Biomedical subjects

M K Sinha

Publications and source records attributed to M K Sinha.

At least 55 records · Page 3Linked to original sources

PubMed1988

Insulin-like growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver.

Insulin-like growth factor-I (IGF-I) in human hepatoma cells (HEP-G2) has, in addition to its effect on cell growth, short-term metabolic effects acting through its own receptor. We have demonstrated that normal human hepatocytes, compared with HEP-G2 cells, have virtually no IGF-I binding sites. Because the rate of growth is the major difference between the hepatoma and the normal liver, we asked if normal liver might express IGF-I binding sites under physiologic growth conditions. Indeed, whereas adult rat hepatocytes have low IGF-I binding sites similar to those in human liver, hepatocytes from regenerating liver after 3 d subtotal hepatectomy have an approximately sixfold increase (P less than 0.005) and those from fetal rat liver a approximately 12-fold increase (P less than 0.005), to levels comparable to those in the HEP-G2 cells. The specificity of 125I IGF-I binding to its receptor was demonstrated by competition studies with monoclonal antibodies directed toward the IGF-I and the insulin receptors, with unlabeled IGF-I and insulin and by affinity labeling experiments. Thus, if IGF-I has any short-term metabolic functions in the adult human liver, it is not through interaction with its own receptor. Autocrine regulation by IGF-I of liver growth appears possible since IGF-I binding sites are expressed under pathological and physiological conditions of growth. The mechanism that couples these two phenomena remains to be elucidated.

Animals↗

PubMed1987

Insulin stimulated protein phosphorylation in human plasma liver membranes: detection of endogenous or plasma membrane associated substrates for insulin receptor kinase.

The present work discloses a procedure for preparation of human liver plasma membranes containing catalytically competent insulin receptor kinase. In addition to insulin promoted phosphorylation of the beta-subunit of insulin receptor kinase, insulin promoted phosphorylation of pp 120 and two other new proteins was demonstrated. The new proteins with molecular weights of 50,000 and 120,000 do not bind to WGA, pp 120 antibody or insulin receptor antibody, but bind to the antiphosphotyrosyl antibody. The identity and physiological significance of these putative substrates for insulin receptor kinase remains to be established.

Cell Membrane↗

PubMed1987

Effect of chronic uremia on fructose 2,6-bisphosphate glycolytic and gluconeogenic enzymes in rat liver.

The level of fructose 2,6-bisphosphate and the maximal activities of key gluconeogenic and glycolytic enzymes were determined in the liver of a rat model of chronic uremia and in ad libitum-fed control and pair-fed control animals. Fructose 2,6-bisphosphate was decreased in uremia and its level negatively correlated with the concentration of blood urea nitrogen. The changes in gluconeogenic enzymes in uremic rats were not different from those in the pair-fed controls. However, pyruvate kinase was decreased in uremia when compared to both controls. These studies offer a possible mechanism for the role of the liver in the carbohydrate intolerance of uremia.

Animals↗

PubMed1987

Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

Adipose Tissue↗

PubMed1987

Insulin receptor binding and protein kinase activity in muscles of trained rats.

Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, we postulated that exercise training would change insulin receptors in muscle and in this study we have investigated this hypothesis. Female rats initially weighing approximately 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise. Insulin binding by the partially purified receptor preparation s was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.

Animals↗

PubMed1987

Insulin receptor kinase in human skeletal muscle from obese subjects with and without noninsulin dependent diabetes.

We have studied the structure and function of the insulin receptors in obese patients with and without noninsulin dependent diabetes mellitus (NIDDM) and in nonobese controls using partially purified receptors from muscle biopsies. Insulin binding was decreased in obesity due to reduced number of binding sites but no differences were observed in insulin binding between obese subjects with or without NIDDM. The structural characteristics of the receptors, as determined by affinity labeling methods and electrophoretic mobility of the beta-subunit, were not altered in obese or NIDDM compared to normal weight subjects. Furthermore, the ability of insulin to stimulate the autophosphorylation of the beta-subunit and the phosphoamino acid composition of the phosphorylated receptor were the same in all groups. However, insulin receptor kinase activity was decreased in obesity using Glu4:Tyr1 as exogenous phosphoacceptor without any appreciable additional defect when obesity was associated with NIDDM. Thus, our data are supportive of the hypothesis that in muscle of obese humans, insulin resistance is partially due to decreased insulin receptors and insulin receptor kinase activity. In NIDDM the defect(s) in muscle is probably distal to the insulin receptor kinase.

Adult↗

PubMed1987

Long-term effect of insulin on glucose transport and insulin binding in cultured adipocytes from normal and obese humans with and without non-insulin-dependent diabetes.

We have tested the hypothesis that in vitro exposure of insulin-resistant adipocytes with insulin results in improved insulin action. A primary culture system of adipocytes from obese subjects with or without non-insulin-dependent diabetes mellitus (NIDDM) and nonobese control subjects has been developed. The adipocytes when cultured in serum-free medium do not lose their original characteristics in regard to insulin binding and glucose transport. The adipocytes from three groups were incubated with insulin (0, 10(-10) M, and 10(-7) M) for 24 h at 37 degrees C, receptor-bound insulin was dissociated, and basal and insulin (1 X 10(-11)-10(-7) M)-stimulated glucose transport and 125I-insulin binding were determined. The 24-h insulin exposure of adipocytes from control subjects decreased basal and insulin-stimulated glucose transport. The effects of 1 X 10(-7) M insulin were more pronounced than 1 X 10(-10) M insulin. Similarly, insulin exposure decreased insulin sensitivity and responsiveness of cultured adipocytes from obese and NIDDM patients. The insulin-induced reduction in insulin sensitivity and responsiveness for glucose transport in three groups were due to alterations at insulin binding and postbinding levels. In conclusion, insulin induces insulin resistance in control adipocytes and further worsens the insulin resistance of adipocytes from obese and NIDDM subjects. For insulin to improve the insulin resistance of adipocytes from NIDDM patients, either more prolonged in vitro insulin exposure and/or other hormonal factors might be required.

Adipose Tissue↗

PubMed1987

Subunit structure, autophosphorylation, and tyrosine-specific protein kinase activity of hepatic insulin receptors in fetal, neonatal, and adult rats.

The ontogeny of the structural and functional characteristics of insulin receptors is determined by examining insulin binding, subunit structure, autophosphorylation, and tyrosine-specific protein kinase activity in partially purified solubilized liver receptors from fetal (approximately 21 days postconception), neonatal (1- and 7-day-old), and adult rats. Specific 125I-labeled insulin binding to these receptor preparations in the presence of different insulin concentrations was higher in fetal and neonatal rats compared with that in the adult rats. The electrophoretic mobilities of the alpha- and beta-subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography were similar at different stages of development. Insulin-stimulated autophosphorylation of insulin receptors was similar in the different groups. With fixed amounts of protein, the tyrosine-specific protein kinase activity in the presence of different insulin concentrations (1 X 10(-8) to 1 X 10(-6) M) was significantly higher in the fetal and neonatal rats than in adult rats. However, when expressed as a function of insulin-binding activity, the insulin-stimulated tyrosine-specific protein kinase activity in fetal and neonatal rats appears to be similar to that in adult rats because of decreased insulin binding in the latter group. These results demonstrate the structural and functional similarities of hepatic insulin receptors in fetal, neonatal, and adult rats. The relative differences in insulin-mediated biological functions in fetal and adult rat livers as reported previously are due to alterations in a step(s) distal to activation of insulin-receptor kinase.

Animals↗

PubMed1987

Insulin-receptor kinase activity of adipose tissue from morbidly obese humans with and without NIDDM.

We have determined glucose transport, insulin binding, and insulin-receptor kinase activity in adipose tissue from morbidly obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM). The insulin sensitivity and responsiveness of glucose transport in freshly isolated adipocytes were significantly reduced in NIDDM subjects compared with nondiabetics. This was due in part to decreased insulin binding in adipocytes. Reduced specific 125I-labeled insulin binding was also observed in crude detergent extracts and partially purified insulin receptors from adipose tissue. In addition, the basal and insulin-stimulated tyrosine-specific protein kinase activity per milligram of protein was significantly decreased in NIDDM patients compared with nondiabetics. The differences between maximally insulin-stimulated and basal kinase activities expressed by insulin-binding activity were also significantly reduced in NIDDM subjects. We conclude that insulin resistance in morbidly obese patients with NIDDM is due to both insulin-binding and postbinding defects. One of the postbinding defects in NIDDM appears to be impaired insulin-receptor kinase activity of fat tissue.

Adipose Tissue↗

PubMed1986

Insulin resistance in uremia: in vitro model in the rat liver using human serum to study mechanisms.

We have previously demonstrated in a rat model of chronic uremia that the liver is resistant to insulin. To further investigate the mechanism(s) of insulin resistance in uremia, primary cultures of normal rat hepatocytes were incubated with varying dilutions (1/10 to 1/10,000) of sera from undialyzed end stage uremic and normal humans for 20 hours. We then studied insulin action, binding, and postbinding events. Dilutions of uremic sera as low as 1/1,000 rendered the hepatocytes resistant to maximal concentrations of insulin with regard to [14C]acetate incorporation into lipids. The dose response curve for insulin-stimulated [14C]aminoisobutyric acid uptake demonstrated a shift to the right in hepatocytes incubated with uremic sera when compared with those incubated with normal sera. The 125I-insulin binding sites and affinity, 125I-insulin internalization and degradation, insulin receptor structure, autophosphorylation of the insulin receptor, and its tyrosine-specific kinase activity were normal in the hepatocytes rendered resistant to insulin by uremic sera. However, these cells failed to generate the chemical mediator or second messenger of insulin action, as assessed by its ability to stimulate pyruvate dehydrogenase (PDH) in liver mitochondria from normal rats. We concluded that uremic sera renders normal rat hepatocytes resistant to insulin. Insulin resistance is a postinsulin receptor kinase defect possibly due to lack of the generation of the chemical mediator of insulin action. This in vitro cell model may be useful to further define the mechanism(s) and the serum factor(s) responsible for insulin resistance in uremia in the absence of complicating hormonal and substrate changes that occur in vivo.

Adult↗

PubMed1986

Studies on the mechanism of insulin resistance in the liver from humans with noninsulin-dependent diabetes. Insulin action and binding in isolated hepatocytes, insulin receptor structure, and kinase activity.

We have developed a method to isolate insulin-responsive human hepatocytes from an intraoperative liver biopsy to study insulin action and resistance in man. Hepatocytes from obese patients with noninsulin-dependent diabetes were resistant to maximal insulin concentration, and those from obese controls to submaximal insulin concentration in comparison to nonobese controls. Insulin binding per cell number was similar in all groups. However, insulin binding per surface area was decreased in the two obese groups because their hepatocytes were larger. In addition, the pool of detergent-extractable receptor was further decreased in diabetics. Insulin receptors in all groups were unaltered as determined by affinity-labeling methods. However, insulin-stimulated insulin receptor kinase activity was decreased in diabetics. Thus, in obesity, decreased surface binding could explain resistance to submaximal insulin concentrations. In diabetes, diminished insulin-stimulated protein kinase activity and decreased intracellular pool of receptors could provide an explanation for postinsulin-binding defect(s) of insulin action in human liver.

Biopsy↗

PubMed1986

Insulin stimulates amino acid and lipid metabolism in isolated fetal rat hepatocytes.

Although fetal hyperinsulinemia is associated with excessive deposition of glycogen in liver, both in vivo and in vitro studies show little effect of insulin on glycogen synthesis from glucose or the activity of the enzyme glycogen synthase in the fetus. To investigate whether lack of insulin effect extends to other fetal metabolic processes, we compared the influence of insulin on amino acid uptake (14C-alpha-aminoisobutyric acid) and lipid synthesis [14C-acetate) in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of F and A hepatocytes was documented by trypan blue exclusion (greater than 90%). In A, insulin stimulated 14C-alpha-aminoisobutyric acid uptake in a dose dependent manner with an apparent Km at 2 ng/ml and a Vmax at 10 ng/ml. When corrected for cell surface area, F cells responded to insulin in a similar dose response manner, although absolute values per 1 X 10(6) cells always remained lower. In contrast, whereas A cells demonstrated a typical dose dependent response of 14C-acetate incorporation into lipid with a Km at 5 ng/ml and Vmax at 10 ng/ml of insulin, F cells remained totally unresponsive when the concentration of acetate was 5 mM or less. However, at higher medium acetate concentrations (15-30 mM) fetal responses were equal to or greater than that of adult, both basally and with insulin. These findings suggest differences in the maturation of insulin-mediated processes in fetal rat hepatocytes; effects on amino acid uptake appear earlier than those on lipid or glycogen synthesis.

Amino Acids↗

PubMed1985

Presence of an activator of pyruvate dehydrogenase in human circulation: elevation following a glucose load and possible relation to an insulin mediator.

Our earlier observation that the chemical mediator of insulin action stimulates lipid synthesis in primary cultures of rat hepatocytes prompted us to examine its presence in human serum and its regulation by changes in insulin levels. Serum samples were obtained from normal subjects following an oral 100 gm glucose tolerance test (GTT; n = 10). An acid soluble, heat stable and charcoal non-absorbable substance was extracted from different sera and tested for their ability to stimulate liver mitochondrial pyruvate dehydrogenase (PDH). This substance obtained from GTT samples at 1/1000 final dilution caused significantly higher stimulation of PDH when compared to that obtained from fasting samples. These results demonstrate the presence of an activator of PDH (molecular weight approximately 1000-2000) in human circulation. Since the activator of PDH is modulated by physiological perturbation such as oral glucose ingestion, known to cause changes in circulating insulin levels, it may possibly be related to insulin mediator.

Adolescent↗

PubMed1984

Is glycosylation in the liver needed for insulin binding, processing, and action? Evidence for heterogeneity.

Incubation of primary cultures of rat hepatocytes with tunicamycin (1 microgram/ml) for 16 h inhibits de novo glycosylation by more than 90%, yet 50% of initial 125I-insulin binding is still present. The remaining insulin-binding activity is to glycosylated receptors, since they are adsorbed and specifically desorbed from wheat germ agglutinin-Sepharose. Furthermore, there is no difference in the molecular weight size of the alpha-subunit of the insulin receptors either in normal or tunicamycin-treated hepatocytes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin receptor cross-linked with disuccinimidyl suberate. This gives us an opportunity to study the function of these glycosylated insulin receptors in cells where de novo glycosylation is inhibited. Inhibition of de novo glycosylation decreases receptor-mediated 125I-insulin internalization and degradation. The tunicamycin-treated cells are totally resistant to insulin with regard to [14C]aminoisobutyric acid uptake. In contrast, there is normal stimulation of [14C] acetate and [14C]glucose incorporation into lipids and glycogen by a maximal concentration of insulin. The interpretation of these data is, however, complicated by the fact that tunicamycin markedly inhibits basal lipid synthesis without altering basal glycogen synthesis or [14C]aminoisobutyric uptake. These results demonstrate the heterogeneity of the postinsulin binding system in the liver. Besides glycosylation of the insulin receptor, which is needed for insulin binding, other cellular glycoproteins are important for insulin processing as well as mediation of some, but not all, of the biological actions of insulin. The nature of these liver glycoproteins and their relationship to the insulin receptor have not yet been defined.

Animals↗

PubMed1984

Modulation of hepatic protein kinase activity by indomethacin.

We previously demonstrated that treatment with indomethacin in vivo significantly blunted the glucagon-induced glycemic response in the rat. This prostaglandin synthetase (cyclo-oxygenase) inhibitor also accentuated the evanescent effect of glucagon on hepatic glucose output in the intact, anesthetized rat. In this report, we present evidence that impairment of glucagon action in the rat liver by indomethacin is mediated through its inhibitory effect on both cAMP-dependent and cAMP-independent hepatic protein kinase. Indomethacin treatment did not have a measurable effect on any of the other components of the glucagon transducer system. Furthermore, infusion with glucagon for two hours that maintained plasma glucagon values at high physiological levels significantly reduced hepatic cAMP-dependent protein kinase activity without altering its Km. Glucagon infusion also down-regulated its own hepatic receptors and glucagon-stimulated cAMP production; prostaglandin E1-stimulated cAMP production was not affected. We concluded that prostaglandins may play a role in the regulation of hepatic protein kinases involved in the glucagon-stimulated glycogenolytic response and that glucagon-induced down-regulation extends at least to the hepatic protein kinases. However, a direct effect of indomethacin or protein kinase and the adenylate cyclase complex cannot be ruled out.

Adenylyl Cyclases↗

PubMed1984

Possible dissociation between insulin binding and insulin action in isolated fetal rat hepatocytes.

To directly examine the relationship between insulin receptors and insulin action in fetal tissue, we compared insulin receptor characteristics and insulin-mediated 14C-glucose incorporation into glycogen, as well as glycogen synthase activity, in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of hepatocytes was documented by trypan blue exclusion (greater than 90%), time-dependent 14C-leucine incorporation into protein, and dose-related incorporation of glucose into glycogen. Percent specific binding of 125I-insulin per unit protein was significantly higher in F than A liver plasma membranes (32.2 +/- 0.3 versus 18 +/- 2.4; P less than 0.01) and Scatchard plots revealed twice the number of receptors in F. Similarly, receptor number per cell surface area was threefold higher in F than in A (150 versus 50 sites/micron2). At a fixed medium glucose concentration of 11.2 mM, insulin stimulated 14C-glucose incorporation into glycogen in a dose-related manner in A with an apparent Km of 1.0 ng/ml and Vmax at 5-10 ng/ml corresponding to 30-40% of total receptor occupancy; no effect was obtained in F with insulin up to 100 ng/ml. Net glucose incorporation into glycogen (nmol/10(6) cells/h) increased progressively with increasing medium glucose concentrations ranging from 1.4 to 27.8 mM; incorporation by F was significantly greater than by A at each glucose concentration. However, whereas insulin at 100 ng/ml significantly augmented net glucose incorporation at each glucose concentration in A, no effect of insulin was apparent in F.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1983

Chemical mediator of insulin action stimulates lipid synthesis and down regulates the insulin receptors in primary cultures of rat hepatocytes.

Incubation of rat liver particulate fraction with insulin causes the release of a substance that stimulates lipid synthesis and down regulates the insulin receptor in primary cultures of isolated rat hepatocytes. This substance may be similar to putative mediator(s) of insulin action which has been shown to modulate the activity of key enzymes of lipid and carbohydrate metabolism in various cell free systems. Our data demonstrate that the mediator of insulin is also biologically active in an intact cell system. Down regulation of the insulin receptor by the mediator supports the concept that this phenomenon is a post binding event of insulin action.

Animals↗

PubMed1983

Ontogeny of hepatic insulin and glucagon receptors and adenylate cyclase in rabbit.

In rabbit liver plasma membranes (LPM), specific binding of 125I-insulin rapidly increased in late gestation and peaked at birth, declining thereafter. In contrast, 125I-glucagon binding was lowest in late gestation, somewhat higher at birth, and increased by 48 h although only to 20-25% of adult. These changes in binding were due to changing numbers of receptors involving predominantly high affinity sites for insulin and low affinity sites for glucagon, with only minor changes in affinity. Despite measurable glucagon receptors by birth, fetal LPM produced no increment above basal in cAMP production with maximal doses of glucagon (10(-6) M), prostaglandin E1 (10(-4) M), or epinephrine (10(-4) M). Near birth only NaF (10 mM) produced a modest but significant increment in cAMP. By 2 h postbirth, all stimuli evoked significant increments in cAMP production that increased progressively but was still only 15-20% of adult at 48 h. Furthermore, although specific binding of cholera toxin was greater in fetal LPM (11 +/- 1 vs. 6 +/- 1%), cholera toxin-stimulated cAMP production increased by only 12-26% above basal in the fetus compared with 220% in adult. Markers of membrane purity including 5'-nucleotidase, phosphodiesterase, and insulin or glucagon degradation were not different in fetus and adult. We conclude that receptors and components of the adenylate cyclase complex mature independently; initial coupling occurs between the G/F regulatory protein and the catalytic unit (NaF but not hormonal activation) followed within hours of birth by coupling to the hormone receptor.

Adenylyl Cyclases↗