Insulin-like action of proinsulin on rat liver carbohydrate metabolism in vitro

Short-term effects of human proinsulin on metabolic rates and its long-term action on enzyme induction were studied in primary cultures of rat hepatocytes and in the perfused rat liver, and compared with the effects of bovine insulin. In the perfused rat liver, proinsulin decreased the glucagon-dependent increase of glycogenolysis. The action of 0.5 nM glucagon was almost completely suppressed by 100 nM proinsulin. Proinsulin and insulin showed similar potency. In cultured rat hepatocytes, proinsulin stimulated glycolysis up to fivefold with a half-maximal effective dose of 30 nM. Proinsulin induced the key glycolytic enzymes glucokinase and pyruvate kinase by twofold and antagonized the glucagon-dependent induction of phosphoenolpyruvate carboxykinase with a half-maximal effective dose at 3 nM. For the effects in cultured hepatocytes, about 100-fold higher concentrations of proinsulin than of insulin were required.     

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of glycogenolysis and glycogen phosphorylase by insulin and proinsulin in rat hepatocyte cultures

The inhibitory action of insulin and proinsulin on basal and glucagon-activated glycogenolysis was studied in cultured rat hepatocytes containing [14C]glycogen. Insulin or proinsulin given as sole hormones in the presence of 5 mM glucose decreased basal release of [14C]glucose from [14C]glycogen to 20%. Half-maximal effective concentration of insulin was approximately 0.15 nM and of proinsulin was approximately 5 nM. Inhibition of [14C]lactate release from [14C]glycogen required slightly higher hormone concentrations with a similar difference in potency for insulin and proinsulin. The glucagon-stimulated release of [14C]glucose was completely blocked by insulin or proinsulin with half-maximal effective concentrations of approximately 0.2 and approximately 8 nM, respectively. In contrast, release of [14C]lactate in the presence of glucagon was increased slightly by insulin and proinsulin. Basal and glucagon-activated phosphorylase activity was inhibited by approximately 50% in a dose-dependent manner by both hormones, with differences in potency similar to those for the inhibition of glycogenolysis. These data point to a direct regulatory role of insulin in the control of hepatic glycogen breakdown even when acting as sole hormone. The results do not support the notion of a preferential inhibitory potency of proinsulin on hepatic glycogenolysis.     

Kinetics of biosynthetic human proinsulin action in isolated rat adipocytes

The binding and biologic potencies of human biosynthetic proinsulin (HPro) were determined in isolated rat adipocytes. At both 16 degrees C and 37 degrees C, proinsulin was found to have 3% (on a molar basis) the potency of porcine insulin for displacing bound [(125I)TyrA14]-insulin from insulin receptors. Human biosynthetic proinsulin also had 3% of the molar potency of insulin for stimulation of deoxyglucose transport (EC50 = 8.8 +/- 0.05 X 10(-11) M for insulin and 2.9 +/- 0.55 nM for HPro). However, both hormones produced the same maximal effect on glucose transport. In order to determine if the delay in onset and persistence of proinsulin action seen in vivo was due to any differences at the cellular level, the time course of HPro action on glucose transport was determined. Biologically equivalent submaximal concentrations of insulin (0.166 nM) and HPro (4.44 nM) gave identical time courses for stimulation of deoxyglucose transport at 37 degrees C with half-maximal effects at 4 min and full effects by 30 min. Maximally stimulating concentrations of insulin (1.66 nM) and HPro (22.2 nM) also had superimposable time courses. Deactivation of stimulated glucose transport was determined by incubating equivalent concentrations of insulin (0.166 and 1.66 nM) and HPro (4.44 and 22.2 nM) until full stimulation was achieved, washing cells free of unbound hormone, and initiating dissociation and deactivation by resuspension in hormone-free buffer. Both the absolute activities of transport and rates of deactivation were the same for insulin and HPro. At the submaximal concentrations, 50% of the hormones' effects were lost by 20 min, while 50 min was required after maximal stimulation for 50% deactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-sectional and prospective association between proinsulin secretion and graft function after clinical islet transplantation

Proinsulin levels as a marker of beta-cell dysfunction have not been described after clinical islet transplantation. Proinsulin secretion was studied in 23 type 1 diabetic patients after islet allotransplantation and in 20 age-matched nondiabetic controls. Fasting serum insulin, total proinsulin (TP), intact proinsulin, proinsulin fragments (PFs) and their ratios to insulin were determined 1 and 12 months after patients became insulin independent. TP, PF, and proinsulin/insulin ratios were lower in transplant recipients compared with controls, in patients who retained long-term insulin independence. Insulin, C-peptide, and intact proinsulin values were similar in transplant recipients and controls. Hormone levels remained stable over time in the group of patients who retained long-term insulin independence, but the TP and PF levels were higher at 12 months compared with 1 month in the group of patients who resumed insulin therapy. TP and PF levels were reduced in transplant recipients compared with controls but increased over time if insulin independence was lost.     

Effects of dietary treatment on serum insulin and proinsulin response in newly diagnosed NIDDM

Serum proinsulin is disproportionately elevated both in the basal state and after an oral glucose load in non-insulin-dependent diabetes mellitus (NIDDM). However, there is no detailed information about the effect of glycemic control on this abnormality. We investigated the effect of glycemic control by dietary treatment on serum proinsulin level in the basal state and in response to an oral glucose load. Ten NIDDM patients (7 men and 3 women), aged 19-60 yr, with mean (+/- SD) body mass index of 28 +/- 6 kg/m2 (range 21-42 kg/m2) and normal renal and liver function were studied. Before and after dietary therapy (25-30 kcal/kg ideal body wt), 100-g oral glucose tolerance tests were performed. Proinsulin was measured with our proinsulin-specific antiserum, which recognizes the connecting site of the B-chain of insulin and C-peptide. After dietary treatment, fasting plasma glucose decreased from 197 +/- 35 to 113 +/- 18 mg/dl (P less than .001). Both serum insulin and proinsulin decreased (insulin from 15 +/- 8 to 10 +/- 4 microU/ml, P less than .02; proinsulin from 31 +/- 18 to 13 +/- 5 pM, P less than .02), and the molar ratio of proinsulin to insulin also tended to decrease (from 0.321 +/- 0.08 to 0.24 +/- 0.10, P less than .10). Insulin response to oral glucose increased after dietary treatment, whereas proinsulin response did not change, resulting in a significant decrease in the molar ratio of the area under the curve of proinsulin to insulin after glucose load (from 0.28 +/- 0.12 to 0.13 +/- 0.07, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnosis and minimally invasive resection of an insulinoma: report of an unusual case and review of the literature

Insulinomas are rare endocrine tumors that are usually diagnosed by inappropriate elevations in insulin and C-peptide during hypoglycemia. We report a case of a surgically confirmed insulinoma diagnosed by a mild elevation in proinsulin with suppressed insulin and C-peptide at the time of hypoglycemia during a supervised fast. A supervised fast with serial measurements of plasma glucose, insulin, and C-peptide was performed in a patient with documented hypoglycemia. Proinsulin was measured at the beginning and end of the fast. Tumor localization was accomplished with spiral CT, magnetic resonance imaging, and endoscopic ultrasound. Minimally invasive tumor resection was performed. The presence of an insulinoma was confirmed on the basis of a minimally elevated proinsulin level with a suppressed insulin level at the time of symptomatic hypoglycemia. Tumor resection was performed without complications, resulting in resolution of the hypoglycemia. This case demonstrates the importance of measuring proinsulin as a routine component of the 72-hour fast for detection of an insulinoma. Even mild elevations in circulating proinsulin can be an independent indicator of aberrant insulin secretion during hypoglycemia. Once the diagnosis of insulinoma is made and tumor localization is achieved, minimally invasive tumor resection is a safe and effective treatment modality.     

Free-fatty acid inhibition of insulin binding, degradation, and action in isolated rat hepatocytes

The effect of free fatty acids (FFAs) on insulin binding and action was investigated in isolated rat hepatocytes. Oleic acid (0.4 mM) added to the cells rapidly (within 45 min) reduced insulin binding and degradation (each by 45%; P less than 0.001, n = 7) without changing the apparent receptor affinity. The effect was concentration dependent; a half-maximal inhibitory effect occurred at 0.150 +/- 0.050 mM (mean +/- SE). Oleic acid exerted no effect on insulin binding in energy-depleted (KCN-treated) cells. Oleic, palmitic, stearic, palmitoleic, and eicosapentaenoic acids were equally effective in reducing insulin binding. FFA did not change insulin binding to partially purified insulin receptors, thus excluding a direct effect on the insulin receptor. Furthermore, binding to partially purified receptors from solubilized cells pretreated with 0.2 mM oleic acid was not changed, indicating the effect of FFA in intact cells is on the rate of receptor internalization and/or recycling. Concomitant with the effect on insulin binding, oleic acid elicited a concentration-dependent reduction in nonstimulated cellular [14C]aminoisobutyric acid uptake (AIB; 29 +/- 8%, P less than 0.05) and decreased the maximal effect of insulin (39 +/- 7%, P less than 0.05). Thus, in a concentration-dependent manner, different fatty acids can reduce the number of binding sites for insulin and the degradation of insulin by isolated liver cells. Basal and insulin-stimulated AIB transport was reduced, suggesting the presence of postbinding perturbations. These data suggest that FFA exerts an important modulating effect on insulin action in the liver.     

Characterization of insulin binding in the UMR-106 rat osteoblastic osteosarcoma cell

The correlation of insulin receptor occupancy with classic insulin effects, such as stimulation of glucose uptake, have not been examined in osteoblastlike cells. Accordingly, we characterized insulin binding and examined its relationship to stimulation of glucose analog transport in the UMR-106 rat osteoblastic osteosarcoma cell line. Insulin binding in UMR-106 cells was found to be pH sensitive, temperature dependent, saturable, and specific. Proinsulin was 100-fold less effective than insulin in displacing specific [125I]insulin binding in these cells, whereas IGF-I at concentrations between 0.1 and 10 nM produced no displacement of [125I]insulin but did produce significant displacement of insulin binding at 100 and 1000 nM. Insulin receptor downregulation was observed after exposure to 100 nM insulin for 6 h at 37 degrees C and was temperature dependent. Insulin binding was reversible after 24 h at 4 degrees C. Insulin binding correlated directly with stimulation of 2-deoxyglucose uptake at insulin concentrations between 0.1 and 100 nM, with a half-maximal concentration (ED50) of 0.9 nM for both [125I]insulin binding displacement and stimulation of 2-deoxyglucose uptake. Hence, there was no evidence for spare insulin receptors with regard to stimulation of glucose analog transport. Scatchard analysis of insulin binding kinetics yielded a curvilinear plot, suggesting negative cooperativity. Analysis of insulin binding kinetics using a two-site model yielded a KD of 0.9 nM for the apparent high-affinity binding site and an estimated 80,000 high-affinity binding sites per cell. These findings demonstrate that osteoblastlike cells exhibit a relationship between insulin binding and glucose transport stimulation that is similar to that in liver cells and other insulin-sensitive tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships among age, proinsulin conversion, and beta-cell function in nondiabetic humans

The aim of the present study was to examine the relationships among beta-cell function, proinsulin conversion to insulin, and age. We studied insulin and proinsulin secretion in nondiabetic subjects during an oral glucose tolerance test (OGTT) using published indexes of beta-cell function (n = 379, age 16--68 years) and a modified hyperglycemic clamp (10 mmol/l, additional glucagon-like peptide [GLP-1] infusion, final arginine bolus; n = 50, age 19--68 years). Proinsulin conversion to insulin was assessed using proinsulin/insulin (PI/I) ratios immediately after an acute stimulus (OGTT, 30 min; hyperglycemic clamp, 2.5-5.0 min after glucose and arginine). There was a negative correlation between age and beta-cell function (adjusted for insulin sensitivity, BMI, and fasting glucose) in the OGTT (r = -0.21, P < 0.001) and first phase of the hyperglycemic clamp (r = -0.30, P = 0.03), but not second phase (r = -0.08, P = 0.6) or arginine-induced insulin secretion (r = 0.06, P = 0.7). There was a positive correlation between age and the PI/I ratio in the OGTT (r = 0.24, P < 0.001). Analogously, there was also a positive correlation between age and the PI/I ratio during first phase (r = 0.37, P = 0.009) and arginine stimulation (r = 0.33, P = 0.01) of the hyperglycemic clamp. First-phase insulin secretion of the hyperglycemic clamp was inversely correlated with the PI/I ratio (r = -0.60, P < 0.001). Interestingly, adjusting first-phase secretion rate for the PI/I ratio abolished the linear relationship with age (r = -0.06, P = 0.7). In conclusion, aging is associated with deteriorating beta-cell function and deteriorating proinsulin conversion to insulin. The age effect on insulin secretion appears to be attributable at least in part to an impairment of proinsulin conversion to insulin.     

Localization of rate-limiting defect for glucose disposal in skeletal muscle of insulin-resistant type I diabetic patients

We searched for metabolic crossover points in muscle glucose metabolite profiles during maintenance of matched glucose fluxes across forearm muscle in insulin-resistant type I (insulin-dependent) diabetic patients and nondiabetic subjects. To classify subjects as insulin sensitive or insulin resistant, whole-body and forearm glucose disposal, oxidative and nonoxidative glucose disposal (indirect calorimetry), and glycogen synthesis (muscle glycogen content in needle biopsies) were measured under euglycemic conditions at two insulin concentrations. Whole-body and forearm muscle glucose disposal were significantly reduced in diabetic patients compared with control subjects. The reduction in total glucose disposal was due to similar relative reductions in oxidative and nonoxidative glucose disposal, pointing toward rate limitation early in glucose metabolism. The defect in nonoxidative glucose disposal was at least partly due to a defect in muscle glycogen synthesis, because muscle glycogen content failed to increase in response to an increase in the plasma insulin concentration in the diabetic patients. The most-insulin-resistant type 1 diabetic patients were restudied under conditions where, by glucose mass action, whole-body glucose disposal was forced to be similar to that in the control subjects. Matching glucose fluxes in the two groups resulted in similar rates of forearm and whole-body oxidative and nonoxidative glucose disposal and muscle glycogen synthesis, but it did not result in accumulation of free intracellular glucose, glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, or lactate in muscle. These data imply that the rate-limiting defect for glucose disposal in skeletal muscle of type I diabetic patients is at the level of glucose transport.     

Serum proinsulin in children and adolescents with chemical diabetes.

Proinsulin levels (PLC) in serum were determined after gel filtration on specimens obtained during oral glucose tolerance testing (OGTT) in seven patients with repeated abnormality in OGTT and in a group of seven control subjects matched for age. Fasting and thirty-, sixty-, and 120-minute postglucose venous samples were analyzed for glucose (PG) and immunocreactive insulin (IRI) as well as PLC, PG and IRI mean concentrations were greater at all testing times in the patient population, but the mean IRI/PG was significantly higher in the patients only at fasting. PLC mean levels were higher in the patients but not to a level of significance. Percentage of total IRI atributable to PLC at each time point was identical between the two groups. The apparcent diminished effectiveness of circulating insulin in chemical diabetes cannot be attributed to an abnormal proportion of proinsulin.     

Impaired insulin biosynthetic capacity in a rat model for non-insulin-dependent diabetes. Studies with dexamethasone

These studies of a rat model for non-insulin-dependent diabetes mellitus (NIDDM) were performed to determine whether hyperglycemia occurs when capacity to synthesize insulin is exceeded. The neonatal streptozocin (STZ)-treated rat has acute hyperglycemia with marked destruction of pancreatic beta-cells, followed by gradual regeneration to 50-70% normal beta-cell number. At age 4 wk, fed serum glucose concentration is only mildly elevated relative to controls. With age, the rats become progressively hyperglycemic, and by 12 wk they have marked impairment of glucose-stimulated insulin release. In these studies, dexamethasone (0.125 mg/kg/day for 4 days) was administered to control and to STZ-treated animals to produce insulin resistance. The relationship between insulin biosynthesis and serum glucose concentrations was assessed. In control rats, response to dexamethasone was similar at both 4 and 12 wk. Serum glucose levels and pancreatic insulin concentration remained unchanged. Both insulin biosynthetic rates (as measured by 3H-leucine incorporation into proinsulin) and proinsulin mRNA levels increased twofold. STZ-treated rats at age 4 wk demonstrated mild hyperglycemia. Dexamethasone injection resulted in an increase in insulin biosynthesis and proinsulin mRNA in these animals, while serum glucose did not increase. STZ-treated rats at 12 wk showed more profound hyperglycemia (serum glucose 315 +/- 38 mg/dl versus control, 187 +/- 12 mg/dl). A marked rise in serum glucose (to 519 +/- 42 mg/dl) was observed after 4 days of dexamethasone injection. Pancreatic insulin content became severely depleted relative to saline-injected, STZ-treated animals, and there was no response of levels of proinsulin mRNA.     

The second activating glucokinase mutation (A456V): implications for glucose homeostasis and diabetes therapy

In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m(2), and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S(0.5) (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant.     

Participation of glucokinase inactivation in inhibition of glucose-induced insulin secretion by 2-cyclohexen-1-one

We assessed our speculation that 2-cyclohexen-1-one (CHX) impairs glucose-induced insulin secretion through inactivation of glucokinase. Treatment of pancreatic islets with CHX at concentrations (0-5 mM) that caused a dose-dependent inactivation of glucokinase activity similarly inhibited glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets was little affected by CHX. CHX-induced inactivation of glucokinase was blocked by the presence of its substrates (glucose and mannose) and an inhibitor (N-acetylglucosamine), all of which also protected against the inhibitory effect of the drug on glucose-induced insulin secretion. CHX also impaired insulin secretion induced by D-glyceraldehyde and dimethyl succinate, which are believed to stimulate the release of the hormone by being directly oxidized by glyceraldehyde-3-phosphate dehydrogenase, by entering the midstream of the glycolytic pathway as glyceraldehyde 3-phosphate, or by entering the tricarboxylic acid cycle in mitochondria after intracellular hydrolysis. The inhibitory effect of CHX on glucose-induced insulin secretion, however, was far more marked than that on insulin secretion evoked by D-glyceraldehyde and dimethyl succinate at any CHX concentrations used. Our study revealed that the inhibitory action of CHX on glucose-induced insulin secretion is exerted mainly, but not solely, through inactivation of glucokinase. This conclusion supports the view that glucokinase is a key enzyme in the recognition of glucose as an insulin secretagogue in pancreatic islets.     

Succinate is a preferential metabolic stimulus-coupling signal for glucose-induced proinsulin biosynthesis translation

The secondary signals emanating from increased glucose metabolism, which lead to specific increases in proinsulin biosynthesis translation, remain elusive. It is known that signals for glucose-stimulated insulin secretion and proinsulin biosynthesis diverge downstream of glycolysis. Consequently, the mitochondrial products ATP, Krebs cycle intermediates, glutamate, and acetoacetate were investigated as candidate stimulus-coupling signals specific for glucose-induced proinsulin biosynthesis in rat islets. Decreasing ATP levels by oxidative phosphorylation inhibitors showed comparable effects on proinsulin biosynthesis and total protein synthesis. Although it is a cofactor, ATP is unlikely to be a metabolic stimulus-coupling signal specific for glucose-induced proinsulin biosynthesis. Neither glutamic acid methyl ester nor acetoacetic acid methyl ester showed a specific effect on glucose-stimulated proinsulin biosynthesis. Interestingly, among Krebs cycle intermediates, only succinic acid monomethyl ester specifically stimulated proinsulin biosynthesis. Malonic acid methyl ester, an inhibitor of succinate dehydrogenase, also specifically increased glucose-induced proinsulin biosynthesis without affecting islet ATP levels or insulin secretion. Glucose caused a 40% increase in islet intracellular succinate levels, but malonic acid methyl ester showed no further effect, probably due to efficient conversion of succinate to succinyl-CoA. In this regard, a GTP-dependent succinyl-CoA synthetase activity was found in cytosolic fractions of pancreatic islets. Thus, succinate and/or succinyl-CoA appear to be preferential metabolic stimulus-coupling factors for glucose-induced proinsulin biosynthesis translation.     

Impaired insulin receptor binding and postbinding defects of adipocytes from normal and diabetic pregnant women

To evaluate the relative contribution of insulin binding and postbinding defects of glucose utilization in peripheral tissue during normal and diabetic pregnancy, we have studied the in vitro insulin action of isolated adipocytes from eight nondiabetic pregnant women and nine pregnant women with insulin-dependent diabetes mellitus who were undergoing cesarian section. The pregnant women were compared with a matched group of normal nonpregnant women undergoing gynecologic surgery. Insulin binding to adipocytes measured at tracer insulin concentration was reduced by 45% (P less than 0.01) in normal pregnant women and by 30% (P less than 0.02) in pregnant women with diabetes. In contrast, no changes were found between the three groups in insulin binding to pure monocytes and erythrocytes. The glucose transport system in fat cells from both groups of pregnant women was characterized by impaired maximal (P less than 0.05) and half-maximal (P less than 0.05) response to insulin. When fat cell glucose metabolism was studied, pregnant diabetic women exhibited decreased basal lipogenesis (P less than 0.05) and decreased maximal responses of lipogenesis and glucose oxidation to insulin stimulation (P less than 0.05). Similar but less pronounced abnormalities were seen in glucose metabolism of adipocytes from nondiabetic pregnant women. In conclusion, both in late normal and diabetic pregnancy, insulin binding to adipocytes is significantly reduced and accompanied by decreased insulin sensitivity and reduced maximal insulin responsiveness of glucose transport and by impaired basal and maximally insulin-stimulated glucose metabolism.     

Stimulation by proinsulin of expression of plasminogen activator inhibitor type-I in endothelial cells.

In patients with non-insulin-dependent diabetes mellitus, concentrations in plasma of insulin and its precursors, proinsulin and split proinsulin, are increased. Because increased concentrations of plasminogen activator inhibitor type-1 (PAI-1) occur also, we hypothesized that proinsulin and split proinsulin may augment endothelial cell PAI-1 expression, thereby potentially attenuating endogenous fibrinolysis and accelerating atherosclerosis. Proinsulin increased PAI-1 activity in conditioned media of endothelial cells as did split proinsulin, paralleled by increased expression of PAI-1 mRNA. These effects of proinsulin were not dependent on its conversion to insulin nor on its interactions with the insulin receptor. The proinsulin stimulation of PAI-1 expression was not attenuated by either anti-insulin receptor antibodies or a 100-fold excess of insulin. Furthermore, proinsulin-mediated increases in PAI-1 expression were not inhibited by a 500-fold excess of insulinlike growth factor I. In addition, inhibition of tyrosine kinase, which mediates many of the diverse effects of insulin and insulinlike growth factor I, did not attenuate the effect of proinsulin. These results indicate that proinsulin augments PAI-1 expression, potentially contributing to vasculopathy in patients with non-insulin-dependent diabetes mellitus.     

Postbinding defects of insulin action in human adipocytes from uremic patients

It is now well established that longstanding human uremia is associated with impaired in vivo insulin action on glucose utilization of peripheral target tissues. In an attempt to define the cellular basis of the uremic insulin resistance we studied insulin action in adipocytes from eight patients with undialyzed chronic uremia and from eight matched healthy controls. (125I)-Insulin binding to fat cells from uremic patients was normal. In contrast (14C)-D-glucose transport exhibited decreased sensitivity to insulin. The concentrations of insulin that elicited half-maximal response was 422 +/- 95 pmoles/liter in uremic patients and 179 +/- 38 pmoles/liter in normal subjects (P less than 0.01). The noninsulin- and the maximal insulin-stimulated glucose transport of adipocytes from uremic patients with normal. (14C)-D-glucose conversion to total lipids was also measured in these cells in the absence and presence of various insulin concentrations. Similar to the findings in transport studies the lipogenesis of fat cells from uremic patients had depressed sensitivity to insulin (half-maximal stimulation at 38 +/- 8 pmoles/liter in uremic patients and at 11 +/- 3 pmoles/liter in normal subjects, P less than 0.01) with unchanged noninsulin and maximal insulin-stimulated lipogenesis. Taken together these results suggest that the insulin resistance of adipocytes from patients with chronic uremia may be accounted for primarily by postbinding defects localized to glucose transport and metabolism.     

Insulin biosynthesis in HIT cells. Effects of glucose, forskolin, IBMX, and dexamethasone

Glucose, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were tested as regulators of proinsulin biosynthesis in HIT T-15 cells, which are glucose-responsive simian virus 40-transformed hamster beta-cells. Rate of [3H]leucine incorporation into proinsulin was increased as glucose concentrations were raised from 0 to 20 mM. Biosynthetic rate increases were significant after 48 but not at 4 or 24 h of glucose and were greater for proinsulin than for total extractable proteins. After 48 h, glucose-stimulated proinsulin biosynthesis was unaffected by 10(-6) M forskolin and/or 3 x 10(-5) M IBMX but was specifically and significantly inhibited by 10(-6) M dexamethasone. Four hours of exposure to dexamethasone had no effect. When cells were incubated for 24 h and then continuously labeled for an additional 24 h, cellular conversion of labeled proinsulin to insulin was increased by glucose, and this increase was reversed or inhibited by 10(-6) M dexamethasone. Therefore, proinsulin biosynthesis in transformed HIT T-15 cells is regulated in several ways by metabolites and hormones in a manner that compares with biosynthetic regulation in normal beta-cells.