Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.     

Diverse effects of Glut 4 ablation on glucose uptake and glycogen synthesis in red and white skeletal muscle

The ability of muscles from Glut 4-null mice to take up and metabolize glucose has been studied in the isolated white EDL and red soleus muscles. In EDL muscles from male or female Glut 4-null mice, basal deoxyglucose uptake was lower than in control muscles and was not stimulated by insulin. In parallel, glycogen synthesis and content were decreased. Soleus muscles from male Glut 4-null mice took up twice more deoxyglucose in the absence of insulin than control muscles, but did not respond to insulin. In females, soleus deoxyglucose uptake measured in the absence of hormone was similar in Glut 4-null mice and in control mice. This uptake was stimulated twofold in Glut 4-null mice and threefold in control mice. Basal glycogen synthesis was increased by 4- and 2.2-fold in male and female null mice, respectively, compared to controls, and insulin had no or small (20% stimulation over basal) effect. These results indicate that while EDL muscles behaved as expected, soleus muscles were able to take up a large amount of glucose in the absence (males) or the presence of insulin (females). Whether this is due to a change in Glut 1 intrinsic activity or targeting and/or to the appearance of another glucose transporter remains to be determined.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.     

Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes

Phosphatidylinositol 3-kinase (PI 3-kinase) activation promotes glucose transporter 4 (Glut 4) translocation in adipocytes. In this study, we demonstrate that protein kinase B, a serine/threonine kinase stimulated by PI 3-kinase, is activated by both insulin and okadaic acid in isolated adipocytes, in parallel with their effects on Glut 4 translocation. In 3T3-L1 adipocytes, platelet-derived growth factor activated PI 3-kinase as efficiently as insulin but was only half as potent as insulin in promoting protein kinase B (PKB) activation. To look for a potential role of PKB in Glut 4 translocation, adipocytes were transfected with a constitutively active PKB (Gag-PKB) together with an epitope tagged transporter (Glut 4 myc). Gag-PKB was associated with all membrane fractions, whereas the endogenous PKB was mostly cytosolic. Expression of Gag-PKB led to an increase in Glut 4 myc amount at the cell surface. Our results suggest that PKB could play a role in promoting Glut 4 appearance at the cell surface following exposure of adipocytes to insulin and okadaic acid stimulation.     

Angiotensin-converting enzyme gene polymorphism in non-insulin dependent diabetes mellitus and its relationship with diabetic nephropathy

The effects of prolactin (PRL) on proliferation of cultured human uterine leiomyoma-derived smooth muscle cells (SMC) and its mechanism of action were investigated. PRL stimulated DNA synthesis and the expression of PRL receptor was identified by ribonuclease protection assay. Moreover, the regulation of mitogen-activated protein (MAP) kinase by PRL in leiomyoma-derived SMC was investigated. PRL stimulated MAP kinase activity, as detected by 32P incorporation into MAP-2, in a dose-dependent manner. PRL also rapidly stimulated MAP kinase phosphorylation as detected by in vivo phosphorylation using 32P labeling and phosphotyrosine immunoblotting. These results suggest that PRL stimulates the proliferation of human leiomyoma cells via the MAP kinase cascade.     

Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes

Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.     

Thiazolidinediones inhibit lipoprotein lipase activity in adipocytes

The thiazolidinediones troglitazone and BRL 49653 improve insulin sensitivity in humans and animals with insulin resistance. Adipose tissue lipoprotein lipase is an insulin-sensitive enzyme. We examined the effects of thiazolidinediones on lipoprotein lipase expression in adipocytes. When added to 3T3-F442A, 3T3-L1, and rat adipocytes in culture, troglitazone and BRL 49653 inhibited lipoprotein lipase activity. This inhibition was observed at concentrations as low as 0.1 microM and within 2 h after addition of the drug. Lipoprotein lipase activity was inhibited in differentiated adipocytes as well as the differentiating cells. Despite this decrease in enzyme activity, these drugs increased mRNA levels in undifferentiated 3T3-F442A and 3T3-L1 cells and had no effect on mRNA expression or synthesis of lipoprotein lipase in differentiated cells. Western blot analysis showed that these drugs did not affect the mass of the enzyme protein. Lipoprotein lipase activity in cultured Chinese hamster ovary cells was not inhibited by troglitazone. Glucose transport, biosynthesis of lipids from glucose or the biosynthesis of proteins were unaffected by thiazolidinediones in differentiated cells, whereas glucose transport and lipid biosynthesis were increased when these drugs were added during differentiation. These results show that troglitazone and BRL 49653 have a specific, post-translational inhibitory effect on lipoprotein lipase in adipocytes, yet they promote lipid accumulation and differentiation in preadipocytes.     

TNF-alpha stimulates glucose uptake in L6 myoblasts

The mechanism of TNF-alpha to regulate glucose metabolism remains unclear. To further delineate the TNF-alpha signal transduction pathway mediating glucose metabolism, we utilized L6 rat myoblasts which contain the receptors for the insulin-like growth factor-I (IGF-I) and TNF-alpha, and the ability of both ligands to stimulate glucose uptake was compared. IGF-I (6.5 nM) maximally stimulated glucose uptake 7-fold after 24 h incubation, while 23 nM TNF-alpha maximally stimulated glucose uptake 3-fold only after 48 h incubation. IGF-I receptor beta-subunit, insulin receptor substrate-1 (IRS-1), and mitogen-activated protein (MAP) kinase were all phosphorylated in response to 6.5 nM IGF-I after 10 min incubation. In contrast, the treatment with 23 nM TNF-alpha failed to phosphorylate either IGF-I receptor beta-subunit or IRS-1 but did phosphorylate MAP kinase as much as IGF-I did. Despite a similar extent to which TNF-alpha induced MAP kinase phosphorylation as IGF-I did, TNF-alpha stimulated glucose uptake less compared to IGF-I. The results indicate that MAP kinase phosphorylation is not sufficient for glucose uptake in L6 myoblasts. TNF-alpha-elicited signal transduction to glucose uptake may utilize a different pathway from that seen with IGF-I.     

Troglitazone prevents the inhibitory effects of inflammatory cytokines on insulin-induced adipocyte differentiation in 3T3-L1 cells

Tumor necrosis factor (TNF) is implicated in wasting syndromes and insulin resistance in chronic infection and obese-linked diabetes. TNF (10 ng/ml) inhibited adipocyte differentiation of 3T3-L1 cells, and in these TNF treated cells little insulin-stimulated glucose uptake was observed. Treatment of 3T3-L1 cells with troglitazone (1-10 microM) partially prevented this inhibitory effect of TNF on adipogenesis, and enhanced expression of C/EBP alpha and GLUT4, even in the presence of TNF. Troglitazone also prevented the inhibitory effects of interleukin-1, interleukin-6, and leukemia inhibitory factor, but not of transforming growth factor beta on adipocyte differentiation of 3T3-L1 cells. These effects might contribute to the antidiabetic effect of troglitazone in obese diabetic animals.     

Chronic effects of glucose on insulin signaling in A-10 vascular smooth muscle cells

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.     

Ethanol inhibition of insulin signaling in hepatocellular carcinoma cells

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyl phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such cells were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyl phosphorylation of the insulin receptor beta-subunit, tyrosyl phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyl phosphorylation of Shc. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on Shc phosphorylation, which activates p21ras through an IRS-1 independent pathway.     

Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro

1. The direct short-term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips. 2. In muscle strips from Sprague-Dawley rats, troglitazone (3.25 micromol l(-1)) increased basal and insulin-stimulated glucose transport by 24% and 41%, respectively (P<0.01 each). 3. In the presence of 5 nmol l(-1) insulin, stimulation of glucose transport by 3.25 micromol l(-1) troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling. 4. Whereas insulin retained its stimulant effect on [3H]-2-deoxy-glucose transport in hypoxia-stimulated muscle (by 44%; c.p.m. mg(-1) h(-1): 852+/-77 vs 1229+/-75, P<0.01), 3.25 micromol l(-1) troglitazone failed to increase glucose transport under hypoxic conditions (789+/-40 vs 815+/-28, NS) suggesting that hypoxia and troglitazone address a similar, non-insulin-like mechanism. 5. No differences between troglitazone and hypoxia were identified in respective interactions with insulin. 6. Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction-like manner, but it remains to be elucidated whether this contributes to the long-term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.     

Reconstitution of insulin signaling pathways in rat 3Y1 cells lacking insulin receptor and insulin receptor substrate-1. Evidence that activation of Akt is insufficient for insulin-stimulated glycogen synthesis or glucose uptake in rat 3Y1 cells

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.     

MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs

The mechanism by which fertilization initiates S-phase in the zygote is examined by manipulating the activity of MAP kinase in mature starfish eggs. These unfertilized eggs, which are arrested at G1-phase after the completion of meiosis, have high MAP kinase activity but undetectable cdc2 kinase activity. Either fertilization or inhibition of protein synthesis causes a decrease in MAP kinase activity, which is followed by DNA synthesis. Inactivation of MAP kinase with its specific phosphatase, CL100, initiates DNA synthesis in the absence of fertilization, while constitutive activation of MAP kinase with MEK represses the initiation of DNA synthesis following fertilization. Thus, in unfertilized mature starfish eggs, a capacity for DNA replication is already acquired, but entry into S-phase is negatively regulated by MAP kinase activity that is supported by a continuously synthesized protein(s) but not by cdc2 kinase. Upon fertilization, downregulation of MAP kinase activity is necessary and sufficient for triggering the G1/S-phase transition.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.