Iria Nieto-Vazquez

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.

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.

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.

Molecular mechanisms involved in obesity-associated insulin resistance: therapeutical approach.

Insulin resistance is an important contributor to the pathogenesis of T2D and obesity is a risk factor for its development. It has been demonstrated that these obesity-related metabolic disorders are associated with a state of chronic low-intensity inflammation. Several mediators released from adipocytes and macrophages, such as the pro-inflammatory cytokines TNF-alpha and IL-6, have been suggested to impair insulin action in peripheral tissues, including fat and skeletal muscle. Such insulin resistance can initially be compensated by increased insulin secretion, but the prolonged presence of the hormone is detrimental for insulin sensitivity. Stress and pro-inflamatory kinases as well as more recent players, phosphatases, seem to be involved in the molecular mechanisms by which pro-inflammatory cytokines and hyperinsulinemia disrupt insulin signalling at the level of IRSs. Pharmacological approaches, such as treatment with PPAR and LXR agonists, overcome such insulin resistance, exerting anti-inflamatory properties as well as controlling the expression of cytokines with tissular specificity.

Liver X receptor agonists ameliorate TNFα-induced insulin resistance in murine brown adipocytes by downregulating protein tyrosine phosphatase-1B gene expression

Aims/hypothesisThe nuclear receptors, including nuclear receptor subfamily 1, group H, member 3 (NR1HR, also known as liver X receptor [LXR]), are sensors of cholesterol metabolism and lipid biosynthesis that have recently been proposed as insulin sensitisers. TNFα has been described as a link between obesity and the development of insulin resistance, an important contributor to the pathogenesis of type 2 diabetes. Therefore, we decided to investigate the ability of NR1HR agonists to ameliorate TNFα-induced insulin resistance in brown adipocytes.MethodsPrimary brown adipocytes from rat fetuses, and from wild-type neonate mice and neonate mice deficient in the gene encoding protein tyrosine phosphatase-1B (Ptpn1, also known as Ptp1b) were cultured in the absence or presence of TNFα and different nuclear receptor agonists. Among them, the unrelated NR1HR ligands T0901317, GW3965 and (22R)-hydroxycholesterol were tested. After insulin stimulation, glucose uptake and solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) translocation were measured. Next the insulin signalling cascade was determined by submitting cells to lysis, immunoprecipitation and immunoblotting.ResultsNR1HR agonists ameliorate TNFα-induced insulin resistance restoring completely insulin-stimulated glucose uptake and SLC2A4 translocation to plasma membrane. This effect is parallel to the recovery of the insulin cascade insulin receptor/IRS-2/phosphatidylinositol 3-kinase/protein kinase B, and could be due to the fact that T0901317 prevents the increase of PTPN1 production and phosphatase activity produced by TNFα. In this regard, Ptpn1-deficient brown adipocytes showed protection against insulin resistance by TNFα. Moreover, we observed that T0901317 produced in itself a significant increase over basal glucose uptake consistent with an increase of SLC2A4 protein content in plasma membrane, attributable to the activation of protein kinase ζ and/or the increase of Slc2a4 expression.Conclusions/interpretationNuclear receptors NR1HR are interesting potential targets for drug treatment of insulin resistance.

Hyperinsulinemia Induces Insulin Resistance on Glucose and Lipid Metabolism in a Human Adipocytic Cell Line: Paracrine Interaction with Myocytes

CONTEXT Adipocytes release a variety of factors which deregulation could provide the basis for complications such as insulin resistance, an early defect on the onset of type 2 diabetes. Such insulin resistance can initially be overcome by compensatory hyperinsulinemia, but the prolonged presence of the hormone can be detrimental for insulin sensitivity. OBJECTIVE The objective of the study was to dissect the molecular mechanisms that may regulate hyperinsulinemia-induced insulin resistance in a human liposarcoma cell line and its paracrine interactions with a human rhabdomyosarcoma cell line. DESIGNS We studied glucose uptake, lipolysis, insulin signaling, and secretion pattern at different days of adipocyte differentiation in the presence of insulin. RESULTS Adipocytes differentiated for 14 d gain insulin sensitivity on glucose uptake and inhibition of lipolysis, but prolonged cultures develop an insulin-resistant state characterized by an increase in phosphatase and tensin homolog-deleted on chromosome 10 expression and defects in insulin signaling at the insulin receptor substrate-1/AKT level. The secretion pattern of nonesterified fatty acids, IL-6, adiponectin, leptin, and monocyte chemotactic protein-1 was in keeping with the changes in insulin sensitivity during differentiation. An inverse biphasic response was also observed in human myocytes when they were cultured with various adipocyte-conditioned media, although insulin resistance was detected earlier than in adipocytes. This behavior mimics hyperinsulinemia because insulin action was restored when adipocytes were cultured in the absence of the hormone. Pharmacological treatment of adipocytes with a liver X receptor agonist reestablishes insulin-stimulated glucose uptake, whereas treatment with a peroxisome proliferator-activated receptor-gamma agonist restored the antilipolytic action of insulin. CONCLUSIONS Hyperinsulinemia deregulates adipocyte secretion pattern, producing insulin resistance in adipocytes and myocytes, a situation that can be ameliorated with nuclear receptor agonists.

GRK2 contribution to the regulation of energy expenditure and brown fat function

Obesity is a major health problem and an important risk factor for the development of multiple disorders. Previous studies in our laboratory have revealed that down‐regulation of GRK2 decreases age‐related adiposity, but the physiological and molecular mechanisms underlying this outcome remain unclear. We evaluate whether the lean phenotype results from a direct effect of GRK2 on energy homeostasis. The study of white adipose tissue (WAT) in wild‐type (WT) and GRK2+/− littermates showed a reduced expression of lipogenic enzymes and enhanced lipolytic rate in adult GRK2+/− mice. Moreover, hemizygous mice display higher energy expenditure and lower respiratory exchange ratio. Analysis of brown adipose tissue (BAT) from adult GRK2+/− mice showed a less deteriorated morphology associated with age compared to WT, which is correlated with a higher basal core temperature. BAT from young GRK2+/− mice showed an increase in gene expression of thermogenesis‐related genes. Accordingly, hemizygous mice displayed better thermogenic capacity and exhibited a more oxidative phenotype in both BAT and WAT than WT littermates. Overexpression of GRK2 in brown adipocytes corroborated the negative effect of this kinase in BAT function and differentiation. Collectively, our data point to GRK2 inhibition as a potential tool for the enhancement of brown fat activity, which may have important therapeutic implications for the treatment of obesity and associated metabolic disorders.—Vila‐Bedmar, R., Garcia‐Guerra, L., Nieto‐Vazquez, I., Mayor, F., Jr., Lorenzo, M., Murga, C., Fernández‐Veledo, S. GRK2 contribution to the regulation of energy expenditure and brown fat function. FASEB J. 26, 3503–3514 (2012). www.fasebj.org

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.