Sensitivity and responsiveness of glucose output to insulin in isolated perfused liver from dexamethasone-treated rats

To elucidate insulin action on hepatic glucose output (glycogenolysis) in the state exposed to an excess glucocorticoid, the fed rat liver was isolated and cyclically perfused with a medium containing 5 mM glucose and various concentrations of insulin. The rat was subcutaneously injected with 1 mg/kg of dexamethasone (Dex) for 7 days. Dex-treated rats showed marked increases of serum insulin and plasma glucose level compared with those in control rats. Hepatic glycogen contents in Dex group were markedly increased compared with those in control (115 +/- 5 and 28 +/- 4 mg/g, respectively). Insulin extraction rate in the perfused liver was not different between control and Dex group. Perfusate glucose level after 60 min perfusion was much higher in the Dex-treated rat liver than that of the control at 0 microU/ml insulin (34.5 +/- 2.5 vs 23.0 +/- 2.0 mM, P less than 0.01), and reduced to the nadir level (19.0 +/- 3.0 and 13.0 +/- 1.5 mM, respectively) at 100 microU/ml insulin in both groups, i.e., the decreasing rate in perfusate glucose level was not different between Dex and control group (43% and 44%, respectively). These results suggest that Dex-treatment augments hepatic glucose output, but does not affect the sensitivity and responsiveness of that to insulin.     

In vitro effects of glycosylated insulin and glucagon in perfused liver of the rat.

Using perfused liver of the rat, the hepatic uptake of glycosylated insulin (GI) and glucagon (GG) and its effects on hepatic glucose output were investigated. Insulin and glucagon were glycosylated in ambient high glucose concentration, and GI80 or GG80 (insulin or glucagon incubated with 0.08% glucose), GI350 or GG350 (incubated with 0.35% glucose), and GI1000 or GG1000 (incubated with 1% glucose) were prepared. The liver was perfused with the medium containing 1000 microU/ml insulin and 200 pg/ml glucagon or 200 microU/ml insulin and 1000 pg/ml glucagon. The fractional uptake of insulin or glucagon by perfused liver was not significantly altered by the glycosylation. In the liver perfused with 1000 microU/ml insulin and 200 pg/ml glucagon, glucose output was not changed by the glycosylation of the hormones, while in the liver perfused with 200 microU/ml insulin and 1000 pg/ml glucagon, GI1000 reduced its biological activity, as reflected by insulin-mediated decrease in glucose output. These results suggest that in the liver insulin incubated with markedly high concentration of glucose reduces its biological activity at a physiological concentration in the presence of high concentration of glucagon.     

Leptin perfusion affects insulin secretion but not insulin receptors in rats

The aim of the study was to investigate acute leptin effects on insulin secretion and liver insulin binding in rats in vitro. In the in situ experiments leptin changed the pattern of insulin secretion from the pancreas but did not influence insulin binding in the liver. Perfusion of the pancreas with leptin (1, 10, and 100 nmol/l, respectively) at physiological and supraphysiological levels of glucose (6.66 and 25.0 mmol/l, respectively) did not evoke the inhibition of insulin output observed by the authors previously in the in vivo manners. On the contrary, leptin perfusion resulted in stimulation of insulin secretion. Simultaneously, liver perfusion with leptin for 30 min did not influence specific insulin binding. Analysis of Scatchard's plots indicated no changes in the number of high- and low-affinity insulin receptors and in their affinity to the hormone. Additionally, leptin did not influence general carbohydrate and lipid metabolism of the perfused liver. After the treatment with leptin, the output of glucose, free fatty acids and triglycerides to perfusate and the final contents of glycogen and triglycerides in liver were comparable to values obtained in control animals. The results indicate that some in vitro effects exerted by leptin differ from those observed in vivo.     

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mm glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-d-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mm glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = ?0.621; n = 29; P < 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P < 0.01). Glucose starvation of cultures for 18 h resulted in several changes: (i) a 49% decrease in specific ¹²⁵I-insulin binding due to a reduction in binding capacity; (ii) elevated basal 2-DG transport; and (iii) an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.     

Changes of insulin binding in rat tissues after exposure to stress.

The effects of various stressors on insulin receptors in adipose, liver and skeletal muscle tissues were studied in rats exposed to acute or repeated stress. Adult male rats were exposed to immobilization (IMO) for 2.5 hours daily for 1, 7 and 42 days, or to hypokinesia (HK) for 1, 7 and 21 days. We determined the values of specific insulin binding (SIB) and insulin receptor binding capacity (IR) of plasma cell membranes from adipose, liver and muscle tissue (IMO groups), or insulin binding to isolated adipocytes and hepatocytes (HK groups). A significant decrease of SIB and IR was observed in rats exposed to acute stress (1x IMO) in muscle, adipose and liver tissues. However, in animals exposed to repeated stress (7x and 42x IMO), SIB and IR were diminished in the muscle tissue, whereas no significant changes were noted in the liver and adipose tissue. When tissue samples were collected 3-24 hours after exposure to IMO stress, no changes of SIB and IR were found in liver and adipose tissue, but insulin binding was lowered in skeletal muscles. In animals exposed to HK for one day, a decrease of SIB and IR was found in isolated adipocytes, but no changes in insulin binding were noted in the liver tissue. In rats exposed to HK for 7 and 21 days, values of IR were similar as in control group. Our results indicate a) the different changes of IR in the liver, fat and muscle tissues after exposure to stress situations, b) a long-term decrease of insulin binding in muscles of rats exposed to repeated IMO stress, and c) the return of reduced SIB and IR (induced by acute stress) to control values in the liver and adipose tissue after a short recovery period.     

Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats

We investigated whether fatty liver preceded insulin resistance or vice versa using a long-term orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) model without the confounding effects of obesity and hyperlipidemia and explored the role of the liver in insulin resistance. Male Wistar rats were fed with or without OA supplementation for 30, 60, and 90 days. The NAFLD group showed increased liver lipid at 30, 60, and 90 days; glucose intolerance was noted at 60 and 90 days. Furthermore, partial liver proteins and gene expressions related to upstream signaling of insulin were decreased. However, the liver glycogen content was elevated, and gluconeogenesis genes expressions were obviously decreased at 90 days. The occurrence of fatty liver preceded insulin resistance in OA-induced NAFLD without the interference of obesity and hyperlipidemia, and hepatic insulin resistance may not play a conclusive role in insulin resistance in this model.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calorie restriction on skeletal muscle and liver insulin binding in growing rat

The effect of specific calorie deprivation was studied in meal-fed growing rats. It resulted in a 50% decrease in growth rate. Blood glucose and most non-essential blood free amino acid levels were depressed. Postprandial plasma insulin was decreased. With insulin ranging from 0.01 to 100 nM, insulin binding to crude Triton X-100 solubilized membranes from liver was higher in calorie restricted rats when compared with control rats. Wheat germ agglutinin (WGA) purified receptor preparations also exhibited higher insulin binding in liver from experimental group but the significance (P less than 0.05) was only visible with low insulin levels; both basal and insulin-stimulated tyrosine-kinase activity were left unchanged. In contrast, whatever the skeletal muscle insulin receptor preparation (enriched plasma membranes, crude Triton X-100 solubilized or wheat-germ agglutinin purified extracts) insulin binding was similar in control and calorie-restricted rats.     

Cellular binding sites for insulin in rat liver.

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin binding capacity did not follow closely the enzyme marker (5'-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5'-nycleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.     

Cellular binding sites for insulin in rat liver

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin-binding capacity did not follow closely the enzyme marker (5′-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinuous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5′-nucleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.     

Effect of short- and long-term chlorpropamide therapy on oral glucose tolerance and erythrocyte insulin receptors in non-obese non-insulin dependent diabetes mellitus

Erythrocyte (RBC) insulin receptors and the insulin response to glucose load (oGTT) were evaluated in ten male, non-obese, non-insulin dependent diabetic patients (NIDDM) before and after 14 and 90 days of 250 mg/day of chlorpropamide administration. In addition, as a control group, twelve healthy non-obese subjects were studied. Diabetic patients with fasting plasma glucose level higher than 14 mmol/l (group A), presented a significant improvement in the incremental glucose area only after 90 days of therapy. There was an evident reduction in insulin secretion, in comparison to normals, which however increased progressively during drug administration. No alterations in insulin binding to RBC receptors were observed either before or after the use of chlorpropamide, but the normalization of the initially low number of receptors per cell (N) and an increased high affinity constant (Ke) were achieved. In group B with fasting glucose less than 14 mmol/l there was a significant reduction in plasma glucose levels during oGTT without changes in glucose areas and a significant improvement of the insulin secretion was noted only in the early samples. Except for transient alterations in N and Ke no significant changes were observed in insulin-RBC binding parameters. We conclude that the improvement in the glucose tolerance in NIDDM is associated both to a greater insulin secretion and to the correction of the alterations in receptor parameters which could be related, at least partially, to proportionate changes in reticulocyte count.     

Effects of short-term insulin deficiency on lipogenesis and cholesterol synthesis in rat small intestine and liver in vivo.

The rate of lipogenesis in rat intestine increased on oral glucose loading and decreased after induction of acute insulin deficiency with streptozotocin. The latter effects could be partially reversed by administration of insulin. Parallel changes in the rate of lipogenesis were found in liver. In contrast, insulin deficiency did not alter the rate of cholesterol synthesis in intestine, but decreased it in liver. The physiological significance of the regulation of intestinal lipogenesis by insulin is discussed.     

Reversal of insulin resistance in type I diabetes after treatment with continuous subcutaneous insulin infusion.

Insulin responsiveness was studied with the euglycaemic glucose clamp technique in seven patients with type I diabetes and in six control subjects matched for age and weight. The glucose disposal rate was significantly reduced in the diabetic subjects when they were receiving conventional insulin treatment compared with the control group, showing insulin resistance in the diabetics. The diabetic patients were again studied after eight days of intensified metabolic control achieved with continuous subcutaneous insulin infusion. During the infusion a more physiological insulin regimen was used compared with their regular treatment, less of the total insulin dose being given as continuous infusion and more as bolus doses before meals. The insulin resistance in the diabetics was largely reversed after this improved metabolic control. Dose response studies showed an increased glucose disposal rate at all plasma insulin concentrations, including the maximum insulin concentration, indicating a predominant effect of the continuous infusion regimen at the postreceptor level. The improved insulin effect seen with continuous subcutaneous insulin infusion could be due to the improved metabolic control achieved as well as the more physiological regimen.     

The effect of magnesium deficiency on glucose stimulated insulin secretion in rats

Weanling Sherman rats were pair-fed for 8 days on a control or a magnesium deficient diet containing 70.5% sucrose. After a 12-hour fast, the rats were injected intraperitoneally with glucose (250 mg/100 g body weight) and arterial blood was drawn at 0, 15, 30, 60, 90 minutes after injection. Before glucose loading, in magnesium deficient rats, plasma magnesium levels were significantly increased. The plasma triglyceride concentration was significantly higher in magnesium deficient rats compared to controls. After glucose loading, in the control group, the plasma insulin concentrations increased to 67.9 +/- 5.8 microU/ml at 15 minutes and returned to pretreatment levels by 30 minutes; in the magnesium-deficient rats, the plasma insulin levels were significantly lower at 15 minutes 32.9 +/- 5.6 microU/ml (P less than 0.01) and returned more slowly to the pre-challenge level. No significant differences were observed in plasma glucose levels between the two groups of rats.     

In vitro effects of glycosylated insulin in perfused liver and hindquarter in rats.

Using the perfused liver and hindquarter of the rat, the uptake of glycosylated insulin and its effect on glucose output were investigated. Insulin was glycosylated in ambient high glucose concentration, and glycosylated insulin GI80 (insulin incubated with 0.08% glucose), GI350 (incubated with 0.35% glucose), and GI1000 (incubated with 1% glucose) were prepared. The liver and hindquarter were perfused with nonglycosylated insulin (N-GI) or glycosylated insulin at a concentration of 100 or 1000 microU/ml. There were no significant differences in the fractional uptake of insulin by perfused liver and hindquarter despite glycosylation. Insulin-induced decrement in glucose output was significantly lower in the liver perfused with GI1000 than that in the liver perfused with N-GI, GI80, and GI350 at an insulin concentration of 100 microU/ml. There were no significant differences in insulin-induced decrement in glucose output between the hindquarter perfused with N-GI, GI80, GI350, and GI1000. These results suggest that when insulin (100 microU/ml) is incubated with a markedly elevated concentration of glucose (1000 mg/dl) its biological activity is reduced in the liver, but not in the hindquarter.     

Hindlimb suspension increases insulin binding and glucose metabolism

After 28 days of hindlimb-suspension, insulin binding, 2-deoxy-D-glucose (2-DG) uptake, and glucose metabolism (glycolysis and glycogenesis) were determined at various insulin concentrations (0.2-30 nM) in soleus muscle of young (18-day-old) and adult (150-day-old) rats. Compared with age-matched controls the young (YS) and adult suspended (AS) rats had lower soleus and body weights and insulin levels (P less than 0.05). Per milligram of protein, insulin binding, 2-DG uptake, and the rate of glycolysis were increased by approximately 200%, and the rate of glycogenesis was increased approximately 100% in the YS group (P less than 0.05). Except for a reduction in glycogenesis (P less than 0.05) all other parameters also increased in the AS rats (P less than 0.05). On the basis of the whole muscle the rate of glucose metabolism (glycogenesis + glycolysis) was reduced in the YS rats (P less than 0.05), but in the AS rats glucose metabolism was similar to the controls. Thus the increased glucose metabolism (i.e., per milligram of protein) in the YS and AS groups may represent a compensatory response by atrophied muscle to attempt to sustain glucose removal from the circulation. Because greater insulin binding occurred in YS muscle [35% slow-twitch (ST)] than in the control group (70% ST), and greater insulin binding occurred in the AS (81% ST) than in the control group (90% ST), higher insulin binding capacities are not always related to a high proportion of ST muscle fibers. In conclusion, after hindlimb suspension, marked increments in insulin binding and glucose metabolism occur in the soleus muscle.     

Computed insulin profiles for normalization of glycemia in diabetic patients

The objective of this preliminary study was to develop a new quantitative method of setting the initial insulin infusion patterns in treatment of diabetic patients. The method is based upon the mathematical estimation of the insulin profile required to maintain the glucose level within the normal range after glucose loading in diabetic patients. Using our previously developed equivalent circuit model of glucose kinetics and the reported data of an intravenous glucose tolerance test (IVGTT) in two groups of normal and diabetic patients, two important physiological parameters of the model (the peripheral tissue's insulin resistivity and the hepatic sensitivity to glucose level) were computed for two clinical groups. Then the insulin profile was obtained by computing the plasma insulin concentrations required to keep the total glucose utilization rate of the tissue and the liver in the diabetic group equal to that of the normal group. The simulation result indicated that the computed insulin profile produced a plasma glucose profile which was more closely matched to the normal group's glucose profile than with the case of emulating the normal group's insulin profile in the diabetic group.     

Adipose tumor necrosis factor-alpha is reduced during onset of insulin resistance in Sprague-Dawley rats

Overexpression of mRNA for tumor necrosis factor-alpha (TNF-alpha) has been observed in adipose tissue in several rodent models of insulin resistance. The purpose of the present study was to examine the expression of TNF-alpha protein during the onset of insulin resistance in maturing Sprague-Dawley (S-D) rats. Compared to 2 months, rats aged 5 and 12 months were glucose intolerant and fasting glucose was elevated at 12 months (p < 0.05). Compared to 2 months, insulin concentrations following glucose loading were elevated at 5 months (p < 0.05) and also at 12 months, but to a lesser degree. In isolated strips of soleus muscle, insulin-stimulated glucose transport was reduced by 38% and 59% between 2 and 5 months and between 2 and 12 months, respectively (p < 0.05), with no changes in basal transport. Insulin resistance was associated with decreased content of TNF-alpha protein in visceral and subcutaneous fat. TNF-alpha protein content was also decreased in tibialis anterior muscle, but was unchanged in soleus and red gastrocnemius muscles. Liver was the only tissue examined that showed an increase in TNF-alpha protein content. In vitro secretion of TNF-alpha protein was markedly reduced in explants of visceral and subcutaneous fat from mature, insulin-resistant animals, but TNF-alpha bioactivity in subcutaneous fat was maintained with age. These results indicate that the onset of insulin resistance in mature S-D rats is associated with reduced adipose expression of TNF-alpha. Our findings do not support the adipose-endocrine model of TNF-alpha in insulin resistance. Our findings do support a paracrine role for TNF-alpha or for a reduction in endogenous TNF-alpha inhibitors in insulin resistance.     

In vivo insulin sensitivity and insulin binding to erythrocytes in children: insulin resistance in obese children

This aim of this study was to determine whether RBC insulin receptor assay represents a clinically useful way of assessing insulin sensitivity in obese children. Steady state plasma glucose (SSPG) was established by a constant infusion of glucose (6 mg/kg/min), insulin (0.8 mU/kg/min) and somatostatin (125 micrograms/m2/h), following the loading dose of somatostatin (125 micrograms/m2). Insulin binding to RBCs was measured by a modified method of Gambhir and was compared with SSPG. Of 21 children with various relative body weight, 8 hyperinsulinemic obese children had a decreased insulin binding to RBCs due to decreased receptor concentrations. The insulin binding was inversely correlated with the fasting serum insulin level and with the insulin area under the O-GTT insulin response curve. In 11 children with various relative body weight, a highly significant inverse relationship was found between SSPG and insulin binding. SSPG was also correlated with the fasting serum insulin level. It was concluded that RBC insulin receptor may quantitatively reflect insulin resistance in obese children, and may be a useful tool for clinical evaluation of tissue insulin sensitivity in children.