Insulin-induced Changes Research Articles

Leptin response to insulin in humans is related to the lipolytic state of abdominal subcutaneous fat.

Insulin-induced leptinemia in humans appears to be blunted by insulin resistance. We therefore examined the relationship between insulin action and plasma leptin by monitoring regional and whole body lipolysis and plasma leptin levels in 15 premenopausal women (body fat range, 14-59%) during a two-stage euglycemic clamp (insulin was infused 90 min each at 6-10 and 12-20 mU/m2 x min). Microdialysis probes were placed in abdominal and femoral sc adipose tissue. Subjects were given a primed, constant infusion of a stable isotope tracer (2H5-glycerol), and plasma glycerol isotope enrichments were analyzed by mass spectrometry to determine glycerol kinetics. Although there was no mean change in plasma leptin during the clamp (baseline, 16.6 +/- 4.5 ng/mL; final, 16.3 +/- 4.3 ng/mL), there was large interindividual variability in the changes in plasma leptin (range, -18% to +19%). Changes in plasma leptin during the clamp stages were correlated with abdominal dialysate glycerol concentrations (r = -0.44; P < 0.05), but not femoral dialysate glycerol concentrations (r = -0.15), the rate of appearance of glycerol in plasma (r = 0.005), or plasma insulin levels (r = 0.16). The results suggest that insulin-induced changes in plasma leptin are more related to the lipolytic state (i.e. low leptin response when lipolysis is high) of abdominal sc adipose tissue than that of other fat depots.

Insulin does not change the intracellular distribution of hexokinase in rat heart.

Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.

Relationship between insulin's haemodynamic effects and insulin-mediated glucose uptake.

Insulin resistance and hyperinsulinaemia are associated with hypertension. The relationship between insulin's metabolic and haemodynamic actions has not been fully elucidated however. We investigated, using the euglycaemic clamp technique, the relationship between insulin-mediated glucose uptake and insulin-induced changes in leg blood flow and cardiac index in 13 healthy subjects. As insulin's effects on blood flow are time dependent, studies were performed during a 4-h insulin (50 mU kg-1 h-1) infusion period. Mean arterial pressure during insulin infusion increased (82.9 +/- 6.7 to 89.8 +/- 7.7 mmHg; P < 0.001), whereas heart rate was unaltered. Leg blood flow gradually increased from 1.09 +/- 0.57 to 1.47 +/- 0.67 mL min-1 dL-1 during the second hour, and to 1.65 +/- 0.68 mL min-1 dL-1 during the fourth hour of the clamp (P = 0.01). Stroke volume index increased from 56.5 +/- 13.3 to 63.7 +/- 16.3 mL m-2 (P = 0.004) and cardiac index from 3.42 +/- 1.02 to 3.73 +/- 1.05 L min-1 m-2 (P = 0.04). The insulin-mediated glucose disposal and the increases in leg blood flow were not correlated during the second hour (r = 0.21, P = 0.51) but showed a strong correlation during the fourth hour of the clamp (r = 0.88, P < 0.001). Insulin-mediated glucose disposal was not correlated with the increases in cardiac index. Thus, insulin-mediated muscle blood flow may be an important contributor to glucose uptake during sustained exogenous hyperinsulinaemia aiming at physiological insulin levels.

Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts.

The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal protein S6 kinase (p70S6k), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimulation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortmannin), p70S6k (rapamycin), and mitogen-activated protein kinase (MAP kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70S6k, an effect that was blocked by rapamycin and wortmannin but not PD-98059. Insulin caused dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70S6k pathway. Insulin also stimulated phosphorylation of 4E-BP1 as well as dissociation of the 4E-BP1.eIF-4E complex. Both rapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G.eIF-4E complex, an effect that was not prevented by any of the inhibitors. Overall, the results suggest that insulin stimulates protein synthesis in L6 myoblasts in part through utilization of both the p70S6k and MAP kinase signal transduction pathways.

Chronic treatment with BRL 35135 potentiates the action of insulin on lipid metabolism

The effects of a β 3-adrenoceptor agonist on insulin-induced changes in lipid metabolism were studied in obese male Zucker ( fa/ fa) rats during euglycaemic clamp. Rats were treated with BRL 35135 ( R *, R *-(±)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)-ethyl-amino]-propyl]-phenoxyacetate hydrobromide) (0.5 mg/kg/day in drinking water) for three weeks before an euglycaemic hyperinsulinaemic clamp was performed. Insulin infusion lowered serum non-esterified fatty acids and plasma glycerol more efficiently in BRL 35135-treated than in control rats although plasma insulin remained significantly lower in the BRL 35135-treated than in the control rats during the clamp. In conclusion, chronic treatment with BRL 35135 potentiates the effect of insulin on lipid metabolism.

Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozocin diabetic rats.

Chronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier-mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats.

Insulin induces rapid and specific rearrangement of the cytoskeleton of rat mesangial cells in vitro.

Mesangial cells (MCs) grown without supplemental insulin (SI-MCs) express a quiescent phenotype and extracellular matrix (ECM) composition similar to MCs in vivo. In contrast, MCs routinely propagated in insulin (SI+MCs) are stimulated to proliferate, change their phenotype, and produce large amounts of collagens I and III. These effects of insulin may in part be mediated through cytoskeletal rearrangement. Differences in cytoskeletal arrangement were compared between SI-MCs and SI+MCs and 1 hr after addition of insulin (1 nM) or IGF-1 (100 nM) to SI-MCs. Cells were examined by light microscopy, electron microscopy, and immunostaining for specific cytoskeletal proteins and fibronectin. Insulin induced rapid rearrangement of stress fibers. Surface ruffling, actin aggregation, vimentin retraction, rearrangement of vinculin in focal adhesions, and fibronectin extraction were apparent. These direct effects of insulin on the SI-MC cytoskeleton occurred before insulin-induced changes in ECM composition. IGF-I induced cytoskeletal reorganization distinct from insulin. These observations demonstrate that insulin and IGF-I have unique effects on the MC cytoskeleton, which is turn may mediate secondary ligand effects on MCs.

The Effects of Brefeldin A on the Glucose Transport System in Rat Adipocytes: IMPLICATIONS REGARDING THE INTRACELLULAR LOCUS Of INSULIN-SENSITIVE Glut4

Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane (PM). To localize intracellular translocating Glut4, we studied the effects of brefeldin A (BFA), which disassembles Golgi and prevents trans-Golgi vesicular budding, on the glucose transport system. Isolated rat adipocytes were treated with and without both BFA (10 micrograms/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methyl-glucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by approximately 2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and high density microsome subfractions. In basal cells, BFA increased PM Glut4 by 58% concomitant with a 18% decrease in LDM (p < 0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a 56% decrease in LDM. BFA did not affect insulin-induced changes in Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut4 to PM. In summary, 1) in basal cells, BFA led to a small increase in glucose transport activity and redistribution of a limited number of transporters from LDM to PM; 2) BFA did not affect insulin's ability to stimulate glucose transport or recruit normal numbers of LDM Glut4 to PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes does not involve trans-Golgi.

Mechanisms of Increased Skeletal Muscle Glucose Transport Activity After an Oral Glucose Load in Rats

It is not known whether the insulin-induced changes in the skeletal muscle glucose transport system occur under physiological circumstances. To clarify whether, by which mechanisms, and for how long skeletal muscle glucose transport activity is increased after an oral glucose load (OGL), we prepared plasma membrane (PM) and microsomal membrane (MsM) vesicles from hindlimb muscles of Sprague-Dawley rats either in the fasting state or 30, 60, 90, or 120 min after an OGL (2 g/kg body wt). In both PM and MsM, we measured the total number of glucose transporters (Ro), GLUT4, and GLUT1. In the PM, we also determined glucose influx (Vmax) and carrier turnover number (TN), an index of average transporter intrinsic activity, (TN = Vmax/Ro). The Vmax significantly increased after OGL, was maximal at 30 min, and returned to baseline at 90 min. The Ro and GLUT4 in the PM also increased significantly, with the maximum level reached at 60 min. The TN was increased only at 30 min. The changes in Ro and GLUT4 in the MsM were opposite to those in the PM, consistent with translocation of GLUT4 from an intracellular pool to the PM. In conclusion, an OGL induces an increase in the skeletal muscle glucose transport activity. This increase is associated with the translocation of GLUT4 from the MsM to the PM and a more transient increase in the average transporter TN. Our results show that transporter translocation and activation occur under physiological circumstances.

Effect of insulin on uric acid excretion in humans.

Although hyperuricemia is a frequent finding in insulin-resistant states, insulin's effect on renal uric acid (UA) handling is not known. In 20 healthy volunteers, diastolic blood pressure, body weight, and fasting plasma insulin were positively (and age was negatively) related to fasting plasma UA concentrations, together accounting for 53% of their variability. During an insulin clamp, urine flow was lower than during fasting conditions (1.01 +/- 0.12 vs. 1.56 +/- 0.32 ml/min, P = 0.04), whereas creatinine clearance was unchanged (129 +/- 7 and 131 +/- 9 ml/min, P = not significant). Hyperinsulinemia did not alter serum UA concentrations (303 +/- 13 vs. 304 +/- 12 microM) but caused a significant decrease in urinary UA excretion [whether expressed as absolute excretion rate (1.66 +/- 0.21 vs. 2.12 +/- 0.23 mumol/min, P = 0.03), clearance rate (5.6 +/- 0.8 vs. 7.3 +/- 0.8 ml/min, P = 0.03), or fractional excretion (4.48 +/- 0.80 ml/min vs. 6.06 +/- 0.64%, P < 0.03)]. Hyperinsulinemia was also associated with a 30% (P < 0.001) fall in urine Na excretion. Fractional UA excretion was related to Na fractional excretion under basal conditions (r = 0.59, P < 0.01) and during the insulin period (r = 0.53, P < 0.02). Furthermore, the insulin-induced changes in fractional UA and Na excretion correlated with one another (r = 0.66, P < 0.001). Physiological hyperinsulinemia acutely reduces urinary UA and Na excretion in a coupled fashion.

Induction of nodular sclerosis by insulin in rat mesangial cells in vitro: Studies of collagen

These studies evaluated the contribution of insulin to the development of the abnormal mesangial matrix that characterizes diabetic nephropathy and is common to mesangial cells in culture. Glomeruli were isolated from a single rat and divided into two aliquots. In one set (SI-MC), the insulin contained in the medium was only that contributed by the fetal calf serum (20%). For the other set, the tissue culture medium was supplemented with 1 microM insulin (SI+MC). Mesangial cell outgrowths from each condition were isolated, cloned, and propagated. At passage 4, mesangial cells were characterized by morphology and cell markers, and compared in terms of composition and appearance of the secreted extracellular matrix. SI-MC grew in nests of cells surrounded by a thin layer of matrix that was rich in collagen IV. In contrast, mesangial cells supplemented with insulin aggregated into macroscopic "hillocks" rich in collagens I and III as described previously. Insulin (1 microM) or IGF-I (0.1 microM) was subsequently added to the medium of SI-MC. Insulin, but not IGF-I, induced a change in culture morphology and collagen accumulation characteristic of SI+MC. In contrast to SI+MC, SI-MC express insulin receptors and at physiologic concentrations insulin is a more potent stimulator of MC proliferation than is IGF-I. Insulin-induced changes in the collagenous composition of the accumulated ECM were directionally correlated with the rate of collagen I synthesis measured by biosynthetic labeling experiments and collagens III and IV as determined by ELISA. These data demonstrate that insulin alters the phenotype of mesangial cells in culture and their expression of interstitial and basement membrane collagens. These observations implicate insulin as a factor in the pathogenesis of mesangial matrix accumulation in diabetic nephropathy. Furthermore, a method for culturing mesangial cells that accumulate an extracellular matrix that is similar in composition to normal mesangial matrix provides a new model system for future studies of mesangial cell biology.

Insulin induces a change in extracellular matrix glycoproteins synthesized by rat mesangial cells in culture

Extracellular matrix (ECM) accumulation within the glomerular mesangium is a hallmark of progressive forms of renal disease. We recently succeeded in propagating mesangial cells (MC) from the time of explant without supplemental insulin which exhibit a matrix profile analogous to normal mesangium in vivo. We used these cells to characterize insulin-induced changes in biosynthesis and accumulation of three important matrix glycoproteins, laminin, fibronectin, and thrombospondin. Two clones of MC derived from glomeruli from a single rat were compared. MC grown in the absence of supplemental insulin (SI-MC) assemble a matrix rich in fibronectin with much smaller accumulations of laminin and thrombospondin. In comparison, MC (SI+MC) grown chronically in the presence of 1 microM insulin have a greatly expanded ECM that immunostains less intensely with antibodies to fibronectin, but, it contains significant accumulations of laminin and thrombospondin. Following metabolic labeling of secreted proteins with 35S-methionine, total protein synthesis was measured, and specific ECM components were identified and quantitated by immunoprecipitation, SDS-PAGE and autoradiography. The rate of total protein synthesis was increased by 50% in SI+MC as compared to SI-MC, yet, individual proteins were increased or decreased. The rate of synthesis of fibronectin was decreased and the rate of synthesis of laminin and thrombospondin was increased by insulin. These changes were directionally correlated with the net accumulation of these proteins as shown by immunostaining. In addition to an increase in laminin synthesis, insulin treatment induced a change in the isoform of laminin expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Poglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKA y mice. Treatment of KKA y mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2—5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulininduced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3–5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10μM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not greatly alter the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKA Y mice. Treatment of KKA Y mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated, using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2- to 5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3–5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 μM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not alter greatly the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Insulin regulation of protein phosphorylation in H4 hepatoma cells. Examination of rapid effects using two-dimensional electrophoresis

Activation of the insulin receptor tyrosine kinase is thought to initiate a chain of phosphorylation changes which regulate various metabolic and growth processes. Insulin has been shown to regulate some kinases by affecting their phosphorylation states; however, direct connections between activation of the receptor tyrosine kinase and subsequent altered phosphorylations of these kinases have yet to be established. To define the constituents of these regulated phosphorylation cascade systems, the time course of insulin-induced phosphorylation changes has been examined. 32P-Labeled H4IIE hepatoma cells were treated with insulin for 5-300 s, and then cell extracts were subjected to two-dimensional gel electrophoresis. Computer analysis of autoradiograms was employed to quantify insulin-induced phosphorylation changes. Phosphotyrosine was assessed by KOH digestion, by anti-phosphotyrosine immunoprecipitation, and by phenylarsine oxide treatment. Insulin induced a hierarchy of phosphorylation changes over the 300-s time course, with most increases being detectable within 10 s. Of these, only four did not undergo enhanced tyrosine phosphorylation. Eight proteins were found to undergo transient phosphorylation changes. Phenylarsine oxide elicited the appearance of novel low molecular weight phosphoproteins. These results support the concept of insulin-directed phosphorylation cascades, but indicate the magnitude of potential targets of the insulin receptor has been underestimated.

Cellular ionic effects of insulin in normal human erythrocytes: a nuclear magnetic resonance study

Elevated erythrocyte cytosolic free calcium, and suppressed free magnesium and pH values are associated with the hyperinsulinaemia and insulin resistance of hypertension, obesity, and Type 2 (non-insulin-dependent) diabetes mellitus. To determine the role of insulin in this process, we utilized 19F- and 31P-nuclear magnetic resonance spectroscopy to study the cellular ionic effects of insulin in vitro on normal human erythrocytes. Insulin elevated cytosolic free calcium levels in a dose- and time-dependent manner. The effect began at 10 microU/ml, peaked at 200 microU/ml, and continued at both the 500 microU/ml and 1000 microU/ml doses. At 200 microU/ml, free calcium levels rose from 24.6 +/- 2.5 nmol/l to a peak value at 120 min of 66.4 +/- 11 nmol/l (p < 0.05 vs basal), levels remaining elevated throughout the incubation (45.7 +/- 5.6 nmol/l at 60 min, and 47.9 +/- 9.1 nmol/l at 180 min, p < 0.05 vs basal, respectively). Similarly, insulin also increased intracellular free magnesium at all time points (basal: 177 +/- 11 mumol/l; 60 min: 209 +/- 19 mumol/l; 120 min: 206 +/- 22 mumol/l; and 180 min: 202 +/- 12 mumol/l; p < 0.05 vs basal at all times). No insulin-induced changes in pH were observed. We conclude: (i) that insulin in physiological concentrations may participate in regulating divalent cations in the mature human erythrocyte, (ii) that insulin per se cannot account for the previously described cellular ionic lesions of hypertension and diabetes, and (iii) that future clinical studies of cell ion metabolism should be conducted in the fasting state, be controlled for ambient circulating insulin levels, or both.

Time-dependent regulation of rat adipose tissue glucose transporter (GLUT4) mRNA and protein by insulin in streptozocin-diabetic and normal rats

Streptozocin (STZ) administration (125 mg/kg) to normal rats resulted in a rapid (24-hour) decrease in circulating insulin levels, marked hyperglycemia, and weight loss. Adipose tissue glucose transporter 4 (GLUT4) mRNA levels decreased approximately eightfold, whereas GLUT4 protein levels were unchanged. However, GLUT4 protein levels decreased approximately 30% by 48 hours and fivefold by 72 hours of insulin deficiency. Although GLUT4 mRNA levels were rapidly restored by insulin therapy (twofold above control levels within 12 hours), GLUT4 protein levels increased only gradually, reaching peak values of 1.5-fold control levels following 7 to 10 days of insulin treatment. Insulin treatment in normal rats increased adipose GLUT4 mRNA levels nearly 100% by 24 hours, while GLUT4 protein levels increased in a more gradual fashion. The delay in GLUT4 protein induction relative to its mRNA was shorter in normal rats treated with insulin than in insulin-treated diabetic rats. These data demonstrate that insulin-induced changes in adipose GLUT4 protein are considerably delayed relative to its mRNA, and that the diabetic state enhances this difference. The known in vivo time-dependent effects of insulin treatment on adipocyte glucose transport activity can be at least partly explained by altered specific expression of GLUT4 protein.

Chronic intrahypothalamic infusions of insulin or insulin antibodies alter body weight and food intake in the rat

In Experiment 1, one-week infusion of insulin (0.15, 1.5, or 15.0 μU/hr) into the ventromedial hypothalamus (VMH) of rats reduced body weight (BW) and nighttime food intake (FI). While 0.15 μU/h decreased daytime FI, 1.5 μU/h increased daytime FI and 15.0 μU/h left daytime FI unchanged. Total daily FI was decreased by the two highest doses. In Experiment 2, intra-VMH infusion of specific insulin antibodies (1.5 μUeq/h) increased BW and FI, while C-peptide antibodies were ineffective. In Experiment 3a, intracerebroventricular infusions of insulin failed to decrease FI and BW comparably to similar intrahypothalamic infusions. In Experiment 3b, intra-VMH insulin was infused via cannulae that bypassed the cerebral ventricles. The decrease in FI and BW was comparable to that observed when insulin was infused via cannulae that penetrated a ventricle. Histology from animals used in Experiments 1–3 indicates that optimum sites for insulin-induced changes in BW and FI in the hypothalamus lie in an area that includes portions of the paraventricular, arcuate, dorsomedial, and ventromedial nuclei.

No Evidence for Short-Term Regulation of Plasminogen Activator Inhibitor Activity by Insulin in Man

In cross-sectional studies a positive correlation has been found between circulating insulin, triglycerides and plasminogen activator inhibitor (PAI-1) activity. To directly examine the effect of insulin on PAI-1 activity in vivo, we determined the response of PAI-1 activity in 17 normal subjects to acute hyperinsulinemia (serum free insulin 92 +/- 8 mU/l) during maintenance of normoglycemia (plasma glucose 5.1 +/- 0.1 mmol/l). In 12 matched control subjects PAI-1 activity was measured during infusion of saline (serum free insulin 3.6 +/- 0.3 mU/l, plasma glucose 5.2 +/- 0.1 mmol/l). Plasma PAI-1 activity decreased during the insulin infusion from 9.0 +/- 1.4 to 5.6 +/- 0.8 U/ml (p less than 0.01), and during saline infusion from 7.0 +/- 1.4 to 4.3 +/- 0.6 U/ml (p less than 0.05). Serum triglyceride concentrations decreased from 1.09 +/- 0.20 to 0.76 +/- 0.09 mmol/l (p less than 0.001) during hyperinsulinemia but remained unchanged during the saline infusion (1.04 +/- 0.11 vs. 1.02 +/- 0.12 mmol/l, NS). We conclude that insulin does not acutely change plasma PAI-1 activity, and that acute insulin-induced changes in serum triglycerides occur independently from those of PAI-1 activity.

Effects of Insulin Deprivation and Replacement on In Vivo Subcutaneous Adipose Tissue Substrate Metabolism in Humans

The effects of insulin deprivation and replacement on adipose tissue metabolism were investigated in vivo with microdialysis in nine insulin-dependent diabetic patients with no residual insulin secretion. Dialysis probes, implanted in abdominal subcutaneous fat, were continuously perfused, and tissue dialysate concentrations of glycerol (lipolysis index), glucose, lactate, and pyruvate were determined. Comparisons were made with respective metabolite levels in venous plasma. After termination of intravenous insulin infusion, free insulin in plasma fell from 130 to 70 pM. At the same time, glucose levels in plasma and adipose tissue rose in parallel. However, the relative increase in glucose levels was greater in adipose tissue than in blood. On the other hand, the increase in glycerol concentration in adipose tissue (35%) was markedly less than that in venous plasma (250%). Lactate and pyruvate levels in adipose tissue and blood remained unchanged. After the resumption of intravenous insulin, free insulin in plasma rose to approximately 600 pM. At the same time, the glucose levels in blood and adipose tissue decreased rapidly, and the glycerol concentration in these tissues decreased to 50% of the baseline levels. The lactate and pyruvate levels in subcutaneous tissue increased briefly after insulin replacement, whereas the lactate but not pyruvate levels in blood showed a similar increase. The alpha- or beta-blocking agents phentolamine and propranolol in the ingoing tissue perfusate did not influence tissue glycerol at any time during the experiment. We concluded that insulin-induced changes in circulating metabolites only partly reflect variations in adipose tissue substrate kinetics. During insulin deprivation, glucose is accumulated in the adipose tissue extracellular compartment, probably because of reduced utilization by the adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)