Preclinical evaluation of tissue-selective gene therapies for congenital generalised lipodystrophy.

Lipodystrophy is a rare disorder which can be life-threatening. Here individuals fail to develop or maintain appropriate adipose tissue stores. This typically causes severe metabolic complications, including hepatic steatosis and lipoatrophic diabetes. There is no cure for lipodystrophy, and treatment options remain very limited. Here we evaluate whether tissue-selective adeno-associated virus (AAV) vectors can provide a targeted form of gene therapy for lipodystrophy, using a preclinical lipodystrophic mouse model of Bscl2 deficiency. We designed AAV vectors containing the mini/aP2 or thyroxine-binding globulin promoter to selectively target adipose or liver respectively. The AAV-aP2 vectors also contained the liver-specific microRNA-122 target sequence, restricting hepatic transgene expression. Systemic delivery of AAV-aP2 vectors overexpressing human BSCL2 restored adipose tissue development and metabolic health in lipodystrophic mice without detectable expression in the liver. High doses (1 × 1012 GCs) of liver-selective vectors led to off target expression and adipose tissue development, whilst low doses (1 × 1010 GCs) expressed selectively and robustly in the liver but did not improve metabolic health. This reveals that adipose tissue-selective, but not liver directed, AAV-mediated gene therapy is sufficient to substantially recover metabolic health in generalised lipodystrophy. This provides an exciting potential new avenue for an effective, targeted, and thereby safer therapeutic intervention.

Protein tyrosine phosphatase 1B in metabolic diseases and drug development.

Protein tyrosine phosphatase 1B (PTP1B), a non-transmembrane phosphatase, has a major role in a variety of signalling pathways, including direct negative regulation of classic insulin and leptin signalling pathways, and is implicated in the pathogenesis of several cardiometabolic diseases and cancers. As such, PTP1B has been a therapeutic target for over two decades, with PTP1B inhibitors identified either from natural sources or developed throughout the years. Some of these inhibitors have reached phase I and/or II clinical trials in humans for the treatment of type 2 diabetes mellitus, obesity and/or metastatic breast cancer. In this Review, we summarize the cellular processes and regulation of PTP1B, discuss evidence from in vivo preclinical and human studies of the association between PTP1B and different disorders, and discuss outcomes of clinical trials. We outline challenges associated with the targeting of this phosphatase (which was, until the past few years, viewed as difficult to target), the current state of the field of PTP1B inhibitors (and dual phosphatase inhibitors) and future directions for manipulating the activity of this key metabolic enzyme.

When a calorie is not just a calorie: Diet quality and timing as mediators of metabolism and healthy aging.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Gene therapy restores adipose tissue and metabolic health in a pre-clinical mouse model of lipodystrophy.

Congenital generalized lipodystrophy type 2 is a serious multisystem disorder with limited treatment options. It is caused by mutations affecting the BSCL2 gene, which encodes the protein seipin. Patients with congenital generalized lipodystrophy type 2 lack both metabolic and mechanical adipose tissue and develop severe metabolic complications including hepatic steatosis, lipoatrophic diabetes, and cardiovascular disease. Gene therapies are becoming viable treatments, helping to alleviate inherited and acquired human disorders. We aimed to determine whether gene therapy could offer an effective form of medical intervention for lipodystrophy. We examined whether systemic adeno-associated virus delivery of human BSCL2 could reverse metabolic disease in seipin knockout mice, where white adipose tissue is absent. We reveal that adeno-associated virus gene therapy targets adipose progenitor cells in vivo and substantially restores white adipose tissue development in adult seipin knockout mice. This resulted in both rapid and prolonged beneficial effects to metabolic health in this pre-clinical mouse model of congenital generalized lipodystrophy type 2. Hyperglycemia was normalized within 2 weeks post-treatment together with normalization of severe insulin resistance. We propose that gene therapy offers great potential as a therapeutic strategy to correct multiple metabolic complications in patients with congenital lipodystrophy.

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization.

OBJECTIVE: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin's lipid-lowering dependent and independent effects on IPA and IPH. APPROACH AND RESULTS: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin's anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs. CONCLUSIONS: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

Effects of dietary restriction on metabolic and cognitive health.

Life expectancy in most developed countries has been rising over the past century. In the UK alone, there are about 12 million people over 65 years old and centenarians have increased by 85% in the past 15 years. As a result of the ageing population, which is due mainly to improvements in medical treatments, public health, improved housing and lifestyle choices, there is an associated increase in the prevalence of pathological conditions, such as metabolic disorders, type 2 diabetes, cardiovascular and neurodegenerative diseases, many types of cancer and others. Statistics suggest that nearly 54% of elderly people in the UK live with at least two chronic conditions, revealing the urgency for identifying interventions that can prevent and/or treat such disorders. Non-pharmacological, dietary interventions such as energetic restriction (ER) and methionine restriction (MR) have revealed promising outcomes in increasing longevity and preventing and/or reversing the development of ageing-associated disorders. In this review, we discuss the evidence and mechanisms that are involved in these processes. Fibroblast growth factor 1 and hydrogen sulphide are important molecules involved in the effects of ER and MR in the extension of life span. Their role is also associated with the prevention of metabolic and cognitive disorders, highlighting these interventions as promising modulators for improvement of health span.

The anxiety and ethanol intake controlling GAL5.1 enhancer is epigenetically modulated by, and controls preference for, high-fat diet.

Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.

The Role of Inflammation in Diabetic Retinopathy.

Inflammation is central to pathogenic processes in diabetes mellitus and the metabolic syndrome and particularly implicates innate immunity in the development of complications. Inflammation is a primary event in Type 1 diabetes where infectious (viral) and/or autoimmune processes initiate disease; in contrast, chronic inflammation is typical in Type 2 diabetes and is considered a sequel to increasing insulin resistance and disturbed glucose metabolism. Diabetic retinopathy (DR) is perceived as a vascular and neurodegenerative disease which occurs after some years of poorly controlled diabetes. However, many of the clinical features of DR are late events and reflect the nature of the retinal architecture and its cellular composition. Retinal microvascular disease is, in fact, an early event pathogenetically, induced by low grade, persistent leukocyte activation which causes repeated episodes of capillary occlusion and, progressive, attritional retinal ischemia. The later, overt clinical signs of DR are a consequence of the retinal ischemia. Metabolic dysregulation involving both lipid and glucose metabolism may lead to leukocyte activation. On a molecular level, we have shown that macrophage-restricted protein tyrosine phosphatase 1B (PTP1B) is a key regulator of inflammation in the metabolic syndrome involving insulin resistance and it is possible that PTP1B dysregulation may underlie retinal microvascular disease. We have also shown that adherent CCR5+CD11b+ monocyte macrophages appear to be selectively involved in retinal microvascular occlusion. In this review, we discuss the relationship between early leukocyte activation and the later features of DR, common pathogenetic processes between diabetic microvascular disease and other vascular retinopathies, the mechanisms whereby leukocyte activation is induced in hyperglycemia and dyslipidemia, the signaling mechanisms involved in diabetic microvascular disease, and possible interventions which may prevent these retinopathies. We also address a possible role for adaptive immunity in DR. Although significant improvements in treatment of DR have been made with intravitreal anti-VEGF therapy, a sizeable proportion of patients, particularly with sight-threatening macular edema, fail to respond. Alternative therapies targeting inflammatory processes may offer an advantage.

Ablation of Bscl2/seipin in hepatocytes does not cause metabolic dysfunction in congenital generalised lipodystrophy.

Mutations affecting the BSCL2 gene cause the most severe form of congenital generalised lipodystrophy (CGL). Affected individuals develop severe metabolic complications including diabetes and hepatic steatosis. Bscl2-deficient mice almost entirely reproduce the CGL phenotype. Adipose tissue-specific loss of Bscl2 is also sufficient to cause early-onset generalised lipodystrophy in mice. However, these mice do not show severe metabolic dysfunction, even when challenged with a high-fat diet. Germline Bscl2 loss in mice and BSCL2 disruption in humans causes severe hepatic steatosis, and the encoded protein, seipin, has acknowledged roles in lipid accumulation. Given the critical role of the liver in glucose regulation, we speculated that intact hepatic Bscl2 expression may protect adipose tissue-specific Bscl2-deficient mice from metabolic disease. To investigate this, we generated a novel mouse model in which Bscl2 has been deleted in both adipose tissue and hepatocytes simultaneously using an adeno-associated viral vector. Despite achieving efficient disruption of Bscl2 in the liver, hepatic lipid accumulation and metabolic homeostasis was unaffected in mice fed a high-fat diet for 4 weeks. We also investigated the consequences of BSCL2 ablation in the human hepatocyte HepG2 cell line using CRISPR/Cas9 genome editing. No significant increases in lipid accumulation were observed in BSCL2 knockout cell lines. Overall, we reveal that Bscl2/BSCL2 does not appear to play a cell-autonomous role in the regulation of lipid accumulation in the liver. Loss of hepatic BSCL2 is therefore unlikely to contribute significantly to the development of hepatic steatosis or metabolic dysfunction in this form of CGL.

Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B-/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia.Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR.

Pharmacological inhibition of protein tyrosine phosphatase 1B protects against atherosclerotic plaque formation in the LDLR-/- mouse model of atherosclerosis.

Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with type 1 or type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance, due to impaired insulin receptor (IR) signalling. Here, we demonstrate that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR prevents and reverses atherosclerotic plaque formation in an LDLR-/- mouse model of atherosclerosis. Acute (single dose) or chronic PTP1B inhibitor (trodusquemine) treatment of LDLR-/- mice decreased weight gain and adiposity, improved glucose homeostasis and attenuated atherosclerotic plaque formation. This was accompanied by a reduction in both, circulating total cholesterol and triglycerides, a decrease in aortic monocyte chemoattractant protein-1 (MCP-1) expression levels and hyperphosphorylation of aortic Akt/PKB and AMPKα. Our findings are the first to demonstrate that PTP1B inhibitors could be used in prevention and reversal of atherosclerosis development and reduction in CVD risk.

Photoperiod- and Triiodothyronine-dependent Regulation of Reproductive Neuropeptides, Proinflammatory Cytokines, and Peripheral Physiology in Siberian Hamsters (Phodopus sungorus).

Seasonal trade-offs in reproduction and immunity are ubiquitous in nature. The mechanisms that govern transitions across seasonal physiological states appear to involve reciprocal switches in the local synthesis of thyroid hormone. In long-day (LD) summer-like conditions, increased hypothalamic triiodothyronine (T3) stimulates gonadal development. Alternatively, short-day (SD) winter-like conditions increase peripheral leukocytes and enhance multiple aspects of immune function. These data indicate that the localized effects of T3 in the hypothalamus and leukocytes are photoperiod dependent. We tested the hypothesis that increased peripheral T3 in SD conditions would increase aspects of reproductive physiology and inhibit immune function, whereas T3 injections in LD conditions would facilitate aspects of immune function (i.e., leukocytes). In addition, we also examined whether T3 regulates hypothalamic neuropeptide expression as well as hypothalamic and splenic proinflammatory cytokine expression. Adult male Siberian hamsters were maintained in LD (15L:9D) or transferred to SD (9L:15D) for 8 weeks. A subset of LD and SD hamsters was treated daily with 5 µg T3 for 2 weeks. LD and SD controls were injected with saline. Daily T3 administration in SD hamsters (SD+T3) resulted in a rapid and substantial decrease in peripheral leukocyte concentrations and stimulated gonadal development. T3 treatment in LD (LD+T3) had no effect on testicular volumes but significantly increased leukocyte concentrations. Molecular analyses revealed that T3 stimulated interleukin 1ß messenger RNA (mRNA) expression in the spleen and inhibited RFamide Related Peptide-3 mRNA expression in the hypothalamus. Moreover, there was a photoperiod-dependent decrease in splenic tumor necrosis factor-α mRNA expression. These findings reveal that T3 has tissue-specific and photoperiod-dependent regulation of seasonal rhythms in reproduction and immune function.

Fenretinide prevents obesity in aged female mice in association with increased retinoid and estrogen signaling.

OBJECTIVE: The synthetic retinoid fenretinide (FEN) inhibits adiposity in male mice fed a high-fat diet (HFD) in association with alterations in retinoic acid (RA) signaling. Young female mice are protected from obesity via estrogen signaling. We, therefore, investigated whether FEN also influences adiposity in aged female mice differing in parity and whether such effects are mediated by retinoid and estrogen signaling. METHODS: Aged nulliparous and parous female mice were maintained on HFD ± FEN, and adiposity was assessed. Quantitative polymerase chain reaction was performed on white adipose tissue (WAT), liver, and 3T3-L1 adipocytes treated with RA or FEN ± estrogen. RESULTS: Parous females were more obese than nulliparous mice independent of age. FEN-HFD prevented the HFD-induced increase in adiposity and leptin levels independently of parity. FEN-HFD induced retinoid-responsive genes in WAT and liver. Parous females had reduced expression of hepatic estrogen-responsive genes, but FEN-HFD up-regulated WAT Cyp19a1 and Esr2 in parous mice. Estrogen and RA acted synergistically to increase RA receptor-mediated gene expression in 3T3-L1 adipocytes. FEN increased Cyp19a1 and Esr2, similar to our findings in vivo. CONCLUSIONS: The prevention of adiposity by FEN in response to HFD in female mice seems to involve increased retinoid signaling in association with induction of local estrogen production and estrogen signaling in WAT.

A key role for PTP1B in dendritic cell maturation, migration, and T cell activation.

Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

Myeloid-cell protein tyrosine phosphatase-1B deficiency in mice protects against high-fat diet and lipopolysaccharide-induced inflammation, hyperinsulinemia, and endotoxemia through an IL-10 STAT3-dependent mechanism.

Protein tyrosine phosphatase-1B (PTP1B) negatively regulates insulin and leptin signaling, rendering it an attractive drug target for treatment of obesity-induced insulin resistance. However, some studies suggest caution when targeting macrophage PTP1B, due to its potential anti-inflammatory role. We assessed the role of macrophage PTP1B in inflammation and whole-body metabolism using myeloid-cell (LysM) PTP1B knockout mice (LysM PTP1B). LysM PTP1B mice were protected against lipopolysaccharide (LPS)-induced endotoxemia and hepatic damage associated with decreased proinflammatory cytokine secretion in vivo. In vitro, LPS-treated LysM PTP1B bone marrow-derived macrophages (BMDMs) displayed increased interleukin (IL)-10 mRNA expression, with a concomitant decrease in TNF-α mRNA levels. These anti-inflammatory effects were associated with increased LPS- and IL-10-induced STAT3 phosphorylation in LysM PTP1B BMDMs. Chronic inflammation induced by high-fat (HF) feeding led to equally beneficial effects of macrophage PTP1B deficiency; LysM PTP1B mice exhibited improved glucose and insulin tolerance, protection against LPS-induced hyperinsulinemia, decreased macrophage infiltration into adipose tissue, and decreased liver damage. HF-fed LysM PTP1B mice had increased basal and LPS-induced IL-10 levels, associated with elevated STAT3 phosphorylation in splenic cells, IL-10 mRNA expression, and expansion of cells expressing myeloid markers. These increased IL-10 levels negatively correlated with circulating insulin and alanine transferase levels. Our studies implicate myeloid PTP1B in negative regulation of STAT3/IL-10-mediated signaling, highlighting its inhibition as a potential anti-inflammatory and antidiabetic target in obesity.

Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice.

Microparticles are membrane vesicles with pro-inflammatory properties. Circulating levels of microparticles have previously been found to be elevated in patients with metabolic syndrome (MetS). The present study aimed to evaluate the effects of in vivo treatment with microparticles, from patients with MetS and from healthy subjects (HS), on ex vivo vascular function in mice. Microparticles isolated from MetS patients or HS, or a vehicle were intravenously injected into mice, following which vascular reactivity in response to vasoconstrictor agonists was assessed by myography with respect to cyclo-oxygenase pathway, oxidative and nitrosative stress. Injection of microparticles from MetS patients into mice induced vascular hypo-reactivity in response to serotonin. Hypo-reactivity was associated with up-regulation of inducible NO-synthase and increased production of NO, and was reversed by the NO-synthase inhibitor (N(G)-nitro-L-arginine). The selective COX-2 inhibitor (NS398) reduced the contractile effect of serotonin in aortas from mice treated with vehicle or HS microparticles; however, this was not observed within mice treated with MetS microparticles, probably due to the ability of MetS microparticles to enhance prostacyclin. MetS microparticle-mediated vascular dysfunction was associated with increased reactive oxygen species (ROS) and enhanced expression of the NADPH oxidase subunits. Neutralization of the pro-inflammatory pathway Fas/FasL completely prevented vascular hypo-reactivity and the ability of MetS microparticles to enhance both inducible NO-synthase and monocyte chemoattractant protein-1 (MCP-1). Our data provide evidence that microparticles from MetS patients induce ex vivo vascular dysfunction by increasing both ROS and NO release and by altering cyclo-oxygenase metabolites and MCP-1 through the Fas/FasL pathway.

Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress.

Obesity is associated with induction of the ER (endoplasmic reticulum)-stress response signalling and insulin resistance. PTP1B (protein tyrosine phosphatase 1B) is a major regulator of adiposity and insulin sensitivity. The aim of the present study was to investigate the role of L-PTP1B (liver-specific PTP1B) in chronically HFD (high-fat diet) and pharmacologically induced (tunicamycin and thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B(-/-)) protected against induction of pharmacologically induced and/or obesity-induced ER stress. The HFD-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) and BIP (binding immunoglobulin protein) mRNA levels were partially inhibited, whereas ATF4 (activated transcription factor 4), GADD34 (growth-arrest and DNA-damage-inducible protein 34), GRP94 (glucose-regulated protein 94), ERDJ4 (ER-localized DnaJ homologue) mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B(-/-) mice relative to control littermates. L-PTP1B(-/-) mice also had increased nuclear translocation of spliced XBP-1 (X box-binding protein-1) via increased p85α binding. We demonstrate that the ER-stress response and L-PTP1B expression are interlinked in obesity- and pharmacologically induced ER stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B(-/-) mice.

Non-invasive ultrasound guided compression repair of post puncture femoral pseudoaneurysm.

BACKGROUND: Pseudo aneurysm (PSA) of femoral artery is the second common post puncture complication. If PSA is less than 18 mm, it can be closed spontaneously and it can be asymptomatic. If not, it needs treatment. The most common non invasive method used in the treatment is ultrasound guided compression (UGC) with duplex color Doppler. In the treatment of PSA, other options are available, such as thrombin and collagen injection in the PSA sack, application of stent, but they are more invasive, with more complications than UGC. MATERIAL AND METHOD: Retrospective study was performed in the period from 2005-2010. During this period, 4575 punctions of femoral artery were performed. Because of suspected PSA of femoral artery, we examined and diagnosed 28 PSA in the ward of ultrasound diagnostic. Data regarding the location and morphologic characteristics of PSA, morbid disease were documented. RESULTS: UGC was performed in 22 PSA patients, 12 men and 9 women, with average age of 48 years. 20 PSA were obliterated, while 2 remained persistent even after second attempt, and they were surgically treated. Efficacy of this method was 90%. Control examination was performed after 12 and 24 hours, and after 1 and 3 months. Intra- and after procedural complications were not observed. CONCLUSION: Non invasive treatment of PSA with UGC is cheap, efficient and easy to perform, with minor complications in well selected patients.

In vivo differential effects of fasting, re-feeding, insulin and insulin stimulation time course on insulin signaling pathway components in peripheral tissues.

BACKGROUND: Components of the insulin receptor signaling pathway are probably some of the best studied ones. Even though methods for studying these components are well established, the in vivo effects of different fasting regimens, and the time course of insulin receptor phosphorylation and that of its downstream components in insulin-sensitive peripheral tissues have not been analyzed in detail. RATIONALE: When assessing insulin signaling, it may be beneficial to drive insulin levels as low as possible by performing an overnight fast before injecting a supra-physiological dose of insulin. Recent studies have shown however that 5 or 6 h fast in mice is sufficient to assess physiological responses to insulin and/or glucose in glucose tolerance tests, insulin tolerance tests and euglycemic hyperinsulinemic clamp studies. Moreover, mice are nocturnal feeders, with ∼70% of their daily caloric intake occurring during the dark cycle, and their metabolic rate is much higher than humans. Therefore, an overnight fast in mice is closer to starvation than just food withdrawal. Thus our aim was to assess insulin signaling components from the insulin receptor to downstream targets IRS1, Akt/PKB, GSK3, Erk1/2 and ribosomal protein S6 in muscle, liver and adipose tissue in 5 h versus 16 h (overnight) fasted mice, and the time course (0-30 min) of these phosphorylation events. We also assessed whether re-feeding under 5 h and 16 h fasting conditions was a more robust stimulus than insulin alone. CONCLUSIONS: Our study determines that a short food withdrawal from mice, for a period of 5 h, results in a similar insulin-stimulated response in phosphorylation events as the long overnight fast, presenting a more physiological experimental set up. We also demonstrate that in vivo, insulin-stimulated phosphorylation of its signaling components is different between different peripheral tissues, and depending on the tissue(s) and protein(s) of interest, an appropriate time course should be chosen.

Altered glucose homeostasis in mice with liver-specific deletion of Src homology phosphatase 2.

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.