Margarita Lorenzo

Insulin resistance associated to obesity: the link TNF-alpha

Abstract Adipose tissue secretes proteins which may influence insulin sensitivity. Among them, tumour necrosis factor (TNF)-alpha has been proposed as a link between obesity and insulin resistance because TNF-alpha is overexpressed in adipose tissue from obese animals and humans, and obese mice lacking either TNF-alpha or its receptor show protection against developing insulin resistance. The activation of proinflammatory pathways after exposure to TNF-alpha induces a state of insulin resistance in terms of glucose uptake in myocytes and adipocytes that impair insulin signalling at the level of the insulin receptor substrate (IRS) proteins. The mechanism found in brown adipocytes involves Ser phosphorylation of IRS-2 mediated by TNF-alpha activation of MAPKs. The Ser307 residue in IRS-1 has been identified as a site for the inhibitory effects of TNF-alpha in myotubes, with p38 mitogen-activated protein kinase (MAPK) and inhibitor kB kinase being involved in the phosphorylation of this residue. Moreover, up-regulation of protein-tyrosine phosphatase (PTP)1B expression was recently found in cells and animals treated with TNF-alpha. PTP1B acts as a physiological negative regulator of insulin signalling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1B expression is increased in peripheral tissues from obese and diabetic humans and rodents. Accordingly, down-regulation of PTP1B activity by treatment with pharmacological agonists of nuclear receptors restores insulin sensitivity in the presence of TNF-alpha. Furthermore, mice and cells deficient in PTP1B are protected against insulin resistance induced by this cytokine. In conclusion, the absence or inhibition of PTP1B in insulin-target tissues could confer protection against insulin resistance induced by cytokines.

DUAL ROLE OF INTERLEUKIN-6 IN REGULATING INSULIN SENSITIVITY IN MURINE SKELETAL MUSCLE

OBJECTIVE—Cytokines are elevated in various insulin-resistant states, including type 2 diabetes and obesity, although the contribution of interleukin-6 (IL-6) in the induction of these diseases is controversial. RESEARCH DESIGN AND METHODS—We analyzed the impact of IL-6 on insulin action in murine primary myocytes, skeletal muscle cell lines, and mice (wild type and protein-tyrosine phosphatase 1B [PTP1B] deficient). RESULTS—IL-6 per se increased glucose uptake by activating serine/threonine protein kinase 11 (LKB1)/AMP-activated protein kinase/protein kinase B substrate of 160 kDa (AS160) pathway. A dual effect on insulin action was observed when myotubes and mice were exposed to this cytokine: additive with short-term insulin (increased glucose uptake and systemic insulin sensitivity) but chronic exposure produced insulin resistance (impaired GLUT4 translocation to plasma membrane and defects in insulin signaling at the insulin receptor substrate 1 [IRS-1] level). Three mechanisms seem to operate in IL-6–induced insulin resistance: activation of c-Jun NH2-terminal kinase 1/2 (JNK1/2), accumulation of suppressor of cytokine signaling 3 (socs3) mRNA, and an increase in PTP1B activity. Accordingly, silencing JNK1/2 with either small interfering RNA or chemical inhibitors impaired phosphorylation of IRS-1 (Ser307), restored insulin signaling, and normalized insulin-induced glucose uptake in myotubes. When using a pharmacological approach, liver X receptor agonists overcome IL-6–induced insulin resistance by producing downregulation of socs3 and ptp1b gene expression. Finally, the lack of PTP1B confers protection against IL-6–induced insulin resistance in skeletal muscle in vitro and in vivo, in agreement with the protection against the IL-6 hyperglycemic effect observed on glucose and insulin tolerance tests in adult male mice. CONCLUSIONS—These findings indicate the important role of IL-6 in the pathogenesis of insulin resistance and further implicate PTP1B as a potential therapeutic target in the treatment of type 2 diabetes.

Insulin produces myogenesis in C2C12 myoblasts by induction of NF-?B and downregulation of AP-1 activities

In the present study, we have examined the insulin‐signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated β‐chain insulin receptor, activated the phosphatidylinositol (PI) 3‐kinase/Akt/p70S6‐kinase signaling pathway, as well as phosphorylated both p44/p42‐ and p38‐mitogen‐activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum‐free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor‐κB (NF‐κB) DNA binding activity and down‐regulated activating protein‐1 (AP‐1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin‐signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3‐kinase), rapamycin (a p70S6‐kinase blocker), and SB203580 or PD169316 (p38‐MAPK inhibitors). These inhibitors abolished insulin induction of NF‐κB DNA binding activity and κB‐chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IκB‐α protein, and increased AP‐1 DNA binding activity and TRE‐CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3‐kinase/p70S6‐kinase and p38‐MAPK pathways, the signaling through p44/p42‐MAPK being inhibited. J. Cell. Physiol. 186:82–94, 2001.

IGF-I : A MITOGEN ALSO INVOLVED IN DIFFERENTIATION PROCESSES IN MAMMALIAN CELLS

The main source of insulin-like growth factor I (IGF-I) postnatally is the liver, under growth hormone stimulation, although IGF-I is already present in embryonic tissues and in fetal serum, when its expression is independent of growth hormone. The extracellular alpha-subunit of the IGF-I receptor (IGF-IR) contains an IGF-I binding domain, and the beta-subunit possesses tyrosine kinase activity, which is greatly enhanced when IGF-I binds to the alpha-subunit and leads to its autophosphorylation. Insulin receptor substrate 1 (IRS-1) is the most well characterized cellular substrate for IGF-I, containing at least 20 potential tyrosine phosphorylation sites. The tyrosine phosphorylated form of IRS-1 acts as a docking protein by associating SH2-containing proteins including the p85 regulatory subunit of phosphatidylinositol-3-kinase (P13-kinase), the protein tyrosine phosphatase SH-PTP2, the SH2- and SH3-containing adaptor protein Nck and the growth factor receptor-bound protein-2 (Grb2/Sem5) protein. Grb2 is found associated with mSOS, a GTP/GDP exchange factor involved in converting the inactive Ras-GDP to the active Ras-GTP. The p85 regulatory subunit of PI3-kinase can be also a direct in vitro substrate of the IGF-IR. Although IRS-1 is the major substrate of the IGF-IR, there is another early phosphotyrosine substrate termed SHC, which also activates Ras via Grb2-mSos complex. Activation of p21-Ras induces a serine/threonine kinase cascade leading to the activation of MAP-kinases. The importance of IGF-I as a mitogen throughout development has been clearly demonstrated in IGF-I and IGF-IR knockout mouse studies and also in transgenic mice over-expressing IGF-I. IGF-I is a mitogen in many cell types in culture such as T lymphocytes, chondrocytes or osteoblasts and it is considered to be a progression factor in mouse fibroblasts. IGF-I is also involved in muscle, neurons and adipogenic differentiation of mesenchymal cells. However, IGF-I induces proliferation and differentiation in fetal brown adipocytes, suggesting that both cellular processes are not necessarily mutually exclusive in fetal cells.

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts.

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3‐kinase/p70S6‐kinase and p38‐mitogen‐activated protein kinase (MAPK) and downregulation of p42/p44‐MAPK. This study investigated the insulin‐signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF‐I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated β‐chain insulin receptor and IRS‐1 tyrosine phosphorylation, IRS‐2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS‐1 was found under insulin stimulation. Insulin stimulated IRS‐1 association with p85α leading to the activation of PI3‐kinase, and, subsequently AKT and p70S6‐kinases. Moreover, both p42/p44‐ and p38‐MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in (3H)‐thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6‐kinase (either by rapamycin or by the PI3‐kinase inhibitor LY294002) as well as by inhibition of p44/p42‐MAPK with PD098059, but was not affected by inhibition of p38‐MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum‐deprived cells from apoptosis in an AKT‐dependent manner, as demonstrated by the inhibition of AKT‐activity by the use of LY294002 and ML‐9, meanwhile neither inhibition of p70S6‐kinase, nor MAPK affected insulin‐induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38‐MAPK, as demonstrated by the use of the p38‐MAPK inhibitors SB203580 or PD169316, but independent of PI3‐kinase and p42/p44‐MAPK.

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3‐kinase‐dependent but p70S6‐kinase‐independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin‐induced glucose uptake by the use of a chemical compound, ML‐9, as well as by transfection with a dominant‐negative form of Akt (ΔAkt). Pretreatment with ML‐9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML‐9 did not affect insulin‐stimulated PI3‐kinase nor PKCζ activities. In consequence, ML‐9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, ΔAkt impaired insulin stimulation of glucose uptake and GFP‐tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML‐9 treatment for 6 h down‐regulated insulin‐induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co‐transfection of brown adipocytes with ΔAkt precluded the transactivation of GLUT4‐CAT promoter by insulin in a similar fashion as a dominant‐negative form of PI3‐kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.

G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

OBJECTIVE Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.

Protein–Tyrosine Phosphatase 1B–Deficient Myocytes Show Increased Insulin Sensitivity and Protection Against Tumor Necrosis Factor-α–Induced Insulin Resistance

Protein–tyrosine phosphatase (PTP)1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. In this study, we have assessed the role of PTP1B in the insulin sensitivity of skeletal muscle under physiological and insulin-resistant conditions. Immortalized myocytes have been generated from PTP1B-deficient and wild-type neonatal mice. PTP1B−/− myocytes showed enhanced insulin-dependent activation of insulin receptor autophosphorylation and downstream signaling (tyrosine phosphorylation of insulin receptor substrate [IRS]-1 and IRS-2, activation of phosphatidylinositol 3-kinase, and serine phosphorylation of AKT), compared with wild-type cells. Accordingly, PTP1B−/− myocytes displayed higher insulin-dependent stimulation of glucose uptake and GLUT4 translocation to the plasma membrane than wild-type cells. Treatment with tumor necrosis factor-α (TNF-α) induced insulin resistance on glucose uptake, impaired insulin signaling, and increased PTP1B activity in wild-type cells. Conversely, the lack of PTP1B confers protection against insulin resistance by TNF-α in myocyte cell lines and in adult male mice. Wild-type mice treated with TNF-α developed a pronounced hyperglycemia along the glucose tolerance test, accompanied by an impaired insulin signaling and increased PTP1B activity in muscle. However, mice lacking PTP1B maintained a rapid clearance of glucose and insulin sensitivity and displayed normal muscle insulin signaling regardless the presence of TNF-α.

Adenosine 5′-Monophosphate-Activated Protein Kinase-Mammalian Target of Rapamycin Cross Talk Regulates Brown Adipocyte Differentiation

Brown adipose tissue (BAT) is considered of metabolic significance in mammalian physiology, because it plays an important role in regulating energy balance. Alterations in this tissue have been associated with obesity and type 2 diabetes. The molecular mechanisms modulating brown adipocyte differentiation are not fully understood. Using a murine brown preadipocyte cell line, primary cultures, and 3T3-L1 cells, we analyzed the contribution of various intracellular signaling pathways to adipogenic and thermogenic programs. Sequential activation of p38MAPK and LKB1-AMPK-tuberous sclerosis complex 2 (TSC2) as well as significant attenuation of ERK1/2 and mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (p70S6K1) activation was observed through the brown differentiation process. This study demonstrates a critical role for AMPK in controlling the mTOR-p70S6K1 signaling cascade in brown but not in 3T3-L1 adipocytes. We observed that mTOR activity is essential in the first stages of differentiation. Nevertheless, subsequent inhibition of this cascade by AMPK activation is also necessary at later stages. An in vivo study showed that prolonged 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced AMPK activation increases uncoupling protein 1 expression and induces an accumulation of brown adipocytes in white adipose tissue (WAT), as revealed by immunohistology. Moreover, the induction of brown adipogenesis in areas of white fat partially correlates with the body weight reduction detected in response to treatment with AICAR. Taken together, our study reveals that differentiation of brown adipocytes employs different signaling pathways from white adipocytes, with AMPK-mTOR cross talk a central mediator of this process. Promotion of BAT development in WAT by pharmacological activation of AMPK may have potential in treating obesity by acting on energy dissipation.

c-Jun N-Terminal Kinase 1/2 Activation by Tumor Necrosis Factor-α Induces Insulin Resistance In Human Visceral But Not Subcutaneous Adipocytes: Reversal by Liver X Receptor Agonists

AIMS Obesity is associated with a chronic systemic low-grade inflammatory state. Markers of inflammation such as TNF-alpha are linked with increased risk for insulin resistance and type 2 diabetes. The objective of the present study was to dissect the molecular mechanisms that may regulate TNF-alpha-induced insulin resistance in human adipose tissue. METHODS We analyzed the impact of TNF-alpha on glucose uptake and insulin action in human visceral and sc adipocytes. The contribution of different intracellular signaling pathways on metabolic effects of TNF-alpha and the reversal of some of these effects with nuclear receptor agonists were also studied. RESULTS TNF-alpha per se increased glucose transporter-4 translocation to the plasma membrane and glucose uptake by activating the AMP-activated protein kinase/AS160 pathway in both visceral and sc adipocytes. Nevertheless, this cytokine induced an insulin-resistant state in visceral adipocytes by impairing insulin-stimulated glucose uptake and insulin signaling at the insulin receptor substrate (IRS)-1/AKT level. Activation of c-Jun N-terminal kinase (JNK) 1/2 seems to be involved in TNF-alpha-induced insulin resistance, causing phosphorylation of IRS1 at the Ser312 residue. Accordingly, silencing JNK1/2 with either small interfering RNA or chemical inhibitors impaired serine phosphorylation of IRS1, restored downstream insulin signaling, and normalized insulin-induced glucose uptake in the presence of TNF-alpha. Furthermore, TNF-alpha increased the secretion of other proinflammatory cytokines such as IL-6. Pharmacological treatment of adipocytes with liver X receptor agonists reestablished insulin sensitivity by impairing TNF-alpha induction of JNK1/2, phosphorylation of IRS1 (Ser312), and stabilizing IL-6 secretion. CONCLUSIONS TNF-alpha induces insulin resistance on glucose uptake in human visceral but not sc adipocytes, suggesting depot-specific effects of TNF-alpha on glucose uptake. Activation of JNK1/2 appears to be involved in serine phosphorylation of IRS1 and subsequently insulin resistance on glucose uptake, a state that can be reversed by liver X receptor agonists.