Y. Le Marchand-Brustel

MAP kinases and mTOR mediate insulin-induced phosphorylation of Insulin Receptor Substrate-1 on serine residues 307, 612 and 632

Aim/hypothesisInsulin-induced IRS-1 serine phosphorylation could be physiologically important to regulate insulin action. In a hyperinsulinaemic state such as obesity or Type 2 diabetes, this phosphorylation could be modified and exacerbate insulin resistance. We aimed at identifying serine residues in IRS-1 phosphorylated in response to insulin stimulation and at determining the involved kinases.Methods3T3-L1 adipocytes, muscle and adipose tissue of mice were subjected to Western Blot analysis with phosphospecific antibodies to identify phosphorylation sites in IRS-1 following insulin treatment. Pharmacological inhibitors were used to determine the serine kinases involved in this phosphorylation.ResultsIn 3T3-L1 adipocytes, insulin promoted the phosphorylation of serine 307, 612 and 632 with Serine612/632 more rapidly phosphorylated than Serine307. Insulin-induced phosphorylation of Serine307 was dependent on the activation of a PI 3-kinase/mTOR pathway. The phosphorylation of Serine612/632 required the activation of the MAP kinase pathway following short-term insulin stimulation and activation of the PI 3-kinase/mTOR pathway following prolonged insulin stimulation. Phosphorylation of Serine307 and Serine632 occurred in vivo in skeletal muscle and white adipose tissue of mice injected with insulin and was dependent on the activation of mTOR. Moreover, inhibition of mTOR led to a persistent PI 3-kinase activation by insulin.Conclusion/InterpretationInsulin-induced IRS-1 serine phosphorylation is a complex process involving different sites and kinases. This complexity could be physiologically important to accurately regulate insulin signalling. Abnormal phosphorylation of these serine residues in hyperinsulinaemic state could participate in the down-regulation of insulin signalling.

Defect in skeletal muscle phosphatidylinositol-3-kinase in obese insulin-resistant mice.

Activation of phosphatidylinositol-3-kinase (PI3K) is one of the earliest postreceptor events in the insulin signaling pathway. Incubation of soleus muscles from lean mice with 50 nM insulin caused a 3-10-fold increase in antiphosphotyrosine-immunoprecipitable PI3K (antiPTyr-PI3K) activity within 2 min in muscle homogenates as well as both the cytosolic and membrane fractions. Insulin did not affect total PI3K activity. Both the antiPTyr-PI3K stimulation and activation of insulin receptor tyrosine kinase were dependent on hormone concentration. In muscles from obese, insulin-resistant mice, there was a 40-60% decrease in antiPTyr-PI3K activity after 2 min of insulin that was present equally in the cytosolic and membrane fractions. A significant reduction in insulin sensitivity was also observed. The defect appears to result from alterations in both insulin receptor and postreceptor signaling. Starvation of obese mice for 48 h, which is known to reverse insulin resistance, normalized the insulin response of both PI3K and the receptor tyrosine kinase. The results demonstrate that: (a) antiPTyr-PI3K activity is responsive to insulin in mouse skeletal muscle, (b) both the insulin responsiveness and sensitivity of this activity are blunted in insulin-resistant muscles from obese mice, (c) these alterations result from a combination of insulin receptor and postreceptor defects, and (d) starvation restores normal insulin responses.

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

Characterization of insulin resistance and NIDDM in transgenic mice with reduced brown fat.

We recently created a new model of murine obesity through transgenic ablation of brown adipose tissue (BAT) using a tissue-specific toxigene (6). The goal of the present study was to further define the altered glucose homeostasis and insulin resistance in these transgenic animals. Despite an ∼30% increase in total body lipid, no abnormalities were observed in 6-week-old transgenic animals. At the age of 22–26 weeks, marked obesity in transgenic mice was associated with significant increases in blood glucose and plasma insulin levels and an abnormal response to both intraperitoneal glucose and insulin tolerance tests. Glucose transport in soleus muscle was reduced, with the response to insulin stimulation blunted by up to 85% in males and 55% in females. The total number of insulin receptors was decreased by 36% in muscle and 59% in adipose tissue of transgenic animals. Insulin receptor tyrosine kinase activity, which was assessed following maximal insulin stimulation in vivo, was reduced in transgenic animals by 59% in muscle and 56% in fat. GLUT4 mRNA and protein was unchanged in muscle of transgenic animals compared with in that of controls but was significantly reduced in adipose tissue. In conclusion, primary BAT deficiency results in the development of glucose intolerance or diabetes and severe insulin resistance with both receptor and postreceptor components. These animals should be a useful model for studies of obesity-linked diabetes and insulin resistance and related complications.

Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes

SummaryInsulin-induced glucose transport stimulation, which results from the translocation of glucose transporter 4 (GLUT 4)-containing vesicles, is completely blocked after prolonged insulin treatment of 3T3-L1 adipocytes. Since GLUT 4 expression was reduced by only 30%, we looked at the insulin signalling pathway in this insulin-resistant model. Insulin-induced tyrosine phosphorylation of the major insulin receptor substrate IRS 1 was reduced by 50±7%, while its expression was decreased by 70±4%. When cells were treated with wortmannin (a PI3-kinase inhibitor) together with insulin, the expression of IRS 1 diminished to a much lower extent. Associated with the decrease in IRS 1 expression and phosphorylation, the activation by insulin of antiphosphotyrosine immunoprecipitable PI3-kinase activity and of p44mapk and p42mapk activities was altered. However, the expression of these proteins was normal and p44mapk activity remained responsive to the tumour promoter TPA. Those results indicate that prolonged insulin treatment of 3T3-L1 adipocytes induces an insulin-resistant state with a reduced ability of insulin to stimulate the PI3-kinase and the MAP-kinases and a blockade of glucose transporter translocation.

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.

Anti-insulin receptor antibodies inhibit insulin binding and stimulate glucose metabolism in skeletal muscle

SummaryAutoantibodies against the insulin receptor are found in the serum of some patients with severe insulin resistance. The effects of one of these sera on insulin binding and on glucose transport and metabolism were investigated in the isolated mouse soleus muscle. Preincubation of muscles with the patients serum resulted in an inhibition of subsequent125I-insulin binding (half-maximal effect at 1∶500 dilution) and in a two to three-fold stimulation of glucose transport and metabolism (half-maximal effect at 1∶2000 dilution). The insulin-like effects were blocked by anti-human IgG, but not by antiinsulin antibodies. The magnitude of the serum effects on 2-deoxyglucose uptake and glycolysis was similar to that of insulin, but the effect on glycogen synthesis was smaller than that of insulin. It is suggested that the patients serum and insulin promote glucose transport and glycolysis through a common pathway, but act differently on glycogen synthesis.

Involvement of TNF-α in abnormal adipocyte and muscle sortilin expression in obese mice and humans

Aims/hypothesisInsulin resistance is caused by numerous factors including inflammation. It is characterised by defective insulin stimulation of adipocyte and muscle glucose transport, which requires the glucose transporter GLUT4 translocation towards the plasma membrane. Defects in insulin signalling can cause insulin resistance, but alterations in GLUT4 trafficking could also play a role. Our goal was to determine whether proteins controlling GLUT4 trafficking are altered in insulin resistance linked to obesity.MethodsUsing real-time RT-PCR, we searched for selected transcripts that were differentially expressed in adipose tissue and muscle in obese mice and humans. Using various adipocyte culture models and in vivo mice treatment, we searched for the involvement of TNF-α in these alterations in obesity.ResultsSortilin mRNA and protein were downregulated in adipose tissue from obese db/db and ob/ob mice, and also in muscle. Importantly, sortilin mRNA was also decreased in morbidly obese human diabetic patients. Sortilin and TNF-α (also known as TNF) mRNA levels were inversely correlated in mice and human adipose tissues. TNF-α decreased sortilin mRNA and protein levels in cultured mouse and human adipocytes, an effect partly prevented by the peroxisome proliferator-activated receptor γ activator rosiglitazone. TNF-α also inhibited adipocyte and muscle sortilin mRNA when injected to mice.Conclusions/interpretationSortilin, an essential player in adipocyte and muscle glucose metabolism through the control of GLUT4 localisation, is downregulated in obesity and TNF-α is likely to be involved in this defect. Chronic low-grade inflammation in obesity could thus contribute to insulin resistance by modulating proteins that control GLUT4 trafficking.

Positive and negative regulation of glucose uptake by hyperosmotic stress

This review will provide insight on the current understanding of the intracellular signaling mechanisms by which hyperosmolarity mimics insulin responses such as Glut 4 translocation and glucose transport but also antagonizes insulin effects. Glucose uptake induced by insulin is largely dependent on the PI 3-kinase/PKB pathway. In both adipocyte and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms which do not require PI 3-kinase/PKB pathway but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMP-Kinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of Gab1-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake. Apart of its insulin-like effects, hyperosmolarity can lead to cellular insulin resistance mediated by both prevention of PKB activation and inhibition of the Insulin Receptor Substrate-1 (IRS1) function. Serine phosphorylation and degradation of IRS1 negatively regulate its functions. Understanding how osmotic stress induces glucose transport or mediates insulin resistance may provide novel targets for strategies to enhance glucose transport or to prevent insulin resistance.

Rab proteins in endocytosis and Glut4 trafficking.

The intracellular trafficking of numerous proteins requires a tight control to fulfil their physiological functions. It is the case of the adipocyte and muscle glucose transporter Glut4 that is retained intracellularly until insulin induces its recruitment to the plasma membrane. Rabs are evolutionarily conserved small GTPases that control intracellular traffic events from yeast to mammalian cells. In the past few decades, considerable progresses have been made in identifying the route of Glut4, the Rabs involved in controlling it, and more recently the connection between insulin signalling and Glut4 trafficking through Rab activity control.