Biomedical subjects

L Mosthaf

Publications and source records attributed to L Mosthaf.

At least 19 recordsLinked to original sources

PubMed1999

Nutritionally induced insulin resistance and receptor defect leading to beta-cell failure in animal models.

Animals with genetically or nutritionally induced insulin resistance and Type 2 diabetes comprise two groups: those with resilient beta-cells, e.g., ob/ob mice or fa/fa rats, capable of longstanding compensatory insulin hypersecretion and those with labile beta-cells in which the secretion pressure leads to beta-cell degranulation and apoptosis, e.g., db/db mice and Psammomys gerbils (sand rats). Psammomys features low insulin receptor density; on a relatively high energy diet it becomes hyperinsulinemic and hyperglycemic. In hyperinsulinemic clamp the hepatic glucose production is only partially suppressed by insulin, even in the normoglycemic state. The capacity of insulin to activate muscle and liver receptor tyrosine kinase is nearly abolished. GLUT4 content and mRNA are markedly reduced. Hyperinsulinemia was also demonstrated to inhibit insulin signaling and glucose transport in several other species. Among the factors affecting the insulin signaling pathway, phosphorylation of serine/threonine appears to be the prominent cause of receptor malfunction as inferred from the finding of overexpression of PKC epsilon isoforms in the muscle and liver of Psammomys. The insulin resistance syndrome progressing in animals with labile beta-cells to overt diabetes and beta-cell failure is a "thrifty gene" characteristic. This is probably also true for human populations emerging from food scarcity into nutritional affluence, inappropriate for their metabolic capacity. Thus, the nutritionally induced hyperinsulinemia, associated with PKC epsilon activation may be looked upon from the molecular point of view as "PKC epsilon overexpression syndrome."

Animals↗

PubMed1999

Insulin signaling is inhibited by micromolar concentrations of H(2)O(2). Evidence for a role of H(2)O(2) in tumor necrosis factor alpha-mediated insulin resistance.

Both hyperglycemia and tumor necrosis factor alpha (TNFalpha) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H(2)O(2) dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 microM H(2)O(2) for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogen-activated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor Gö6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H(2)O(2) is involved in hyperglycemia- and/or TNFalpha-induced insulin resistance, we preincubated the cells with the H(2)O(2) scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNFalpha, or 500 microM H(2)O(2), respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H(2)O(2) and TNFalpha on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNFalpha.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.

Binding Sites↗

PubMed1997

Impact of mutations at different serine residues on the tyrosine kinase activity of the insulin receptor.

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.

Amino Acid Substitution↗

PubMed1997

Protein kinase C isoforms beta 1 and beta 2 inhibit the tyrosine kinase activity of the insulin receptor.

Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor beta-subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms alpha, beta 1, beta 2, gamma, delta, epsilon, eta, theta and zeta were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10(-7) mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms beta 1 and beta 2, a 50 +/- 15.7 and 45 +/- 10.1% inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms beta 1 and beta 2 might be candidates for insulin receptor inhibition.

Cell Line↗

PubMed1996

Insulin leads to a parallel translocation of PI-3-kinase and protein kinase C zeta.

Protein kinase C consists of a family of at least 12 isoforms which exhibit clear differences in their cofactor dependence and responsiveness to phospholipids. Insulin effects on PKC translocation/activation are now clearly established but responsiveness to this hormone was observed so far only for the classical PKC-isoforms alpha and beta. While activation of the classical PKC's requires Ca2+ and occurs mainly through Diacylglycerol (DAG), stimulation of the atypical isoform PKC-zeta appears to function through a different mechanism involving PI-3-kinase activation. In the present study we used rat-1 fibroblasts stably over-expressing human insulin receptor to investigate whether insulin can activate PKC-zeta and whether such an effect might be related to insulin's effect on PI-3-kinase. After stimulation of the cells with insulin (10(-7) mol/l) for one to ten minutes, a rapid translocation of PKC-zeta to the plasma membrane was detectable, as determined by immunoblotting of plasma membrane proteins with antibodies against PKC-zeta. In parallel immunoblots applying antibodies against the regulatory subunit of PI-3-kinase (p85), an insulin-induced translocation of p85 was detectable within one minute after stimulation. The translocation of p85 was associated with an increase in PI-3-kinase activity at the plasma membrane. The data show that insulin stimulates translocation of PKC-zeta in rat-1 fibroblasts. The parallel kinetics of PI-3-kinase translocation/activation and PKC-zeta translocation are compatible with the idea that the insulin effect on PKC-zeta is transduced through PI-3-kinase activation.

Animals↗

PubMed1996

Modulation of insulin receptor signaling. Potential mechanisms of a cross talk between bradykinin and the insulin receptor.

Insulin resistance of the skeletal muscle plays a key role in the development of the metabolic endocrine syndrome and its further progression to type II diabetes. Impaired signaling from the insulin receptor to the glucose transport system and to glycogen synthase is thought to be the cause of skeletal muscle insulin resistance. An incomplete activation of the insulin receptor tyrosine kinase, which is found in type II diabetes, appears to contribute to the pathogenesis of the signaling defect. Available data suggest that the impaired tyrosine kinase function of the insulin receptor is not due to an inherited defect but rather is caused by a modulation of insulin receptor function. We used rat-1 fibroblasts and NIH-3T3 cells stably overexpressing human insulin receptor and 293 cells transiently overexpressing human insulin receptor to characterize conditions modulating the signaling function of the insulin receptor kinase. Using these cell models, we could demonstrate that activation of different protein kinase C (PKC) isoforms by high glucose levels or phorbol esters causes a rapid inhibition of the receptor tyrosine kinase activity. This effect is most likely mediated through serine phosphorylation of the receptor beta-subunit. It can be prevented by PKC inhibitors and the new oral antidiabetic agent thiazolidindione. The data suggest that PKC might be an important negative regulator of insulin receptor function. Because we have recently shown that bradykinin activates different isoforms of PKC in these cell types, an inhibitory cross talk between the bradykinin receptor and the insulin receptor through PKC activation seemed possible. However, we were unable to observe an insulin receptor tyrosine kinase inhibition through bradykinin, suggesting that different isoforms of PKC are activated by hyperglycemia and bradykinin. On the other hand, a modulation of bradykinin signals by insulin could be demonstrated in these cells. Bradykinin-induced tyrosine phosphorylation of proteins of approximately 130 and 70 kDa was inhibited by insulin treatment of rat-1 fibroblasts. These data suggest that signals from the insulin receptor modify signaling from the bradykinin receptor to tyrosine phosphorylation of different cellular proteins.

Animals↗

PubMed1995

Insulin- and insulin-like growth-factor-I receptor tyrosine-kinase activities in human renal carcinoma.

We studied expression and functional characteristics of the insulin- and insulin-like-growth-factor-I (IGF-I) receptors in human renal carcinoma. Ligand-binding properties and tyrosine-kinase activity of both receptors, as well as the expression of the 2 isoforms of the human insulin receptor (HIR-A and -B) were analyzed in renal carcinoma and normal adjacent kidney tissue of 8 adult patients. Partially purified insulin- and IGF-I receptors from normal and renal cell carcinoma tissue possessed identical affinities for their ligands. Renal cell carcinoma, however, contained 3- to 4-fold more specific insulin-binding sites and 2-fold more IGF-I binding sites than adjacent normal kidney tissue. In addition, we determined the relative content of insulin/IGF-I receptor hybrids in both tissues. Renal cell carcinoma and adjacent normal tissue revealed similar amounts of insulin/IGF-I receptor hybrids, i.e., 44 +/- 8.2% of tracer IGF-I binding in normal tissue and 46 +/- 12.0% in renal cell carcinoma. When equal amounts of insulin- and IGF-I receptor protein were studied, we found significantly increased receptor autophosphorylation and elevated substrate phosphorylation in carcinoma tissue. To assess whether the differences in insulin-receptor tyrosine-kinase activity were caused by an altered pattern of insulin receptor isoform expression, we determined mRNA levels for HIR-A and -B. The 2 insulin receptor isoforms were, however, expressed in highly variable ratios in both normal and tumor tissue. Our experiments show that renal carcinoma expresses an elevated amount of insulin- and IGF-I receptor protein with increased specific autophosphorylation and tyrosine-kinase activity each. The increase of insulin-receptor tyrosine-kinase activity in renal carcinoma cannot be explained by an altered expression pattern of insulin receptor isoforms.

Carcinoma↗

PubMed1995

C-terminus or juxtamembrane deletions in the insulin receptor do not affect the glucose-dependent inhibition of the tyrosine kinase activity.

We have previously shown, in rat-1 fibroblasts which stably overexpress high levels of human insulin receptor (HIR), that high glucose levels induce an inhibition of insulin receptor tyrosine kinase (IRK) activity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., Seffer, E., Seedorf, K., Häring, H. (1994) J. Biol. Chem. 269, 3381-3386]. This effect appears to be mediated through activation of protein kinase C and phosphorylation of the receptor beta-subunit on threonine or serine residues. The aim of the present study was to determine whether the juxtamembrane region or the C-terminus tail of the receptor are involved in the IRK modulation by glucose. In these domains increased serine and threonine phosphorylation was observed after phorbol ester or insulin stimulation of cells, and a regulatory function for IRK activity seems conceivable. We used an antibody directed against one potential regulatory site in the C-terminus tail, i.e. PSer1315, to study the effect of glucose. An increased signal was detected in HIR from rat-1 fibroblasts treated with phorbol 12-myristate 13-acetate or glucose (25 mM). To investigate whether this site in the C-terminus is essential for glucose-dependent IRK inhibition, rat-1 fibroblasts stably overexpressing a C-terminus-truncated human insulin receptor lacking 43 amino acids (HIR delta CT) were studied in parallel with cells expressing the wild-type receptor. As described earlier, HIR delta CT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibited the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of Ser1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtamembrane region of the insulin receptor are involved in the inhibitory effect of glucose, 293 cells transiently transfected either with wild-type HIR or HIR with a juxtamembrane deletion spanning amino acids 954-965 [des-(954-965)-HIR] were studied in parallel. As described earlier, the des-(954-965)-HIR has lost the ability to stimulate PI-3 kinase. However, 25 mM glucose equally inhibited the insulin effect on tyrosine phosphorylation of the receptor. Together, the data suggest that the regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C-terminus nor in the juxtamembrane region of the insulin receptor beta subunit.

3-O-Methylglucose↗

PubMed1995

Mechanism of insulin receptor kinase inhibition in non-insulin-dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered.

The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs. 3.2 +/- 1.2% control). Moreover, within each group there was no correlation between the level of tyrosine kinase activity and the extent of serine/threonine phosphorylation. It is concluded that the stoichiometry of serine/threonine phosphorylation of insulin receptor in vivo is low, and that increased phosphorylation of Ser 1327 or Thr 1348 is not responsible for the decreased insulin receptor tyrosine kinase activity observed in the skeletal muscle of NIDDM patients.

Aged↗

PubMed1995

Acute hyperglycemia provides an insulin-independent inducer for GLUT4 translocation in C2C12 myotubes and rat skeletal muscle.

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381-3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10(-7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose +/- insulin (1.6 x 10(-9) mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme alpha 1 Na(+)-K(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Bradykinin induces translocation of the protein kinase C isoforms alpha, epsilon, and zeta.

Bradykinin exerts a broad spectrum of cellular effects on different tissues. It is believed that these effects are predominantly mediated by the recently cloned B2 receptor. The mechanism of post-receptor signal transduction is not known in detail. Involvement of protein kinase C (PKC) was suggested and activation of the classical PKC isoforms alpha and beta was recently demonstrated. The aim of the present study was to investigate whether the B2 receptor also activates new (delta, epsilon) and atypical (zeta) PKC isoforms. To investigate this, chinese hamster ovary (CHO) cells, stably transfected with human B2 receptor, were used. In these cells the PKC isoforms alpha, delta, epsilon and zeta were detected by immunoblotting with specific antibodies. To monitor hormone-induced PKC translocation plasma membranes were prepared. Stimulation of the cells with bradykinin resulted in a rapid (30-60 s) translocation of the PKC isoforms alpha, epsilon, and zeta. Translocation of PKC delta was not detected. The effect of bradykinin was reduced by simultaneous addition of the receptor antagonist HOE 140, a bradykinin-related decapeptide. The data show that the B2 receptor in this cell model is able to activate, in addition to the classical PKC isoform alpha, the new PKC isoform epsilon and the atypical PKC isoform zeta. To test whether these effects are as well observed in a non-transfected cell, the experiments were repeated in human foreskin fibroblasts which naturally express high levels of B2 receptors. In this cell system similar results on PKC alpha, epsilon, and zeta were observed, suggesting that all three PKC isoforms are involved in signal transduction of the B2 receptor.

Animals↗

PubMed1994

Mutagenesis of Phe381 and Phe382 in the extracellular domain of the insulin receptor: effects on receptor biosynthesis, processing, and ligand-dependent internalization.

Mutations of the extracellular domain of the insulin receptor impair processing and transport of receptors to the plasma membrane. We have previously reported that a mutation substituting Val for Phe382 in the alpha-subunit of the insulin receptor impairs intracellular processing and insulin-induced autophosphorylation of the mutant receptor. In this investigation, we have generated two independent mutations of amino acids Phe381 and Phe382 of the insulin receptor: Val for Phe381 and Leu for Phe382. These substitutions cause a slight impairment of intracellular processing and transport of the mutant receptors. Furthermore, insulin-dependent internalization of the mutant receptors is unaffected by these mutations. Thus, of the three substitutions studied to date, Val for Phe382 is the only mutation of the Phe381-Phe382 sequence that causes a major defect in post-translational processing of the receptor.

3T3 Cells↗

PubMed1994

Glucose-induced translocation of protein kinase C isoforms in rat-1 fibroblasts is paralleled by inhibition of the insulin receptor tyrosine kinase.

Rat-1 fibroblasts stably overexpressing high levels of human insulin receptor were used as a model system to study the effects of hyperglycemia on insulin receptor tyrosine kinase (IRK) activity and protein kinase C (PKC) translocation in parallel in the intact cell. Glucose (10-25 mM) induced a significant reduction of IRK activity (tyrosine phosphorylation of IR-beta-subunit and IR-substrate-1) within 10 min. This effect was paralleled by a rapid translocation of several PKC isoforms (cPKC alpha, nPKC delta, nPKC epsilon, nPKC zeta) to the plasma membrane within 1 min. Kinetics of IRK inhibition and PKC translocation are consistent with the idea that the glucose effect on IRK is mediated by PKC activation. This hypothesis is supported by further observations. Addition of the protein kinase C inhibitor H-7 can prevent the effect of glucose on IRK. Inhibition of IRK is also observed after stimulation of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which can substitute for a physiological activator of PKC. Glucose (25 mM) increases the 32P incorporation in serine residues of the beta-subunit of IRK. We conclude that high levels of glucose induce inhibition of IRK in vivo. There is indirect evidence that this effect is mediated by a glucose-induced PKC translocation/activation and serine phosphorylation of the insulin receptor.

3-O-Methylglucose↗

PubMed1994

Modulation of insulin receptor signalling: significance of altered receptor isoform patterns and mechanism of hyperglycaemia-induced receptor modulation.

Insulin resistance of the skeletal muscle plays a key role in the development of the metabolic endocrine syndrome and its further progression to non-insulin dependent diabetes (NIDDM). Available data suggest that insulin resistance is caused by an impaired signal from the insulin receptor to the glucose transport system and to glycogen synthase. The impaired response of the insulin receptor tyrosine kinase which is found in NIDDM appears to contribute to the pathogenesis of the signalling defect. The reduced kinase activation is not caused by mutations within the insulin receptor gene. We investigated two potential mechanisms that might be relevant for the abnormal function of the insulin receptor in NIDDM, i.e. changes in the expression of the receptor isoforms and the effect of hyperglycaemia on insulin receptor tyrosine kinase activity. The insulin receptor is expressed in two different isoforms (HIR-A and HIR-B). We found that HIR-B expression in the skeletal muscle is increased in NIDDM. However, the characterisation of the functional properties of HIR-A and HIR-B revealed no difference in their tyrosine kinase activity in vivo. The increased expression of HIR-B might represent a compensatory event. In contrast, hyperglycaemia might directly inhibit insulin-receptor function. We have found that in rat-1 fibroblasts which overexpressing human insulin receptor an inhibition of the tyrosine kinase activity of the receptor may be induced by high glucose levels. This appears to be mediated through activation of certain protein kinase C isoforms which form stable complexes with the insulin receptor and modulate the tyrosine kinase activity of the insulin receptor through serine phosphorylation of the receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Insulin-induced translocation of GLUT 4 in skeletal muscle of insulin-resistant Zucker rats.

The genetically obese Zucker rat (fa/fa) is an animal model with severe insulin resistance of the skeletal muscle. We investigated whether a defect of insulin-dependent glucose transporter (GLUT 4) translocation might contribute to the pathogenesis of the insulin-resistant state. fa/fa rats, lean controls (Fa/Fa) as well as normal Wistar rats were injected intraperitoneally with insulin and were killed after 2 or 20 min, respectively. Subcellular fractions were prepared from hind-limb skeletal muscle and were characterized by determination of marker-enzyme activities and immunoblotting applying antibodies against alpha 1 Na+/K+ ATPase. The relative amounts of GLUT 1 and GLUT 4 were determined in the fractions by immunoblotting with the respective antibodies. Insulin induced an approximately two-fold increase of GLUT 4 in a plasma membrane and transverse tubule enriched fraction and a decrease in the low density enriched membrane fraction in all three groups of rats. There was a high individual variation in GLUT 4 translocation efficiency within the groups. However, no statistically significant difference was noted between the groups. No effect of insulin was detectable on the distribution of GLUT 1 or alpha 1 Na+K+ ATPase. The data suggest that skeletal muscle insulin resistance of obese Zucker rats is not associated with a lack of GLUT 4 translocation.

Animals↗

PubMed1994

Modulation of insulin signalling in non-insulin-dependent diabetes mellitus: significance of altered receptor isoform patterns and mechanisms of glucose-induced receptor modulation.

Insulin resistance in skeletal muscle plays a key role in the development of the metabolic-endocrine syndrome and its further progression to non-insulin-dependent diabetes mellitus (NIDDM). Available data suggest that insulin resistance is caused by impaired signalling from the insulin receptor to the glucose transport system and to glycogen synthase. The impaired response of the insulin receptor tyrosine kinase, which is found in NIDDM, appears to contribute to the pathogenesis of the signalling defect. The reduced kinase activation is not caused by mutation of the receptor. Two potential mechanisms were investigated that might be relevant to the abnormal function of the insulin receptor in NIDDM. That is, changes of the receptor isoforms and the effect of hyperglycaemia. The insulin receptor is expressed in two different isoforms (HIR-A and HIR-B). HIR-B expression in skeletal muscle is increased in NIDDM. Characterization of the functional properties of HIR-B, however, revealed that increased HIR-B expression did not cause impaired tyrosine kinase activity, but more probably represented a compensatory event. In contrast, hyperglycaemia is able to inhibit insulin receptor function. In a rat-1 fibroblast cell line overexpressing human insulin receptor, inhibition of the tyrosine kinase activity of the receptor can be induced by high glucose levels. This effect appears to be mediated through activation of certain protein kinase C isoforms, which are able to form stable complexes with the insulin receptor and modulate its tyrosine kinase activity through serine phosphorylation of the receptor beta-subunit. This mechanism might also be relevant in human skeletal muscle and thereby contribute to the pathogenesis of insulin resistance.

Animals↗

PubMed1994

Troglitazone prevents glucose-induced insulin resistance of insulin receptor in rat-1 fibroblasts.

Troglitazone (CS045), a compound belonging to the thiazolidine diones, is being tested as a new oral antidiabetic agent. Evidence exists from animal studies and clinical trials with non-insulin-dependent diabetes mellitus patients that Troglitazone might reduce insulin resistance. The molecular mechanism of this effect is not understood. In this study, we investigated whether Troglitazone might interfere with the mechanism of glucose-induced insulin resistance. Several studies indicate that hyperglycemia reduces the kinase activity of the insulin receptor in different cell types. This effect is paralleled by translocation of several protein kinase C (PKC) isoforms, and it can be prevented by PKC inhibitors, which suggests that glucose-induced receptor desensitization is mediated by activation of PKC. We studied the effect of hyperglycemia on the insulin receptor kinase activity and its modulation by Troglitazone in rat-1 fibroblasts that stably overexpress the human insulin receptor. Before stimulation with insulin (10(-7) M), cells were acutely exposed to hyperglycemic conditions in the absence or presence of Troglitazone (0.01-2 micrograms/ml). The insulin receptor was solubilized from a plasma membrane fraction or whole cell lysates, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted against antiphosphotyrosine and anti-insulin receptor beta-subunit (CT 104) antibodies. Acute hyperglycemia (25 mM glucose) induced a significant inhibition of the insulin receptor kinase (IRK) activity within 30 min (inhibition to 30 +/- 12.5% of maximal insulin-stimulated beta-subunit phosphorylation, n = 9, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗