Rocio Vila-Bedmar

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.

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.

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.

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.

Increased Nitric Oxide Bioavailability in Adult GRK2 Hemizygous Mice Protects Against Angiotensin II–Induced Hypertension

G protein–coupled receptor kinase 2 (GRK2) is a ubiquitous serine/threonine protein kinase able to phosphorylate and desensitize the active form of several G protein–coupled receptors. Given the lack of selective inhibitors for GRK2, we investigated the effects elicited by GRK2 inhibition in vascular responses using global adult hemizygous mice (GRK2+/−). The vasodilator responses to acetylcholine or isoproterenol were increased in aortas and mesenteric resistance arteries from GRK2+/− mice compared with wild-type (WT) littermates. After angiotensin II (AngII) infusion, GRK2+/− mice were partially protected against hypertension, vascular remodeling, and mechanical alterations, even when resting basal blood pressures were not significantly different. AngII infusion also (1) increased GRK2 levels in WT but not in GRK2+/− vessels; (2) increased vasoconstrictor responses to phenylephrine in WT but not in GRK2+/− mice; and (3) decreased vasodilator responses to acetylcholine and vascular pAkt and eNOS levels more in WT than in GRK2+/− animals. Vascular NO production and the modulation of vasoconstrictor responses by endothelial-derived NO remained enhanced in GRK2+/− mice infused with AngII. Thus, GRK2+/− mice are resistant to the development of vascular remodeling and mechanical alterations, endothelial dysfunction, increased vasoconstrictor responses, and hypertension induced by AngII at least partially through the preservation of NO bioavailability. In conclusion, our results describe an important role for GRK2 in systemic hypertension and further establish that an inhibition of GRK2 could be a beneficial treatment for this condition.

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.

A new era for brown adipose tissue: New insights into brown adipocyte function and differentiation.

Until quite recently, brown adipose tissue was considered of metabolic significance only in small mammals and human newborns, since it was thought to disappear rapidly after birth in humans. However, nowadays this tissue is known to play a role in the regulation of energy balance not only in rodents, but also in humans. In this review we highlight new features regarding brown adipose tissue origin and function and revise old paradigms about brown adipocyte differentiation.

Concerted expression of the thermogenic and bioenergetic mitochondrial protein machinery in brown adipose tissue

Brown adipose tissue (BAT) is specialized in non‐shivering thermogenesis through the expression of the mitochondrial uncoupling protein‐1 (UCP1). In this paper, we describe the relationship between UCP1 and proteins involved in ATP synthesis. By the use of BATIRKO mice, which have enhanced UCP1 expression in BAT, an increase in ATP synthase as well as in ubiquinol cytochrome c reductase levels was observed. Alterations in mitochondrial mass or variations in ATP levels were not observed in BAT of these mice. In addition, using a protocol of brown adipocyte differentiation, the concerted expression of UCP1 with ATP synthase was found. These two scenarios revealed that increases in the uncoupling machinery of brown adypocites must be concomitantly followed by an enhancement of proteins involved in ATP synthesis. These concerted changes reflect the need to maintain ATP production in an essentially uncoupling cell type. J. Cell. Biochem. 114: 2306–2313, 2013.