PTP1B deficiency enhances liver growth during suckling by increasing the expression of insulin‐like growth factor‐I

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin and tyrosine kinase growth factor signaling. We have recently demonstrated that PTP1B deficiency increases GLUT2/insulin receptor (IR) A complexes and glucose uptake in suckling, but not adult, primary hepatocytes. Herein we have investigated intrahepatic glucose utilization in 3–5 days old wild‐type and PTP1B^?/? mice. PTP1B deficiency decreased glycogen, lactate, and pyruvate content in the livers from suckling mice. Conversely, the activity of glucose 6‐phosphate dehydrogenase (G6PD), the rate limiting enzyme of the pentose phosphate cycle (PPC) which provides substrates for DNA synthesis, was enhanced in the liver of PTP1B^?/? animals. Liver weight, liver‐to‐body mass ratio, DNA content, and PCNA expression were increased in PTP1B^?/? suckling mice compared to the wild‐type controls. At the molecular level, STAT 5B phosphorylation, IGF‐I mRNA, and protein levels as well as IGF‐IR tyrosine phosphorylation were increased in the livers of PTP1B‐deficient neonates. Unexpectedly, hepatic and serum triglycerides (TG) were increased by PTP1B deficiency, although the expression of lipogenic enzymes remained as in the wild‐type controls. However, the analysis of milk composition revealed higher TG content in lactating females lacking PTP1B. The effects of PTP1B deficiency on G6PD activity, STAT 5B/IGF‐I/IGF‐IR axis, PCNA expression and liver growth during suckling were maintained by transferring PTP1B^?/? embryos (PTP1B^?/?T) to a wild‐type female. Conversely, PTP1B^?/?T mice did not show hepatic fat accumulation. In conclusion, the present study suggests that PTP1B plays a unique role in the control of the physiological liver development after birth. J. Cell. Physiol. 225: 214–222, 2010. © 2010 Wiley‐Liss, Inc.     

Role of insulin receptor and balance in insulin receptor isoforms A and B in regulation of apoptosis in simian virus 40-immortalized neonatal hepatocytes

We have investigated the unique role of the insulin receptor (IR) and the balance of its isoforms A and B in the regulation of apoptosis in simian virus 40 (SV40)-immortalized neonatal hepatocytes. Immortalized hepatocytes lacking (HIR KO) or expressing the entire IR (HIR LoxP), and cells expressing either IRA (HIR RecA) or IRB (HIR RecB) have been generated. IR deficiency in hepatocytes increases sensitivity to the withdrawal of growth factors, because these cells display an increase in reactive oxygen species, a decrease in Bcl-x(L), a rapid accumulation of nuclear Foxo1, and up-regulation of Bim. These events resulted in acceleration of caspase-3 activation, DNA laddering, and cell death. The single expression of either IRA or IRB produced a stronger apoptotic phenotype. In these cells, protein complexes containing IRA or IRB and Fas/Fas-associating protein with death domain activated caspase-8, and, ultimately, caspase-3. In hepatocytes expressing IRA, Bid cleavage and cytochrome C release were increased whereas direct activation of caspase-3 by caspase-8 and a more rapid apoptotic process occurred in hepatocytes expressing IRB. Conversely, coexpression of IRA and IRB in IR-deficient hepatocytes rescued from apoptosis. Our results suggest that balance alteration of IRA and IRB may serve as a ligand-independent apoptotic trigger in hepatocytes, which may regulate liver development.     

Protein-tyrosine phosphatase 1B deficiency protects against Fas-induced hepatic failure

Genetic disruption of protein-tyrosine phosphatase 1B (PTP1B) in mice leads to increased insulin sensitivity and resistance to weight gain. Although PTP1B has been implicated as a regulator of multiple signals, its function in other physiological responses in vivo is poorly understood. Here we demonstrate that PTP1B-null mice are resistant to Fas-induced liver damage and lethality, as evident by reduced hepatic apoptosis in PTP1B-null versus wild type mice and reduced levels of circulating liver enzymes. Activation of pro-apoptotic caspases-8, -9, -3, and -6 was attenuated in livers from PTP1B-null mice following Fas receptor stimulation, although components of the death-inducing signaling complex were intact. Activation of anti-apoptotic regulators, such as the hepatocyte growth factor/Met receptor tyrosine kinase, as well as Raf, ERK1/2, FLIP(L), and the NF-kappaB pathway, was elevated in response to Fas activation in livers from PTP1B-null mice. Using PTP1B-deficient primary hepatocytes, we show that resistance to Fas-mediated apoptosis is cell autonomous and that signals involving the Met, ERK1/2, and NF-kappaB pathways are required for cytoprotection. This study identifies a previously unknown physiological role for PTP1B in Fas-mediated liver damage and points to PTP1B as a potential therapeutic target against hepatotoxic agents.     

Levels of protein tyrosine phosphatase 1B determine susceptibility to apoptosis in serum-deprived hepatocytes

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. To assess the importance of PTP1B in the balance between death and survival in the liver, we have developed immortalized neonatal hepatocyte cell lines lacking (PTP1B(-/-)) or overexpressing (PTP1B(+/+PTP1B)) PTP1B. Early activation of caspase-3 occurred in PTP1B(+/+PTP1B) hepatocytes but was nearly abolished in PTP1B(-/-) cells. At the molecular level, PTP1B overexpression/deficiency altered the balance of pro-(Bim) and anti-(Bcl-x(L)) apoptotic members of the Bcl-2 family upon serum withdrawal. Likewise, cytosolic cytochrome C increased rapidly in PTP1B(+/+PTP1B) hepatocytes whereas it was retained in the mitochondria of PTP1B(-/-) cells. DNA fragmentation and the increase of apoptotic cells induced by serum withdrawal in wild-type (PTP1B(+/+)) hepatocytes were absent in PTP1B(-/-) cells. Conversely, overexpression of PTP1B accelerated DNA laddering and increased the number of apoptotic cells. In serum-deprived PTP1B(+/+PTP1B) hepatocytes, a rapid entry of Foxo1 into the nucleus and an earlier activation of caspase-8 was observed. However, both events were suppressed in PTP1B(-/-) hepatocytes. Moreover, PTP1B deficiency conferred resistance to apoptosis induced by activation of Fas and constitutively active Foxo1. Rescue of PTP 1B in deficient hepatocytes recovered the phenotype of wild-type cells whereas reduction of PTP1B by siRNA suppressed apoptosis. Our results reveal a unique role for PTP1B as a mediator of the apoptotic pathways triggered by trophic factors withdrawal in hepatocytes. This novel mechanism may represent an important target in the design of therapeutic strategies for human liver regeneration after pathological damage as well as for treatment of hepatocarcinomas.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatase-1B

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.     

Protein-tyrosine phosphatase 1B (PTP1B) deficiency confers resistance to transforming growth factor-β (TGF-β)-induced suppressor effects in hepatocytes

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, mediating both tumor suppressor and promoter effects. The suppressor effects of the cytokine can be negatively regulated by activation of survival signals, mostly dependent on tyrosine kinase activity. The aim of our work was to study the role of the protein-tyrosine phosphatase 1B (PTP1B) on the cellular responses to TGF-β, using for this purpose immortalized neonatal hepatocytes isolated from both PTP1B(+/+) and PTP1B(-/-) mice. We have found that PTP1B deficiency conferred resistance to TGF-β suppressor effects, such as apoptosis and growth inhibition, correlating with lower Smad2/Smad3 activation. Both responses were recovered in the presence of the general tyrosine kinase inhibitor genistein. PTP1B(-/-) cells showed elevated NF-κB activation in response to TGF-β. Knockdown of the NF-κB p65 subunit increased cell response in terms of Smads phosphorylation and apoptosis. Interestingly, these effects were accompanied by inhibition of Smad7 up-regulation. In addition, lack of PTP1B promoted an altered NADPH oxidase (NOX) expression pattern in response to TGF-β, strongly increasing the NOX1/NOX4 ratio, which was reverted by genistein and p65 knockdown. Importantly, NOX1 knockdown inhibited nuclear translocation of p65, promoted Smad phosphorylation, and decreased Smad7 levels. In summary, our results suggest that PTP1B deficiency confers resistance to TGF-β through Smad inhibition, an effect that is mediated by NOX1-dependent NF-κB activation, which in turn, increases the level of the Smad inhibitor Smad7 and participates in a positive feedback loop on NOX1 up-regulation.     

Protein-tyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance

Mice heterozygous for insulin receptor (IR) and IR substrate (IRS)-1 deficiency provide a model of polygenic type 2 diabetes in which early-onset, genetically programmed insulin resistance leads to diabetes. Protein-tyrosine phosphatase 1B (PTP1B) dephosphorylates tyrosine residues in IR and possibly IRS proteins, thereby inhibiting insulin signaling. Mice lacking PTP1B are lean and have increased insulin sensitivity. To determine whether PTP1B can modify polygenic insulin resistance, we crossed PTP1B-/- mice with mice with a double heterozygous deficiency of IR and IRS-1 alleles (DHet). DHet mice weighed slightly less than wild-type mice and exhibited severe insulin resistance and hyperglycemia, with approximately 35% of DHet males developing diabetes by 9-10 weeks of age. Body weight in DHet mice with PTP1B deficiency was similar to that in DHet mice. However, absence of PTP1B in DHet mice markedly improved glucose tolerance and insulin sensitivity at 10-11 weeks of age and reduced the incidence of diabetes and hyperplastic pancreatic islets at 6 months of age. Insulin-stimulated phosphorylation of IR, IRS proteins, Akt/protein kinase B, glycogen synthase kinase 3beta, and p70(S6K) was impaired in DHet mouse muscle and liver and was differentially improved by PTP1B deficiency. In addition, increased phosphoenolpyruvate carboxykinase expression in DHet mouse liver was reversed by PTP1B deficiency. In summary, PTP1B deficiency reduces insulin resistance and hyperglycemia without altering body weight in a model of polygenic type 2 diabetes. Thus, even in the setting of high genetic risk for diabetes, reducing PTP1B is partially protective, further demonstrating its attractiveness as a target for prevention and treatment of type 2 diabetes.     

Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B

Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle triglycerides and serum adipokines are also affected similarly by HFD in both groups. Nevertheless, muscle-specific PTP1B(-/-) mice exhibit increased muscle glucose uptake, improved systemic insulin sensitivity, and enhanced glucose tolerance. These findings correlate with and are most likely caused by increased phosphorylation of the insulin receptor and its downstream signaling components. Thus, muscle PTP1B plays a major role in regulating insulin action and glucose homeostasis, independent of adiposity. In addition, rosiglitazone treatment of HFD-fed control and muscle-specific PTP1B(-/-) mice revealed that rosiglitazone acts additively with PTP1B deletion. Therefore, combining PTP1B inhibition with thiazolidinediones should be more effective than either alone for treating insulin-resistant states.     

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α₁-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α₁-AR stimulation protected against increased lactate dehydrogenase release or Annexin V⁺ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α₁-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α₁-AR stimulation increased ³H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α₁-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α₁-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α₁-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α_1A-AR but not α_1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α_1A-AR but not α_1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α₁-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α_1A-AR subtype.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action

Previous studies implicate protein-tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related phosphatase (LAR) as negative regulators of insulin signaling. The expression and/or activity of PTP1B and LAR are increased in muscle of insulin-resistant rodents and humans. Overexpression of LAR selectively in muscle of transgenic mice causes whole body insulin resistance. To determine whether overexpression of PTP1B also causes insulin resistance, we generated transgenic mice overexpressing human PTP1B selectively in muscle at levels similar to those observed in insulin-resistant humans. Insulin-stimulated insulin receptor (IR) tyrosyl phosphorylation and phosphatidylinositol 3'-kinase activity were impaired by 35% and 40-60% in muscle of PTP1B-overexpressing mice compared with controls. Insulin stimulation of protein kinase C (PKC)lambda/zeta activity, which is required for glucose transport, was impaired in muscle of PTP1B-overexpressing mice compared with controls, showing that PTP1B overexpression impairs activation of these PKC isoforms. Furthermore, hyperinsulinemic-euglycemic clamp studies revealed that whole body glucose disposal and muscle glucose uptake were decreased by 40-50% in PTP1B-overexpressing mice. Overexpression of PTP1B or LAR alone in muscle caused similar impairments in insulin action; however, compound overexpression achieved by crossing PTP1B- and LAR-overexpressing mice was not additive. Antibodies against specific IR phosphotyrosines indicated overlapping sites of action of PTP1B and LAR. Thus, overexpression of PTP1B in vivo impairs insulin sensitivity, suggesting that overexpression of PTP1B in muscle of obese humans and rodents may contribute to their insulin resistance. Lack of additive impairment of insulin signaling by PTP1B and LAR suggests that these PTPs have overlapping actions in causing insulin resistance in vivo.     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Neuronal PTP1B regulates body weight, adiposity and leptin action

Obesity is a major health problem and a risk factor for type 2 diabetes. Leptin, an adipocyte-secreted hormone, acts on the hypothalamus to inhibit food intake and increase energy expenditure. Most obese individuals develop hyperleptinemia and leptin resistance, limiting the therapeutic efficacy of exogenously administered leptin. Mice lacking the tyrosine phosphatase PTP1B are protected from diet-induced obesity and are hypersensitive to leptin, but the site and mechanism for these effects remain controversial. We generated tissue-specific PTP1B knockout (Ptpn1(-/-)) mice. Neuronal Ptpn1(-/-) mice have reduced weight and adiposity, and increased activity and energy expenditure. In contrast, adipose PTP1B deficiency increases body weight, whereas PTP1B deletion in muscle or liver does not affect weight. Neuronal Ptpn1(-/-) mice are hypersensitive to leptin, despite paradoxically elevated leptin levels, and show improved glucose homeostasis. Thus, PTP1B regulates body mass and adiposity primarily through actions in the brain. Furthermore, neuronal PTP1B regulates adipocyte leptin production and probably is essential for the development of leptin resistance.     

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.     

A role for hepatic scavenger receptor class B, type I in decreasing high density lipoprotein levels in mice that lack phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt(+/+) and Pemt(-/-) mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt(-/-) mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt(+/+) mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt(-/-) compared with Pemt(+/+) livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [(3)H]cholesteryl ethers and [(3)H]PC by hepatocytes derived from Pemt(-/-) compared with Pemt(+/+) mice. Furthermore, we observed an increased association of [(3)H]cholesteryl ethers in livers of Pemt(-/-) compared with Pemt(+/+) mice after tail vein injection of [(3)H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI.     

Stearoyl-CoA desaturase 1 deficiency increases insulin signaling and glycogen accumulation in brown adipose tissue

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of oleate (C18:1) and palmitoleate (C16:1), which are the main monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Previously, we showed that SCD1 deficiency elevates insulin-signaling components and downregulates protein-tyrosine phosphatase-1B (PTP-1B) in muscle, a major insulin-sensitive tissue. Here we found that, in brown adipose tissue (BAT), another insulin-sensitive tissue, the basal tyrosine phosphorylations of insulin receptor (IR) and IR substrates (IRS-1 and IRS-2) were upregulated in SCD1(-/-) mice compared with wild-type mice. The association of IRS-1 and IRS-2 with the alpha-p85 subunit of phosphatidylinositol 3-kinase as well as Akt-Ser(473) and Akt-Thr(308) phosphorylation is also elevated in the SCD1(-/-) mice. The mRNA expression, protein levels, and activity of PTP-1B implicated in the attenuation of the insulin signal are reduced in the SCD1(-/-) mice. The content of GLUT4 in the plasma membrane increased 2.5-fold, and this was accompanied by a 6-fold increase in glucose uptake in BAT of SCD1(-/-) mice. The increased glucose uptake was associated with higher glycogen synthase activity and glycogen accumulation. In the presence of insulin, [U-(14)C]glucose incorporation into glycogen was increased in BAT of SCD1(-/-) mice. Taken together, these studies illustrate increased insulin signaling and increased glycogen metabolism in BAT of SCD1(-/-) mice.     

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.     

Trivalent arsenicals and glucose use different translocation pathways in mammalian GLUT1

Rat glucose transporter isoform 1 or rGLUT1, which is expressed in neonatal heart and the epithelial cells that form the blood-brain barrier, facilitates uptake of the trivalent arsenicals arsenite as As(OH)? and methylarsenite as CH?As(OH)?. GLUT1 may be the major pathway for arsenic uptake into heart and brain, where the metalloid causes cardiotoxicity and neurotoxicity. In this paper, we compare the translocation properties of GLUT1 for trivalent methylarsenite and glucose. Substitution of Ser(66), Arg(126) and Thr(310), residues critical for glucose uptake, led to decreased uptake of glucose but increased uptake of CH?As(OH)?. The K(m) for uptake of CH?As(OH)? of three identified mutants, S66F, R126K and T310I, were decreased 4-10 fold compared to native GLUT1. The osmotic water permeability coefficient (P(f)) of GLUT1 and the three clinical isolates increased in parallel with the rate of CH?As(OH)? uptake. GLUT1 inhibitors Hg(II), cytochalasin B and forskolin reduced uptake of glucose but not CH?As(OH)?. These results indicate that CH?As(OH)? and water use a common translocation pathway in GLUT1 that is different to that of glucose transport.     

Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/-)) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/-) mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B(-/-) mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.     

Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B

Common obesity is primarily characterized by resistance to the actions of the hormone leptin. Mice deficient in protein tyrosine phosphatase 1B (PTP1B) are resistant to diabetes and diet-induced obesity, prompting us to further define the relationship between PTP1B and leptin in modulating obesity. Leptin-deficient (Lep(ob/ob)) mice lacking PTP1B exhibit an attenuated weight gain, a decrease in adipose tissue, and an increase in resting metabolic rate. Furthermore, PTP1B-deficient mice show an enhanced response toward leptin-mediated weight loss and suppression of feeding. Hypothalami from these mice also display markedly increased leptin-induced Stat3 phosphorylation. Finally, substrate-trapping experiments demonstrate that leptin-activated Jak2, but not Stat3 or the leptin receptor, is a substrate of PTP1B. These results suggest that PTP1B negatively regulates leptin signaling, and provide one mechanism by which it may regulate obesity.