiate insulinoma cell lines. However, sodium has been shown to decrease glucose phosphorylation in the liver and heart and decrease the expression of glucose transporter. Since these mechanisms are essential for glucose-induced insulin secretion, the ultimate function of the pancreatic beta-cell, we investigated the effect of sodium butyrate on both glucose-phosphorylating enzymes as well as glucose transport in the pancreatic cell line RIN-m5F. Treatment of RIN-m5F cells with 2.5 mM sodium butyrate for 72 h increased by twofold both hexokinase and glucokinase (GK) activities, as well as the gene expression of GK. Sodium butyrate treatment had no effect on GLUT-1 mRNA levels but increased the GLUT-2 mRNA 3.7-fold. Kinetic analysis of 2-deoxyglucose transport displayed a single curve with Km = 1.2 mM and Vmax = 10.9 pmol/micrograms protein/min in the untreated cells, values similar to the low Km glucose transport reported in the pancreatic beta-cells. This low Km transport component markedly decreased with sodium butyrate treatment, and interestingly a second component with a higher Km appeared, consistent with the increase in GLUT-2 mRNA. We conclude that the differentiating action of sodium butyrate involves increases in GK and GLUT-2 gene expression, which characterizes the differentiated state of the pancreatic beta-cell. However, the inhibitory effect of sodium butyrate on low Km glucose transport needs to be considered in the use of this compound to promote differentiation.

In vivo insulin antagonism but evanescent in vitro tissue effect in rats with growth hormone-secreting tumors

Rats bearing mammosomatotropic tumors have raised insulin but lowered glucose concentrations. To determine if growth hormone (GH) secreted by these tumors causes insulin antagonism, pancreatic suppression tests utilizing infusions (per kg per min) of glucose (8 mg), insulin (200 ng) and somatostatin (1.4 μg) for 130 min were performed. Although the steady state plasma glucose and insulin levels (mean of 90, 100, 110, 120 and 130 min samples) were similar in 8 control and 13 tumor-bearing rats, the decrease from the already depressed basal glucose concentration (mmoles/l±SE) in the tumor animals was less than in the controls (0.90 ± 0.30 vs. 2.56 ± 0.040, p < 0.005). Since the interpretation of these results was not entirely clear, glucose and insulin-glucose tolerance tests were performed. The glucose disappearance rates (%/min ± SE) in the glucose tolerance test were lower in 17 tumor rats (2.00 ± 0.13) compared to 17 control animals (2.51 ± 0.22). This difference just missed statistical significance (t = 2.00, value of 2.04 necessary for p = 0.05). The decrease occurred in the presence of increased insulin (nmoles/l ×16 min) levels (4.29 ± 0.38 vs. 2.58 ± 0.29, p < 0.005) suggesting insulin antagonism. The glucose disappearance rates (%/min ± SE) in the insulin-glucose tolerance test were less in 12 tumor-bearing rats compared to 11 control animals (2.80 ± 0.29 vs. 4.12 ± 0.35, p < 0.02). Thus, these GH-secreting tumors cause insulin antagonism in vivo. Freshly isolated hepatocytes from these tumor-bearing animals manifest decreased insulin binding and action (Diabetologia 25: 60, 1983). In the present study, however, insulin binding and action (net glucose-C14 incorporation into glycogen) were normal after the hepatocytes were cultured for two days. This suggests that the changes induced by GH in vivo that lead to insulin antagonism are short-lived.