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.     

Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance

Insulin resistance contributes to a number of metabolic disorders, including type II diabetes, hypertension, and atherosclerosis. Cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, and hormones, such as growth hormone, are known to cause insulin resistance, but the mechanisms by which they inhibit the cellular response to insulin have not been elucidated. One mechanism by which these agents could cause insulin resistance is by inducing the expression of cellular proteins that inhibit insulin receptor (IR) signaling. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling pathways, the expression of which is regulated by certain cytokines. SOCS proteins are therefore attractive candidates as mediators of cytokine-induced insulin resistance. We have found that SOCS-1 and SOCS-6 interact with the IR when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing the human IR. In SOCS-1-expressing cells, insulin treatment increases the extent of interaction with the IR, whereas in SOCS-6-expressing cells the association with the IR appears to require insulin treatment. SOCS-1 and SOCS-6 do not inhibit insulin-dependent IR autophosphorylation, but both proteins inhibit insulin-dependent activation of ERK1/2 and protein kinase B in vivo and IR-directed phosphorylation of IRS-1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signaling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.     

Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells

The main purpose of this study is to examine the effect of caffeine on lipid accumulation in human hepatoma HepG2 cells. Significant decreases in the accumulation of hepatic lipids, such as triglyceride (TG), and cholesterol were observed when HepG2 cells were treated with caffeine as indicated. Caffeine decreased the mRNA level of lipogenesis-associated genes (SREBP1c, SREBP2, FAS, SCD1, HMGR and LDLR). In contrast, mRNA level of CD36, which is responsible for lipid uptake and catabolism, was increased. Next, the effect of caffeine on AMP-activated protein kinase (AMPK) signaling pathway was examined. Phosphorylation of AMPK and acetyl-CoA carboxylase were evidently increased when the cells were treated with caffeine as indicated for 24 h. These effects were all reversed in the presence of compound C, an AMPK inhibitor. In summary, these data indicate that caffeine effectively depleted TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP signaling pathways. [BMB Reports 2013; 46(4): 207-212]     

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes. Here, we demonstrate that the activation of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) depends critically on the structures of membrane sterols. IR and IGF1R autophosphorylation in vivo was inhibited by cholesterol depletion, and autophosphorylation was restored by the replacement with exogenous cholesterol. We next screened a variety of sterols for effects on IR activation. The ability of sterols to support IR autophosphorylation was strongly correlated to the propensity of the sterols to form ordered domains. IR autophosphorylation was fully restored by the incorporation of ergosterol, dihydrocholesterol, 7-dehydrocholesterol, lathosterol, desmosterol, and allocholesterol, partially restored by epicholesterol, and not restored by lanosterol, coprostanol, and 4-cholesten-3-one. These data support the hypothesis that the ability to form ordered domains is sufficient for a sterol to support ligand-induced activation of IR and IGF1R in intact mammalian cells.     

Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells.

Fish oil supplementation in humans is often associated with an expanded low density lipoprotein (LDL) pool that is not thought to reflect increased production. Since data on clearance of LDL after fish oil supplementation (FO-LDL) are equivocal, normal volunteers (four men and three women) received ten capsules containing 3.6 g eicosapentaenoic acid and 2.9 g docosahexaenoic acid (approximately 2.5% total calories as methyl esters) for 2 weeks. Total plasma cholesterol was unchanged, but triglycerides decreased 30%. Low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were unchanged. Analysis of the LDL particles revealed that increased esterified cholesterol caused the FO-LDL core/surface ratio to be greater than baseline LDL (BL-LDL), resulting in a shift in mean LDL density from 1.060 to 1.056. N-3 fatty acids in FO-LDL were also increased greater than 40% at the expense of n-6 and n-9 fatty acids. Human hepatoma HepG2 cells were used to study the effects of FO-LDL on LDL receptor activity and mRNA abundance for the LDL receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and various apolipoproteins associated with cholesterol metabolism. In this system FO-LDL reduced LDL receptor activity compared to BL-LDL. Scatchard analysis revealed that LDL receptor number (Bmax) was reduced to one-third normal (P less than 0.001) whereas particle binding affinity was unchanged. The mRNA abundance for the LDL receptor and apoA-I were also depressed, even by low concentrations (10 micrograms/ml and 20 micrograms/ml LDL protein) of FO-LDL as compared to BL-LDL. HepG2 cells incubated with FO-LDL had decreased cellular free cholesterol but increased cholesteryl esters. Thus, moderate supplementation with fish oil n-3 fatty acids in normal humans enriches their LDL particles in cholesteryl esters and n-3 fatty acids. These particles depress both LDL receptor activity and LDL receptor mRNA abundance in HepG2 cells.     

Selected cholesterol biosynthesis inhibitors produce accumulation of the intermediate FF-MAS that targets nucleus and activates LXRα in HepG2 cells

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.     

Inhibition of the insulin receptor tyrosine kinase by sphingosine.

Sphingosine inhibits autophosphorylation of the insulin receptor tyrosine kinase in vitro and in situ. This lysosphingolipid has been shown previously to inhibit the Ca2+/lipid-dependent protein kinase C. Here we show that insulin-dependent autophosphorylation of partially purified insulin receptor is half-maximally inhibited by 145 microM sphingosine (9 mol %) in Triton X-100 micelles. Half-maximal inhibition of protein kinase C autophosphorylation occurs with 60 microM sphingosine (3.4 mol %) in Triton X-100 mixed micelles containing phosphatidylserine and diacylglycerol. Sphingomyelin does not inhibit significantly the insulin receptor, suggesting that, as with protein kinase C, the free amino group may be essential for inhibition. Similar to the effects observed for protein kinase C, inhibition of the insulin receptor kinase by sphingosine is reduced in the presence of other lipids. However, the reduction displays a marked dependence on the lipid species: phosphatidylserine, but not a mixture of lipids compositionally similar to the cell membrane, markedly reduces the potency of sphingosine inhibition. The inhibition occurs at the level of the protein/membrane interaction: a soluble form of the insulin receptor comprising the cytoplasmic kinase domain is resistant to sphingosine inhibition. Lastly, sphingosine inhibits the insulin-stimulated rate of tyrosine phosphorylation of the insulin receptor in NIH 3T3 cells expressing the human insulin receptor. These results suggest that sphingosine alters membrane function independently of protein kinase C.     

Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control

Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Str?lfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.     

Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.     

Characterization of phosphatidylinositol kinase activity associated with the insulin receptor

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.     

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.     

AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.     

Effect of cell cholesterol content on apolipoprotein B secretion and LDL receptor activity in the human hepatoma cell line, HepG2

Human hepatoma HepG2 cells were used to study the effects of cholesterol loading and depletion on apolipoprotein B (apoB) secretion and low-density lipoprotein (LDL) receptor activity. Exposure of HepG2 cells to cholesterol and oleic acid, which elevated intracellular cholesterol levels, stimulated apoB secretion and reduced receptor-mediated uptake of LDL, whereas recombinant complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine, which depleted the cellular cholesterol pool, inhibited apoB secretion and up-regulated LDL receptors. Significant negative correlation (r = -0.92, P less than 0.001) between the levels of apoB secretion and LDL uptake was found. These data suggest that the cholesterol content of the cells may induce concomitant changes in apoB secretion and LDL receptor activity.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Age‐associated cholesterol reduction triggers brain insulin resistance by facilitating ligand‐independent receptor activation and pathway desensitization

In the brain, insulin plays an important role in cognitive processes. During aging, these faculties decline, as does insulin signaling. The mechanism behind this last phenomenon is unclear. In recent studies, we reported that the mild and gradual loss of cholesterol in the synaptic fraction of hippocampal neurons during aging leads to a decrease in synaptic plasticity evoked by glutamate receptor activation and also by receptor tyrosine kinase (RTK) signaling. As insulin and insulin growth factor activity are dependent on tyrosine kinase receptors, we investigated whether the constitutive loss of brain cholesterol is also involved in the decay of insulin function with age. Using long‐term depression (LTD) induced by application of insulin to hippocampal slices as a read‐out, we found that the decline in insulin function during aging could be monitored as a progressive impairment of insulin‐LTD. The application of a cholesterol inclusion complex, which donates cholesterol to the membrane and increases membrane cholesterol levels, rescued the insulin signaling deficit and insulin‐LTD. In contrast, extraction of cholesterol from hippocampal neurons of adult mice produced the opposite effect. Furthermore, in vivo inhibition of Cyp46A1, an enzyme involved in brain cholesterol loss with age, improved insulin signaling. Fluorescence resonance energy transfer (FRET) experiments pointed to a change in receptor conformation by reduced membrane cholesterol, favoring ligand‐independent autophosphorylation. Together, these results indicate that changes in membrane fluidity of brain cells during aging play a key role in the decay of synaptic plasticity and cognition that occurs at this late stage of life.     

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.     

Aflatoxin B1 Up-Regulates Insulin Receptor Substrate 2 and Stimulates Hepatoma Cell Migration

Aflatoxin B1 (AFB1) is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor p53. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR) signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS) 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.