Hepatocyte TLR4 triggers inter-hepatocyte Jagged1/Notch signaling to determine NASH-induced fibrosis.

Aberrant hepatocyte Notch activity is critical to the development of nonalcoholic steatohepatitis (NASH)-induced liver fibrosis, but mechanisms underlying Notch reactivation in developed liver are unclear. Here, we identified that increased expression of the Notch ligand Jagged1 (JAG1) tracked with Notch activation and nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in human liver biopsy specimens and mouse NASH models. The increase in Jag1 was mediated by hepatocyte Toll-like receptor 4 (TLR4)-nuclear factor κB (NF-κB) signaling in pericentral hepatocytes. Hepatocyte-specific Jag1 overexpression exacerbated fibrosis in mice fed a high-fat diet or a NASH-provoking diet rich in palmitate, cholesterol, and sucrose and reversed the protection afforded by hepatocyte-specific TLR4 deletion, whereas hepatocyte-specific Jag1 knockout mice were protected from NASH-induced liver fibrosis. To test therapeutic potential of this biology, we designed a Jag1-directed antisense oligonucleotide (ASO) and a hepatocyte-specific N-acetylgalactosamine (GalNAc)-modified siRNA, both of which reduced NASH diet-induced liver fibrosis in mice. Overall, these data demonstrate that increased hepatocyte Jagged1 is the proximal hit for Notch-induced liver fibrosis in mice and suggest translational potential of Jagged1 inhibitors in patients with NASH.

Notch activity characterizes a common hepatocellular carcinoma subtype with unique molecular and clinicopathologic features.

BACKGROUND & AIMS: The hepatocyte Notch pathway is a pathogenic factor in non-alcoholic steatohepatitis (NASH)-associated fibrosis, but its role in hepatocellular carcinoma (HCC) is less well defined. Herein, we aimed to characterize the molecular and clinical features of Notch-active human HCC, and to investigate the mechanisms by which Notch affects NASH-driven HCC. METHODS: Using a 14-gene Notch score, we stratified human HCCs from multiple comprehensively profiled datasets. We performed gene set enrichment analyses to compare Notch-active HCCs with published HCC subtype signatures. Next, we sorted Notch-active hepatocytes from Notch reporter mice for RNA sequencing and characterized Notch-active tumors in an HCC model combining a carcinogen and a NASH-inducing diet. We used genetic mouse models to manipulate hepatocyte Notch to investigate the sufficiency and necessity of Notch in NASH-driven tumorigenesis. RESULTS: Notch-active signatures were found in ~30% of human HCCs that transcriptionally resemble cholangiocarcinoma-like HCC, exhibiting a lack of activating CTNNB1 (ß-catenin) mutations and a generally poor prognosis. Endogenous Notch activation in hepatocytes is associated with repressed ß-catenin signaling and hepatic metabolic functions, in lieu of increased interactions with the extracellular matrix in NASH. Constitutive hepatocyte Notch activation is sufficient to induce ß-catenin-inactive HCC in mice with NASH. Notch and ß-catenin show a pattern of mutual exclusivity in carcinogen-induced HCC; in this mouse model, chronic blockade of Notch led to ß-catenin-dependent tumor development. CONCLUSIONS: Notch activity characterizes a distinct HCC molecular subtype with unique histology and prognosis. Sustained Notch signaling in chronic liver diseases can drive tumor formation without acquiring specific genomic driver mutations. LAY SUMMARY: The Notch signaling pathway is known to be involved in the pathogenesis of liver fibrosis. However, its role in liver cancer has not been well defined. Herein, we show that Notch activity is increased in a subset of liver cancers and is associated with poor outcomes. We also used a mouse model to show that aberrant Notch activity can drive cancer progression in obese mice.

GCN2 regulates pancreatic ß cell mass by sensing intracellular amino acid levels.

EIF2AK4, which encodes the amino acid deficiency-sensing protein GCN2, has been implicated as a susceptibility gene for type 2 diabetes in the Japanese population. However, the mechanism by which GCN2 affects glucose homeostasis is unclear. Here, we show that insulin secretion is reduced in individuals harboring the risk allele of EIF2AK4 and that maintenance of GCN2-deficient mice on a high-fat diet results in a loss of pancreatic ß cell mass. Our data suggest that GCN2 senses amino acid deficiency in ß cells and limits signaling by mechanistic target of rapamycin complex 1 to prevent ß cell failure during the consumption of a high-fat diet.

Essential role of glucokinase in the protection of pancreatic ß cells to the glucose energetic status.

Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic ß cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic ß cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic ß cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic ß cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic ß cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic ß cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic ß cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

Evidence for a Non-leptin System that Defends against Weight Gain in Overfeeding.

Weight is defended so that increases or decreases in body mass elicit responses that favor restoration of one's previous weight. While much is known about the signals that respond to weight loss and the central role that leptin plays, the lack of experimental systems studying the overfed state has meant little is known about pathways defending against weight gain. We developed a system to study this physiology and found that overfed mice defend against increased weight gain with graded anorexia but, unlike weight loss, this response is independent of circulating leptin concentration. In overfed mice that are unresponsive to orexigenic stimuli, adipose tissue is transcriptionally and immunologically distinct from fat of ad libitum-fed obese animals. These findings provide evidence that overfeeding-induced obesity alters adipose tissue and central responses in ways that are distinct from ad libitum obesity and activates a non-leptin system to defend against weight gain.

MTORC1 Regulates both General Autophagy and Mitophagy Induction after Oxidative Phosphorylation Uncoupling.

Mechanistic target of rapamycin complex 1 (MTORC1) is a critical negative regulator of general autophagy. We hypothesized that MTORC1 may specifically regulate autophagic clearance of damaged mitochondria. To test this, we used cells lacking tuberous sclerosis complex 2 (TSC2-/- cells), which show constitutive MTORC1 activation. TSC2-/- cells show MTORC1-dependent impaired autophagic flux after chemical uncoupling of mitochondria, increased mitochondrial-protein aging, and accumulation of p62/SQSTM1-positive mitochondria. Mitochondrial autophagy (mitophagy) was also deficient in cells lacking TSC2, associated with altered expression of PTEN-induced putative kinase 1 (PINK1) and PARK2 translocation to uncoupled mitochondria, all of which were recovered by MTORC1 inhibition or expression of constitutively active forkhead box protein O1 (FoxO1). These data prove the necessity of intact MTORC1 signaling to regulate two synergistic processes required for clearance of damaged mitochondria: (i) general autophagy initiation and (ii) PINK1/PARK2-mediated selective targeting of uncoupled mitochondria to the autophagic machinery.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic ß cells.

Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic ß cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic ß cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2) expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic ß cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s) involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding gelsolin to modulate the oligomerization of VDAC complexes and cell survival.

Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.

TSC2 N-terminal lysine acetylation status affects to its stability modulating mTORC1 signaling and autophagy.

There is a growing evidence of the role of protein acetylation in different processes controlling metabolism. Sirtuins (histone deacetylases nicotinamide adenine dinucleotide-dependent) activate autophagy playing a protective role in cell homeostasis. This study analyzes tuberous sclerosis complex (TSC2) lysine acetylation, in the regulation of mTORC1 signaling activation, autophagy and cell proliferation. Nicotinamide 5mM (a concentration commonly used to inhibit SIRT1), increased TSC2 acetylation in its N-terminal domain, and concomitantly with an augment in its ubiquitination protein status, leading to mTORC1 activation and cell proliferation. In contrast, resveratrol (RESV), an activator of sirtuins deacetylation activity, avoided TSC2 acetylation, inhibiting mTORC1 signaling and promoting autophagy. Moreover, TSC2 in its deacetylated state was prevented from ubiquitination. Using MEF Sirt1 +/+ and Sirt1 -/- cells or a SIRT1 inhibitor (EX527) in MIN6 cells, TSC2 was hyperacetylated and neither NAM nor RESV were capable to modulate mTORC1 signaling. Then, silencing Tsc2 in MIN6 or in MEF Tsc2-/- cells, the effects of SIRT1 modulation by NAM or RESV on mTORC1 signaling were abolished. We also observed that two TSC2 lysine mutants in its N-terminal domain, derived from TSC patients, differentially modulate mTORC1 signaling. TSC2 K599M variant presented a lower mTORC1 activity. However, with K106Q mutant, there was an activation of mTORC1 signaling at the basal state as well as in response to NAM. This study provides, for the first time, a relationship between TSC2 lysine acetylation status and its stability, representing a novel mechanism for regulating mTORC1 pathway.

Pancreatic ß-cell failure mediated by mTORC1 hyperactivity and autophagic impairment.

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in ß-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in ß-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with ß-cell-specific deletion of Tsc2 (ßTsc2(-/-)) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on ß-cell failure. mTORC1 hyperactivation drove an early increase in ß-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older ßTsc2(-/-) mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in ß-cells of ßTsc2(-/-) mice, but mitophagy was also impaired under these circumstances. We provide evidence of ß-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to ß-cell failure.

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.

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic ß cell death.

There is a growing evidence of the role of autophagy in pancreatic ß cell homeostasis. During development of type 2 diabetes, ß cells are required to supply the increased demand of insulin. In such a stage, ß cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, ß cell death and overt diabetes. In this study, we used insulin secretion-deficient ß cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived ß cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased ß cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient ß cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in ß cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.

DPP4 inhibitor vildagliptin preserves ß-cell mass through amelioration of endoplasmic reticulum stress in C/EBPB transgenic mice.

The development of type 2 diabetes is accompanied by a progressive decline in ß-cell mass and function. Vildagliptin, a dipeptidyl peptidase 4 inhibitor, is representative of a new class of antidiabetic agents that act through increasing the expression of glucagon-like peptide-1. The protective effect of this agent on ß cells was studied in diabetic mice. Diabetic pancreatic ß cell-specific C/EBPB transgenic (TG) mice exhibit decreased ß-cell mass associated with increased apoptosis, decreased proliferation, and aggravated endoplasmic reticulum (ER) stress. Vildagliptin was orally administered to the TG mice for a period of 24 weeks, and the protective effects of this agent on ß cells were examined, along with the potential molecular mechanism of protection. Vildagliptin ameliorated hyperglycemia in TG mice by increasing the serum concentration of insulin and decreasing the serum concentration of glucagon. This agent also markedly increased ß-cell mass, improved aggravated ER stress, and restored attenuated insulin/IGF1 signaling. A decrease in pancreatic and duodenal homeobox 1 expression was also observed in ß cells isolated from our mouse model, but this was also restored by vildagliptin treatment. The expression of C/EBPB protein, but not mRNA, was unexpectedly downregulated in vildagliptin-treated TG mice and in exenatide-treated MIN6 cells. Activation of the GLP1 pathway induced proteasome-dependent C/EBPB degradation in ß cells as the proteasome inhibitor MG132 restored the downregulation of C/EBPB protein by exenatide. Vildagliptin elicits protective effects on pancreatic ß cells, possibly through C/EBPB degradation, and has potential for preventing the progression of type 2 diabetes.

Role of the TSC1-TSC2 complex in the integration of insulin and glucose signaling involved in pancreatic beta-cell proliferation.

Tuberous sclerosis complex proteins 1-2 (TSC1-TSC2) complex integrates both nutrient and hormonal signaling and is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1. The use of different beta-cell lines expressing or not the insulin receptor (IR(+/+) and IR(-/-)) or with a reconstituted expression of IR isoform A or B (Rec A and Rec B) revealed that both phosphatidylinositol 3-kinase/Akt/TSC/mTOR complex 1 and MAPK kinase/ERK pathways mediate insulin signaling in IR(+/+)-, IRA-, or IRB-expressing cells. However, glucose signaling was mediated by MAPK kinase/ERK and AMP-activated protein kinase pathways as assessed in IR(-/-) cells. The effect of insulin on Akt phosphorylation was completely inhibited by the use of the phosphatidylinositol 3-kinase inhibitor wortmannin in IR(+/+) and Rec B cells, a partial inhibitory effect being observed in Rec A cell line. The knockdown of TSC2 expression up-regulated the downstream basal phosphorylation of 70-kDa ribosomal protein S6 kinase (p70S6K) and mTOR. More importantly, upregulation of p70S6K signaling impaired insulin-stimulated phosphorylation of Akt Ser(473) and p70S6K in IR(+/+) and Rec B but not in Rec A cell lines. In fact, insulin receptor substrate-1 Ser(307) phosphorylation signal in Rec B was stronger than in Rec A cell line during insulin action. Rec A cells induced a higher proliferation rate compared with Rec B or IR(+/+) during serum stimulation. Thus, we propose that the regulation of TSC2 phosphorylation by insulin or glucose independently integrates beta-cell proliferation signaling, the relative expression of IRA or IRB isoforms in pancreatic beta cells playing a major role.

Complicaciones vasculares en el síndrome del opérculo torácico / Vascular complications in thoracic outlet compression syndrome

Se presentan cuatro pacientes con complicaciones vasculares en miembros superiores provocadas por síndrome comprensivo del opérculo torácico que fueron sometidos a tratamiento quirúrgico. Se realizó una revisión histórica del tema así como de los temas controvertidos referidos a su diagnóstico, tratamiento y en el caso que éste sea quirúrgico los distintos procedimientos efectuados sobre elementos blamdos, óseos, cartilaginosos y musculo-tendinosos. Referente al diagnóstico se promueve la utilización de los nuevos métodos incruentes como el Ecodoppler. Con respecto al tratamiento quirúrgico creemos que los mejores resultados se obtienen con la resección de la primera costilla. La evolución de cuatro pacientes fue satisfactoria incluso en uno de ellos que requirió reiteradas intervenciones

Tumores del corpúsculo carotídeo / Carotid body tumors

Se presenta 1 caso de tumor del corpúsculo carotídeo debido a su baja frecuencia. Se realiza una breve revisión histórica del tema y de la bibliografía nacional y extranjera. Se los ubica dentro de los tumores derivados del neuroectodermo. Son generalmente poco sintomáticos y su diagnóstico se realiza por ecografía, tomografía computada y fundamentalmente angiografía carotídea. El tratamiento es la resección quirúrgica completa subadventicial o con reconstrucción carotídea