okadaic-acid and Insulin-Resistance

The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser(171) and Ser(275) were found to be phosphorylated in pancreatic beta-cells. Calcineurin (Cn) is proposed as the Ser(275) phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser(275) and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser(171) has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser(307). The Ser(307)-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser(307), suggesting that differential dephosphorylation at Ser(171) and Ser(307) cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser(307) and Ser(171) for TORC2's function in mouse liver. Levels of phosphorylation at Ser(171) and Ser(307) changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.

Topics: Animals; Chlorocebus aethiops; COS Cells; Cyclic AMP Response Element-Binding Protein; Cyclosporine; Diabetes Mellitus; Enzyme Inhibitors; Insulin Resistance; Liver; Mice; Mice, Inbred C57BL; Mice, Obese; Mutagenesis, Site-Directed; Okadaic Acid; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Trans-Activators; Transcription Factors

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.

Topics: Adipocytes; Aged; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunoblotting; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Kinetics; Male; Middle Aged; Okadaic Acid; Phosphoproteins; Phosphorylation; Serine; Signal Transduction; Sirolimus; Time Factors; Tyrosine

Insulin receptor substrate (IRS) proteins are major substrates of the insulin receptor (IR). IRS-1 associates with an insoluble multiprotein complex, possibly the cytoskeleton, in adipocytes. This localization may facilitate interaction with the IR at the cell surface. In the present study, we examined the hypothesis that the release of IRS proteins from this location may be a mechanism for insulin desensitization. We show that a second IRS protein, IRS-2, is associated with a multiprotein complex in adipocytes with similar characteristics to the IRS-1 complex. Insulin treatment (15-60 min) caused the release of IRS-1 and IRS-2 from this complex (high speed pellet; HSP) into the cytosol, whereas the level of tyrosyl-phosphorylated IRS proteins remained constant. Chronic insulin treatment resulted in a dramatic reduction in IRS-1 and IRS-2 in the HSP, eventually (>2 h) leading to IRS protein degradation and decreased levels of tyrosyl-phosphorylated IRS proteins. Okadaic acid, which rapidly induces insulin resistance in adipocytes independently of IR function, caused an almost quantitative release of IRS-1 into the cytosol commensurate with a significant reduction in tyrosyl-phosphorylated IRS proteins. Platelet-derived growth factor, a factor known to compromise insulin signaling, caused a more moderate release of IRS proteins from the HSP. Collectively, these results suggest that the assembly of IRS-1/IRS-2 into a multiprotein complex facilitates coupling to the IR and that the regulated release from this location may represent a novel mechanism of insulin resistance.

Topics: 3T3 Cells; Androstadienes; Animals; Base Sequence; CHO Cells; Cricetinae; Glucose; Glucose Transporter Type 4; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Macromolecular Substances; Mice; Molecular Sequence Data; Monosaccharide Transport Proteins; Multiprotein Complexes; Muscle Proteins; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Platelet-Derived Growth Factor; Receptor, Insulin; Wortmannin

Glucose transport in skeletal muscle can be mediated by two separate pathways, one stimulated by insulin and the other by muscle contraction. High-fat feeding impairs glucose transport in muscle, but the mechanism remains unclear. FVB mice (3 weeks old) were fed a high-fat diet (55% fat, 24% carbohydrate, 21% protein) or standard chow for 3-4 weeks or 8 weeks. Insulin-stimulated glucose transport, assessed with either 2-deoxyglucose or 3-O-methylglucose was decreased 35-45% (P < 0.001) in isolated soleus muscle, regardless of diet duration. Similarly, glucose transport stimulated by okadaic acid, a serine/threonine phosphatase inhibitor, was also 45% lower with high-fat feeding, but the glucose transport response to hypoxia or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (which are stimulators of the "contraction pathway") was intact. Hexokinase I, II, and total activity were normal in soleus muscle from high-fat-fed mice. GLUT4 expression in soleus muscle from the high-fat-fed mice was also normal, but the insulin-stimulated cell surface recruitment of GLUT4 assessed by exofacial photolabeling with [3H]-ATB bis-mannose was reduced by 50% (P < 0.001). Insulin-receptor substrate 1 (IRS-1) associated phosphatidylinositol (PI) 3-kinase activity stimulated by insulin was also reduced by 36% (P < 0.001), and expression of p85 and p110b subunits of PI 3-kinase was normal. In conclusion, high-fat feeding selectively impairs insulin-stimulated, but not contraction-pathway-mediated, glucose transport by reducing GLUT4 translocation to the plasma membrane. This appears to result from an acquired defect in insulin activation of PI 3-kinase. Since effects of okadaic acid on glucose transport are independent of PI 3-kinase, a second signaling defect may also be induced.

Topics: Animals; Biological Transport; Blood Glucose; Cell Compartmentation; Dietary Fats; Enzyme Inhibitors; Female; Glucose Transporter Type 4; Hexokinase; Hypoxia; Insulin; Insulin Resistance; Male; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Muscle, Skeletal; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sulfonamides

Topics: Animals; Hexokinase; Insulin; Insulin Resistance; Kinetics; Muscle, Skeletal; Obesity; Okadaic Acid; Phosphorylation; Protein Tyrosine Phosphatases; Rats; Rats, Zucker; Thinness; Vanadates

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.

Topics: Animals; Binding Sites; Enzyme Inhibitors; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Rats; Receptor, Insulin; Sphingomyelin Phosphodiesterase; Tumor Necrosis Factor-alpha; Tyrosine

To further elucidate the mechanisms for short-term regulation of the receptor for insulin-like growth factor II (IGF-II), we investigated effects of insulin, cAMP and phosphatase inhibitors on cell surface 125I-IGF-II binding in rat adipocytes. Preincubation with the serine/threonine phosphatase inhibitor okadaic acid (OA, 1 microM) or the non-hydrolysable cAMP analogue N6-mbcAMP (4 mM) markedly impaired insulin-stimulated 125I-IGF-II binding. Furthermore, addition of OA enhanced the inhibitory effect exerted by N6-mbcAMP. N6-mbcAMP also induced an insensitivity to insulin which was normalized by concomitant addition of the tyrosine phosphatase inhibitor vanadate (0.5 mM). In contrast, vanadate did not affect the impairment in maximal insulin-stimulated 125I-IGF-II binding produced by either OA or N6-mbcAMP. Phospholipase C (PLC), which cleaves phospholipids at the cell surface, markedly enhanced cell surface 125I-IGF-II binding in a concentration-dependent manner. Scatchard analysis demonstrated that the effect of PLC was due to an increased number of binding sites suggesting that "cryptic' IGF-II receptors are associated with the plasma membrane (PM). PLC (5 U/ml) also reversed the N6-mbcAMP-induced decrease of 125I-IGF-II binding at a low insulin concentration (10 microU/ml). Taken together, these data indicate that cAMP, similar to its effects on the glucose transporter GLUT 4 and the insulin receptor, may increase the proportion of functionally cryptic IGF-II receptors in the PM through mechanisms involving serine phosphorylation, possibly of a docking or coupling protein. Tyrosine phosphorylation appears to exert an opposite effect promoting the full cell surface expression of receptors.

Topics: Adipocytes; Animals; Bucladesine; Cell Membrane; Enzyme Inhibitors; Ethers, Cyclic; Insulin; Insulin Resistance; Insulin-Like Growth Factor II; Iodine Radioisotopes; Male; Okadaic Acid; Phosphoric Monoester Hydrolases; Phosphoserine; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 2; Type C Phospholipases; Vanadates

In response to insulin, several proteins are phosphorylated on tyrosine and on serine/threonine residues. Decreased phosphorylation of signaling peptides by a defective insulin receptor kinase may be a cause of insulin resistance. Accordingly, inhibition of the appropriate phosphatases might increase the phosphorylation state of these signaling peptides and thereby elicit increased glucose transport. The purpose of this study was to examine the effect of the serine/threonine phosphatase inhibitor okadaic acid and the tyrosine phosphatase inhibitors phenylarsine oxide and vanadate on 2-deoxyglucose transport in insulin-resistant human skeletal muscle. All three phosphatase inhibitors stimulated 2-deoxyglucose transport in insulin-resistant skeletal muscle. These data suggest that these compounds have bypassed a defect in at least one of the signaling pathways leading to glucose transport. Furthermore, maximal transport rates induced by the simultaneous presence of insulin and phosphatase inhibitor in insulin-resistant muscle were equal to insulin-stimulated rates in lean control subjects. However, both vanadate alone and vanadate plus insulin stimulated 2-deoxyglucose transport significantly more in insulin-sensitive tissue than in insulin-resistant tissue. These results demonstrate that although vanadate is able to stimulate glucose transport in insulin-resistant muscle, it is not able to normalize transport to the same rate achieved in insulin-sensitive muscle.

Topics: Adult; Animals; Arsenicals; Biological Transport; Deoxyglucose; Ethers, Cyclic; Humans; Insulin Resistance; Male; Muscle, Skeletal; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley; Vanadates

Insulin-stimulated glycogen synthase activity in human muscle is reduced in insulin-resistant subjects. Insulin regulation of human muscle glycogen synthase may require activation of a type-1 protein phosphatase (PP-1). We investigated the change of phosphorylase phosphatase and glycogen synthase activities in muscle biopsies obtained during a 2-h hyperinsulinemic euglycemic clamp in 12 insulin-sensitive (group S) and 8 insulin-resistant (group R) subjects. Fasting phosphorylase phosphatase activity was lower in group R than in group S, and did not increase significantly with insulin infusion in group R until 20 min. In group S, phosphorylase phosphatase was significantly stimulated by 10 min, remaining significantly higher than in group R at all time points. The insulin-mediated changes in phosphatase activities were not decreased by 3 nM okadaic acid but were completely inhibited by 1 microM okadaic acid, thereby verifying that insulin-stimulated phosphorylase phosphatase is accounted for by a PP-1. Subcellular fractionation demonstrated reduced fasting PP-1 activities in both the glycogen and cytosolic fractions of muscle obtained from subjects in group R compared to those in group S. These results suggest that insulin activation of PP-1 could contribute to the stimulation of glycogen synthase by this hormone in human muscle. Lower fasting PP-1 activity in cytosol and glycogen fractions plus lower insulin-stimulated PP-1 activity could explain, in part, reduced insulin-stimulated glycogen synthase in skeletal muscle of insulin-resistant subjects.

Topics: Adult; Ethers, Cyclic; Female; Glycogen Synthase; Humans; Insulin; Insulin Resistance; Male; Muscles; Okadaic Acid; Phosphorylase Phosphatase; Phosphorylation

The insulin-like effects of okadaic acid (OKA) in rat adipocytes were further characterized. Okadaic acid did not alter insulin receptor function. This includes undisturbed insulin binding and receptor-mediated ligand internalization in OKA-treated cells. Also, the tyrosine kinase activity of the insulin receptor was not modified in a cell-free system. The stimulating effects of OKA were significantly increased by preincubating (40 min) the cells at 37 C. At lower temperatures (i.e. 26-30 C), OKA did not mimic insulin. Maximal stimulation of lipogenesis occurred at 0.5 microM and then declined at higher concentrations. The insulin-like effects of OKA on lipogenesis did not persist after removal of the agent by washing at 37 C. Okadaic acid maximally stimulated the incorporation of [1-14C]glucose into lipids and the oxidation of [6-14C]glucose into 14CO2, but unlike insulin, it had little if any effect of oxidizing [1-14C]glucose to 14CO2 or incorporating [6-14C]glucose into lipids. Okadaic acid was equivalent to insulin in stimulating 3-O-methyl-glucose uptake. Since the insulin-like effects of OKA did not persist after preincubation and washing, the effects of insulin in OKA-treated cells could be evaluated. The adipocytes were found to be fully refractory to the modulating actions of insulin. Thus, insulin did not stimulate glucose transport, its oxidation, or its incorporation into lipids, and failed to reverse lipolysis. Unresponsiveness was fully developed after 40-min preincubation at 37 C with 3 microM OKA and was half-maximal at 0.13 microM OKA. It persisted at least over a period of 150 min. The effect of OKA was restricted to the stimulating actions of insulin and vanadate. Basal activities were not altered, nor was the ability of the desensitized cells to respond to isoproterenol. The lack of an insulin-like effect of OKA on some metabolic pathways enabled us to demonstrate that OKA (0.25 microM) also rendered adipocytes fully unresponsive to insulin in the continuous presence of the agent. Western blotting of the 40,000 x g pellets with antibodies to phosphotyrosine revealed the appearance of a protein with an apparent mol wt of 43,000 in OKA-desensitized cells. In summary, OKA mimics some of insulin bioeffects, but concomitantly renders the cells tolerant to the modulating action of the hormone itself.

Topics: Adipose Tissue; Animals; Drug Resistance; Ethers, Cyclic; Glucose; Hexoses; Insulin; Insulin Resistance; Lipid Metabolism; Lipolysis; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Vanadates

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.

Topics: Animals; Biological Transport; Carrier Proteins; Ethers, Cyclic; Glucose; Glycogen; Glycolysis; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Mice; Muscle Proteins; Muscles; Obesity; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Receptor, Insulin