Biological Actions Of Insulin Research Articles

MiRNA-27a mediates insulin resistance in 3T3-L1 cells through the PPARγ.

The biological actions of insulin have been originated by activation of membrane receptors, which trigger a diversity of signaling pathways in facilitating their biological activities. Insulin homeostasis functions in promoting metabolism balance and promotes cell growth and proliferation. If these mechanisms are reformed, this could lead to insulin resistance as a result of defective insulin signaling triggered by mutations in receptors or effector molecules located downstream or by abnormal posttranslational modifications. The purpose of this is to preliminarily investigate the mechanism of miRNA-27a-mediating insulin resistance in 3T3-L1 cells. Insulin resistance in 3T3-L1 adipocytes as a cell model was induced by tumor necrosis factor-alpha (TNF-α) and the miRNA-27a expression in 3T3-L1 adipocytes had been experiential. The regulation of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA by miRNA-27a had been studied by reverse transcription receptor polymerase chain reaction (RT-PCR). MiRNA-27a was up-regulated in 3T3-L1 cells, miRNA-27a mimics reserved expression of PPARγ mRNA, and miRNA-27a inhibitors up-regulated the expression of PPARγ mRNA. The insulin resistance in 3T3-L1 cells mediated by miRNA-27a may be achieved by targeting PPARγ.

Low molecular weight regulators of the intracellular insulin signal transduction as a correction method of the insulin resistance in the treatment of type 2 diabetes

Insulin resistance is the characteristic feature of type 2 diabetes. This condition is manifested in the reduction of peripheral tissues sensitivity to the biological action of insulin and is expressed in the inhibition of cellular glucose absorption and metabolism in response to hormonal stimulation. At the cellular level, disorders which are realized both at the receptor and the postreceptor levels can serve a prerequisite to the formation of insulin resistance and are associated with a change in the amount or dysfunction of major molecular signaling cascade. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and thus low molecular weight effectors which act on the individual links of insulin signaling cascade may be positioned as a new generation of anti-diabetic agents. This report provides information on the regulators of insulin receptor cascade, main advantages and disadvantages of their impact on biological targets and prospects for their therapeutic use as anti-diabetic drugs.

Low molecular weight regulators of the intracellular insulin signal transduction as the method of correction of insulin resistance in the treatment of type 2 diabetes mellitus

Insulin resistance is the characteristic feature of type 2 diabetes mellitus. This condition is manifested in decreased sensitivity of peripheral tissues to the biological action of insulin and is expressed in the inhibition of cell glucose uptake and metabolism in response to the hormonal stimulation. At the cellular level, impairments realized both at the receptor and the postreceptor levels and associated with changes in the content or dysfunction of the main molecules of the signal cascade can serve a molecular prerequisite to the formation of insulin resistance. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and low molecular weight effectors, which act on the individual links of the insulin signaling cascade, may be positioned as a new generation of anti-diabetic agents. This review summarizes current knowledge on regulators of the insulin receptor cascade, main advantages and disadvantages of their effects on biological targets and prospects for their therapeutic use as anti-diabetic agents.

Interaction of Filamin A with the Insulin Receptor Alters Insulin-dependent Activation of the Mitogen-activated Protein Kinase Pathway

The biological actions of insulin are associated with a rapid reorganization of the actin cytoskeleton within cells in culture. Even though this event requires the participation of actin-binding proteins, the effect of filamin A (FLNa) on insulin-mediated signaling events is still unknown. We report here that human melanoma M2 cells lacking FLNa expression exhibited normal insulin receptor (IR) signaling, whereas FLNa-expressing A7 cells were unable to elicit insulin-dependent Shc tyrosine phosphorylation and p42/44 MAPK activation despite no significant defect in IR-stimulated phosphorylation of insulin receptor substrate-1 or activation of the phosphatidylinositol 3-kinase/AKT cascade. Insulin-dependent translocation of Shc, SOS1, and MAPK to lipid raft microdomains was markedly attenuated by FLNa expression. Coimmunoprecipitation experiments and in vitro binding assays demonstrated that FLNa binds constitutively to IR and that neither insulin nor depolymerization of actin by cytochalasin D affected this interaction. The colocalization of endogenous FLNa with IR was detected at the surface of HepG2 cells. Ectopic expression of a C-terminal fragment of FLNa (FLNaCT) in HepG2 cells blocked the endogenous IR-FLNa interaction and potentiated insulin-stimulated MAPK phosphorylation and transactivation of Elk-1 compared with vector-transfected cells. Expression of FLNaCT had no major effect on insulin-induced phosphorylation of the IR, insulin receptor substrate-1, or AKT, but it elicited changes in actin cytoskeletal structure and ruffle formation in HepG2 cells. Taken together, these results indicate that FLNa interacts constitutively with the IR to exert an inhibitory tone along the MAPK activation pathway.

Insulin-induced Akt activation is inhibited by angiotensin II in the vasculature through protein kinase C-alpha.

Insulin resistance is an important risk factor in the development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. Angiotensin (Ang) II is a potent vasculotrophic and vasoconstricting factor. We hypothesize that in vascular smooth muscle cells (VSMCs), Ang II interferes with insulin action by inhibiting Akt, a major signaling molecule implicated in the biological actions of insulin. By immunoblotting with a phospho-specific antibody for Akt, we found that Ang II inhibits insulin-induced Akt phosphorylation in a time- and concentration-dependent manner. The inhibitory effect of Ang II was blocked by a Ang II type 1 receptor antagonist, RNH6270. A protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, also inhibited insulin-induced Akt phosphorylation. PKC inhibitors, including Go6976 (specific for alpha- and beta-isoforms), blocked the Ang II- and PMA-induced inhibition of Akt phosphorylation by insulin. Moreover, overexpression of PKC-alpha but not PKC-beta isoform by adenovirus inhibited insulin-induced Akt phosphorylation. By contrast, an epidermal growth factor receptor inhibitor (AG1478), a p42/44 mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 598,059), and a p38 MAPK inhibitor (SB 203,580) did not block the Ang II-induced inhibition of Akt phosphorylation. From these data, we conclude that Ang II negatively regulates the insulin signal, Akt, in the vasculature specifically through PKC-alpha activation, providing an alternative molecular mechanism that may explain the association of hyperinsulinemia with cardiovascular diseases.

Clinical review 125: The insulin receptor and its cellular targets.

The pleiotropic actions of insulin are mediated by a single receptor tyrosine kinase. Structure/function relationships of the insulin receptor have been conclusively established, and the early steps of insulin signaling are known in some detail. A generally accepted paradigm is that insulin receptors, acting through insulin receptor substrates, stimulate the lipid kinase activity of phosphatidylinositol 3-kinase. The rapid rise in Tris-phosphorylated inositol (PIP(3)) that ensues triggers a cascade of PIP(3)-dependent serine/threonine kinases. Among the latter, Akt (a product of the akt protooncogene) and atypical protein kinase C isoforms are thought to be involved in insulin regulation of glucose transport and oxidation; glycogen, lipid, and protein synthesis; and modulation of gene expression. The presence of multiple insulin-regulated, PIP(3)-dependent kinases is consistent with the possibility that different pathways are required to regulate different biological actions of insulin. Additional work remains to be performed to understand the distal components of insulin signaling. Moreover, there exists substantial evidence for insulin receptor substrate- and/or phosphatidylinositol 3-kinase-independent pathways of insulin action. The ultimate goal of these investigations is to provide clues to the pathogenesis and treatment of the insulin resistant state that is characteristic of type 2 diabetes.

Protein Kinase C-ζ Phosphorylates Insulin Receptor Substrate-1 and Impairs Its Ability to Activate Phosphatidylinositol 3-Kinase in Response to Insulin

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.

A phosphotyrosyl mimetic peptide reverses impairment of insulin-stimulated translocation of GLUT4 caused by overexpression of PTP1B in rat adipose cells.

Biological actions of insulin are initiated by activation of the insulin receptor tyrosine kinase. Protein tyrosine phosphatases (PTPases) PTP1B and PTPalpha are known to dephosphorylate the insulin receptor and may contribute to insulin resistance in diseases such as diabetes. We previously reported that overexpression of PTP1B in rat adipose cells significantly impairs insulin-stimulated translocation of GLUT4 [Chen, H., et al. (1997) J. Biol. Chem. 272, 8026]. In the present study, we treated adipose cells with a PTPase inhibitor containing the phosphotyrosyl mimetic difluorophosphonomethyl phenylalanine (F2Pmp) to determine whether we could improve the insulin resistance caused by overexpression of PTP1B or PTPalpha. Rat adipose cells transfected by electroporation with either PTP1B or PTPalpha were treated without or with the inhibitor, and effects on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. The IC50 of the F2Pmp-containing inhibitor is 180 nM for PTP1B and 10 mM for PTPalpha in vitro. As expected, in the absence of the inhibitor, overexpression of either PTP1B or PTPalpha caused a significant decrease in the amount of GLUT4 at the cell surface both in the absence and in the presence of insulin when compared with control cells transfected with epitope-tagged GLUT4 alone. Interestingly, the insulin resistance caused by overexpression of PTP1B (but not PTPalpha) was reversed by treating the transfected cells with the F2Pmp-containing inhibitor. Furthermore, the inhibitor blocked the insulin-stimulated association of PTP1B with the insulin receptor. We conclude that the F2Pmp-containing compound is a potent and specific inhibitor of overexpressed PTP1B that may be useful for designing rational therapies for treating insulin resistant diseases such as diabetes.

Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells.

Alpha2-Heremans Schmid glycoprotein (alpha2-HSG) is a member of the fetuin family of serum proteins whose biological functions are not completely understood. There is a consensus that alpha2-HSG plays a role in the regulation of tissue mineralization. However, one aspect of alpha2-HSG function that remains controversial is its ability to inhibit the insulin receptor tyrosine kinase and the biological actions of insulin. Interestingly, some studies suggest that alpha2-HSG differentially inhibits mitogenic, but not metabolic, actions of insulin. However, these previous studies were not carried out in bona fide insulin target cells. Therefore, in the present study we investigate the effects of alpha2-HSG in the physiologically relevant rat adipose cell. We studied insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in transfected rat adipose cells overexpressing human alpha2-HSG. In addition, we measured insulin-stimulated glucose transport in adipose cells cultured with conditioned medium from the transfected cells as well as in freshly isolated adipose cells treated with purified human alpha2-HSG. Compared with control cells, we were unable to demonstrate any significant effect of alpha2-HSG on insulin-stimulated translocation of GLUT4 or glucose transport. In contrast, we did demonstrate that overexpression of alpha2-HSG in adipose cells inhibits both basal and insulin-stimulated phosphorylation of Elk-1 (a transcription factor phosphorylated and activated by mitogen-activated protein kinase and other related upstream kinases). Interestingly, we did not observe any major effects of alpha2-HSG to inhibit insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate-1, -2, or -3, in either transfected or freshly isolated adipose cells. We conclude that alpha2-HSG inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in adipose cells by a mechanism that may involve effector molecules downstream of insulin receptor substrate proteins.

Insulin-like Effects of Vanadium: Mechanisms of Action, Clinical and Basic Implications

Most or all mammalian cells contain vanadium at a concentration of 20 nM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the years 1975–1980 the vanadate ion was shown to act as an efficient inhibitor of Na+,K+-ATPase and of other related phosphohydrolases as well. In 1980 it was observed that vanadate and vanadyl, when added to intact rat adipocytes, mimic the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.

PC-1 content in skeletal muscle of non-obese, non-diabetic subjects: relationship to insulin receptor tyrosine kinase and whole body insulin sensitivity.

Insulin sensitivity varies widely in non-obese, non-diabetic subjects, and we have previously reported that in vivo insulin action correlates with in vitro insulin stimulated insulin receptor tyrosine-kinase activity in skeletal muscle. Plasma membrane glyco-protein PC-1 content is elevated in fibroblasts of insulin-resistant subjects, and expression of PC-1 cDNA in cultured cells reduces both insulin receptor tyrosine-kinase activity and the biological actions of insulin. In the present study we investigated non-obese, non-diabetic subjects and found a significant negative correlation between muscle PC-1 content and both in vivo insulin action as measured by the intravenous insulin tolerance test (r = -0.51, p = 0.035) and the sensitivity (ED50) of in vitro insulin stimulation of insulin receptor tyrosine-kinase activity (r = 0.66, p = 0.027). These studies indicate, therefore, that increased muscle PC-1 content is associated with reduced insulin action both in vivo and in vitro. Moreover, they suggest a possible role for PC-1 in regulating insulin receptor function in human skeletal muscle.

Signal transduction mechanism of insulin and insulin-like growth factor-1.

Insulin and insulin-like growth factor-1 (IGF-1) are two structurally related hormones which produce similar biological activities such as metabolic and growth promoting actions. Their receptors, insulin and IGF-1 receptors, also share similarities in both structure and functions such as tyrosine-specific protein kinase. We identified insulin receptor substrate-1 (IRS-1) as a common substrate for insulin and IGF-1 receptor tyrosine kinases. We generated IRS-1 knockout mice and showed that IRS-1 plays a physiological role in signal transduction and biological actions of insulin and IGF-1. We also identified pp190 (IRS-2) as an alternative substrate for IRS-1.

Insulin as an Amplifier of Gonadotropin Action on Steroid Production: Mechanisms and Sites of Action in Goldfish Prematurational Full-Grown Ovarian Follicles

The present study examines the effects of mammalian insulin and insulin-like growth factor-1 (IGF-I) on steroid production by goldfish vitellogenic and prematurational full-grown ovarian follicles in vitro. Insulin (0.4-40 μg/ml) had no effect on its own, but potentiated human chorionic gonadotropin (hCG) and 25-hydroxycholesterol-stimulated testosterone production by prematurational full-grown follicles in both a dose- and time-dependent manner. By comparison, in vitellogenic follicles insulin (4 μg/ml) had no effect on hCG- or 25-hydroxycholesterol-stimulated testosterone and 17β-estradiol production. IGF-I (11-100 ng/ml) neither affected basal nor hCG- and 25-hydroxycholesterol-stimulated testosterone production by vitellogenic or prematurational full-grown follicles. Studies to determine the site(s) of action of insulin showed that insulin had no effect on basal cyclic adenosine 3′.5′-monophosphate (cAMP) generation, but it enhanced hCG-stimulated cAMP and testosterone production by prematurational full-grown follicles. Insulin also enhanced conversion of 25-hydroxycholesterol to testosterone, but had no effect on the metabolism of pregnenolone, suggesting that insulin either enhances mobilization of cholesterol to the mitochondria or increases the activity of the cytochrome P450 side-chain cleavage enzyme. The ability of insulin to enhance hCG and/or 25-hydroxycholesterol-stimulated testosterone production was blocked by the addition of the protein kinase A (PKA) inhibitor H89, which confirmed that these actions of insulin are exerted, at least in part, by the cAMP/PKA pathway. These findings suggest that insulin, but not IGF-I, is capable of participating in the regulation of ovarian steroid biosynthesis through effects at multiple steps within the steroid cascade and that ovarian responsiveness to insulin changes during the course of follicular development in the goldfish. Together, these results provide new insights into the biological action of insulin in the fish ovary.

Biological actions of insulin are differentially regulated by glucose and insulin in primary cultured adipocytes. Chronic ability to increase glycogen synthase activity.

We have shown previously that prolonged exposure to insulin and glucose impairs the insulin-responsive glucose transport system in primary cultured adipocytes. To assess the ability of insulin and glucose to regulate other cellular insulin actions, epididymal rat adipocytes were cultured in media containing 0-15 mM D-glucose and with or without insulin (50 ng/ml). After 24 h, cells were washed and basal and maximally insulin-stimulated rates of 2-deoxy-D-glucose uptake, L-leucine incorporation into protein, glucose oxidation to CO2, glucose incorporation into lipids, and glycogen synthase activity were measured. The results confirmed that glucose potentiates insulin's chronic ability to decrease basal and maximal glucose transport rates by approximately 50% at 5 mM glucose and by approximately 70% at 15 mM glucose compared with control cells. However, neither glucose nor insulin, alone or in combination, affected rates of leucine incorporation into protein. In addition, basal and maximal rates of glucose oxidation and of glucose incorporation into lipids were not regulated by glucose, and maximal responses declined approximately 50% over 24 h only when insulin was not present during preincubation (i.e., chronic insulin exposure was necessary to maintain full maximal responses). Glycogen synthase activity was measured in a cell-free system (0.5 mM UDP-glucose, with 10 or 0.01 mM glucose-6-phosphate) after exposing intact cells to glucose and insulin. Both short-term (1 h) and long-term (24 h) exposure to glucose alone led a dose-dependent increase in I-form and D-form glycogen synthase activity. Chronic exposure to insulin also increased total glycogen synthase activity (I- plus D-form) but did not affect absolute rates of maximally stimulated I-form activity. Glucose (but not insulin) increased the cellular content of immunoreactive glycogen synthase by 70% after 1 h. These results show that 1) chronic exposure to glucose and insulin impairs insulin responsiveness of the glucose transport system but does not affect rates of amino acid incorporation into protein; 2) the chronic presence of insulin is necessary for the maintenance of normal maximally stimulated rates of glucose oxidation and of glucose incorporation into lipids in cultured cells; and 3) glucose increases both D-form and I-form glycogen synthase activity, in part by increasing the amount of synthase protein, whereas chronic insulin exposure increases total glycogen synthase activity without altering maximal absolute rates of I-form activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological actions of insulin are differentially regulated by glucose and insulin in primary cultured adipocytes. Chronic ability to increase glycogen synthase activity

Biological actions of insulin are differentially regulated by glucose and insulin in primary cultured adipocytes. Chronic ability to increase glycogen synthase activity

Inhibitors such as staurosporine, H-7 or polymyxin B cannot be used in skeletal muscle to prove the role of protein kinase C on insulin action.

The precise role of protein kinase C in insulin action in skeletal muscle is not well defined. Based on the fact that inhibitors of protein kinase C block some insulin effects, it has been concluded that some of the biological actions of insulin are mediated via protein kinase C. In this study, we present evidence that inhibitors of protein kinase C such as staurosporine, H-7 or polymyxin B cannot be used to ascertain the role of protein kinase C in skeletal muscle. This is based on the following experimental evidences: a) staurosporine, H-7 and polymyxin B markedly block in muscle the effect of insulin on System A transport activity; however, this effect of insulin is not mimicked in muscle by TPA-induced stimulation of protein kinase C, b) H-7 and polymyxin B block insulin action on System A transport activity in an additive manner to the inhibitory effect of phorbol esters, c) staurosporine, H-7 and polymyxin B block the effect of insulin on lactate production, a process that is activated by insulin and TPA in an additive fashion, and d) staurosporine completely blocks the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle.

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.

Molecular mechanisms of insulin secretion and insulin action

This article presents information and current ideas on the factors regulating insulin secretion, the mechanisms underlying the secretion and biological actions of insulin and the main characteristics of diabetes mellitus.

Insulin as a mitogenic factor: Role in the pathogenesis of cardiovascular disease

Evidence has been accumulating that insulin has actions that may promote the development of atherosclerosis. Research has involved three broad areas: actions of insulin on cultured arterial cells, the effect of insulin on isolated artery preparations, and the development of lipid-containing lesions in the arteries of experimental animals. Insulin, in concentrations similar to those found in physiologic conditions, stimulates proliferation of cultured arterial smooth muscle cells from a number of species, including humans. Insulin also stimulates migration of smooth muscle cells. Cholesterol synthesis and low-density lipoprotein interaction with its receptor in smooth muscle cells are stimulated by insulin. Insulin's mitogenic action appears to be mediated by the insulin-like growth factor receptor. Endothelial cells cultured from large vessels are resistant to the actions of insulin, but hyperglycemia inhibits their proliferation. Insulin deficiency protects animals from experimental atherosclerosis; this protection is lost with insulin treatment. Insulin administration results in lipid-containing lesions in chickens and rats fed a normal diet, and in increased lipid synthesis in the arteries of pigs and dogs. Isolated artery preparations from insulin-deficient or insulin-treated animals undergo lipid metabolism at a rate that correlates with the insulin concentrations in the donor animals. The biological actions of insulin (and glucose) on arterial tissue suggest that hyperglycemia and hyperinsulinemia may promote the development of atherosclerosis.

Lipid-induced insulin resistance in cultured hepatoma cells is associated with a decreased insulin receptor tyrosine kinase activity.

We have shown previously that experimental modifications of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (Zajdela Hepatoma Culture, ZHC) affect both binding and biological actions of insulin. Discrepancies between insulin binding and actions implied a postbinding defect, responsible for the observed insulin resistance in lipid-treated cells. To elucidate the mechanism for this defect, we have studied insulin binding and insulin receptor kinase activity in partially purified receptor preparations from ZHC cells grown either in normal medium or in medium supplemented with linoleic acid or 25-hydroxycholesterol. Insulin binding to the lectin-purified insulin receptor showed only a small alteration in receptor affinity for the preparations from lipid-treated cells. Insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor, as well as insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr)4:1, was significantly decreased in the preparations from lipid-modified cells. Although differences in basal levels were observed, the magnitude of the insulin-stimulated kinase activity was significantly decreased in receptor preparations from lipid-treated cells. These findings indicate that experimental modification of the lipids of cultured hepatoma cells can produce in insulin receptor kinase activity changes that are proportional to the reduced insulin action observed in these cells.