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Background & Aims Increasing evidence highlights dietary fructose as a major driver of non-alcoholic fatty liver disease (NAFLD) pathogenesis, the majority of which is cleared on first pass through the hepatic circulation by enzymatic phosphorylation to fructose-1-phosphate via the ketohexokinase (KHK) enzyme. Without a current approved therapy, disease management emphasises lifestyle interventions, but few patients adhere to such strategies. New targeted therapies are urgently required. Methods We have used a unique combination of human liver specimens, a murine dietary model of NAFLD and human multicellular co-culture systems to understand the hepatocellular consequences of fructose administration. We have also performed a detailed nuclear magnetic resonance-based metabolic tracing of the fate of isotopically labelled fructose upon administration to the human liver. Results Expression of KHK isoforms is found in multiple human hepatic cell types, although hepatocyte expression predominates. KHK knockout mice show a reduction in serum transaminase, reduced steatosis and altered fibrogenic response on an Amylin diet. Human co-cultures exposed to fructose exhibit steatosis and activation of lipogenic and fibrogenic gene expression, which were reduced by pharmacological inhibition of KHK activity. Analysis of human livers exposed to 13C-labelled fructose confirmed that steatosis, and associated effects, resulted from the accumulation of lipogenic precursors (such as glycerol) and enhanced glycolytic activity. All of these were dose-dependently reduced by administration of a KHK inhibitor. Conclusions We have provided preclinical evidence using human livers to support the use of KHK inhibition to improve steatosis, fibrosis, and inflammation in the context of NAFLD. Lay summary We have used a mouse model, human cells, and liver tissue to test how exposure to fructose can cause the liver to store excess fat and become damaged and scarred. We have then inhibited a key enzyme within the liver that is responsible for fructose metabolism. Our findings show that inhibition of fructose metabolism reduces liver injury and fibrosis in mouse and human livers and thus this may represent a potential route for treating patients with fatty liver disease in the future., Graphical abstract, Highlights • Mice deficient in ketohexokinase (KHK) show reduced features of NASH on a high-fat/high-fructose diet. • KHK inhibition reduces lipogenic gene expression in the presence of high fructose/glucose. • Activated myofibroblasts show reduced expression of fibrogenic genes when treated with KHK inhibitor. • KHK inhibitor reduced hepatic accumulation of lipogenic fructose derivatives and glycolysis in human liver.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western world, affecting about 1/3 of the US general population and remaining as a significant cause of morbidity and mortality. The hallmark of the disease is the excessive accumulation of fat within the liver cells (hepatocytes), which eventually paves the way to cellular stress, injury and apoptosis. NAFLD is strongly associated with components of the metabolic syndrome and is fast emerging as a leading cause of liver transplant in the USA. Based on clinico-pathologic classification, NAFLD may present as isolated lipid collection (steatosis) within the hepatocytes (referred to as non-alcoholic fatty liver; NAFL); or as the more aggressive phenotype (known as non-alcoholic steatohepatitis; NASH). There are currently no regulatory agency- approved medication for NAFLD, despite the enormous work and resources that have gone into the study of this condition. Therefore, there remains a huge unmet need in developing and utilizing pre-clinical models that will recapitulate the disease condition in humans. In line with progress being made in developing appropriate disease models, this review highlights the cutting-edge preclinical in vitro and animal models that try to recapitulate the human disease pathophysiology and/or clinical manifestations.

The progressive disease spectrum of non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), is a rapidly emerging public health crisis with no approved therapy. The diversity of various therapies under development highlights the lack of consensus around the most effective target, underscoring the need for better translatable preclinical models to study the complex progressive disease and effective therapies. Areas covered: This article reviews published literature of various mouse models of NASH used in preclinical studies, as well as complex organotypic in vitro and ex vivo liver models being developed. It discusses translational challenges associated with both kinds of models, and describes some of the studies that validate their application in NAFLD. Expert opinion: Animal models offer advantages of understanding drug distribution and effects in a whole body context, but are limited by important species differences. Human organotypic in vitro and ex vivo models with physiological relevance and translatability need to be used in a tiered manner with simpler screens. Leveraging newer technologies, like metabolomics, proteomics, and transcriptomics, and the future development of validated disease biomarkers will allow us to fully utilize the value of these models to understand disease and evaluate novel drugs in isolation or combination.

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo-like phenotype. iECs and iSMCs were cocultured under vascular region-specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel-derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow-induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated-low-density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized-LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor β (TGFβ) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761-mediated transforming growth factor β receptor I/II (TGFβRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC-derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches. Stem Cells Translational Medicine 2017;6:1673–1683

In this paper, a new approach to assess the skew angle for scanned/printed document images has been proposed. This is significant for an automatic document processing system (as text and image segmentation) to avert errors in auxiliary stages. The proposed tactic is based on the statistical analysis of the slope of the connected lines in the document. The proposed technique detects skew and corrects it by initial letter (X1, Y1+150) from left margin of the resized (800X800) image. Final letter (X2, Y2-150) were chosen from right margin of the same image. The skew angle estimation is done for standard skewed dataset and effective correction of the same is performed with minimum errors.

The heterogeneity of biological processes driving the severity of nonalcoholic fatty liver disease (NAFLD) as reflected in the transcriptome and the relationship between the pathways involved are not well established. Well-defined associations between gene expression profiles and disease progression would benefit efforts to develop novel therapies and to understand disease heterogeneity. We analyzed hepatic gene expression in controls and a cohort with the full histological spectrum of NAFLD. Protein-protein interaction and gene set variation analysis revealed distinct sets of coordinately regulated genes and pathways whose expression progressively change over the course of the disease. The progressive nature of these changes enabled us to develop a framework for calculating a disease progression score for individual genes. We show that, in aggregate, these scores correlate strongly with histological measures of disease progression and can thus themselves serve as a proxy for severity. Furthermore, we demonstrate that the expression levels of a small number of genes (~20) can be used to infer disease severity. Finally, we show that patient subgroups can be distinguished by the relative distribution of gene-level scores in specific gene sets. While future work is required to identify the specific disease characteristics that correspond to patient clusters identified on this basis, this work provides a general framework for the use of high-content molecular profiling to identify NAFLD patient subgroups.

Objective: To evaluate the effects of Heartfulness self-development program in improving psychological flourishing of nursing students. Methodology: The study was designed as a prospective interventional study to examine the effects on positive psychological flourishing parameters such as empathy, optimism, self-confidence, positive outlook towards future, and social responsibilities. Participants of this study are students of a four-year undergraduate program in a nursing college in Chennai, Tamilnadu, India. Psychological flourishing scores [Ed Diener and Robert Biswas-Diener, 2009] were evaluated before and after a 24-week Heartfulness self-development program. Results: Data from 174 students was collected and analyzed using SPSS version 21.There was a significant overall increase in the psychological flourishing of nursing students after the intervention [P < 0.000*]. This improvement persisted even in the subgroup analysis based on academic year and family background. Conclusion: Significant improvements in psychological flourishing of nursing students were observed with the Heartfulness self-development program. Stress is a salient facet of nursing professionals, due to the excessive work load and long working hours they spend in tending to and taking care of patients. Nursing students experience substantial stress when they undergo training as nurses, and additionally endure anxiety from their academic pressures, financial concerns, family problems, and uncertainties of their future. In this study the intervention has substantially increased the self-confidence of the students, facilitating them to look within and explore their inner potential to successfully fulfil their goals and expectations.

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.

Nonalcoholic steatohepatitis (NASH) is an emerging health crisis with no approved therapies. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, shows promise in NASH trials. However, the precise mechanisms mediating OCA effects and impact on cholesterol metabolism are not fully understood. We explored the pharmaco-toxicological effects of OCA on patho-physiological pathways in hepatocytes using a previously described perfused organotypic liver system that allows culture in near-physiological insulin/glucose milieus, and exhibits drug responses at clinically-relevant concentrations. Primary hepatocytes experienced 48-hour exposure to OCA at concentrations approximating therapeutic (0.5μM) and supratherapeutic (10μM) levels. Global transcriptomics by RNAseq was complimented by cellular viability (MTT), CYP activity assays, and secreted FGF19 levels in the media. Dose-dependent, transcriptional effects suggested suppression of bile acid synthesis (↓CYP7A1, ↓CYP27A1) and increased bile efflux (↑ABCB4, ↑ABCB11, ↑OSTA, ↑OSTB). Pleiotropic effects included suppression of TGFβ and IL-6 signaling pathways, and signatures suggestive of HDL suppression (↑SCARB1, ↓ApoAI, ↓LCAT) and LDL elevation (↑ApoB, ↓CYP7A1). OCA exhibited direct FXR-mediated effects with increased FGF19 secretion. Transcriptomics revealed regulation of metabolic, anti-inflammatory, and anti-fibrotic pathways beneficial in NASH, and predicted cholesterol profiles consistent with clinical findings. Follow-up studies under lipotoxic/inflammatory conditions would corroborate these effects in a disease-relevant environment.

An experimental in vitro model of the hemodynamics that occur in atrial fibrillation (AFib) in the left atrial appendage (LAA) was developed to study changes in human endothelial cell thrombotic potential. We applied human-derived sinus rhythm and AFib hemodynamic shear stress patterns to primary human endothelial cells (ECs) in culture. We found that ECs exposed to AFib hemodynamics have increased thrombotic potential as measured by increased expression of pro-thrombotic gene markers and fibrin deposition on the endothelium. Treatment with the factor Xa inhibitor, apixaban, attenuated fibrin deposition thickness while increasing fibrin density at the endothelial cell surface. This study suggests that altered hemodynamics associated with AFib play a key role in driving the thrombotic potential of the LAA endothelium.

Central obesity is associated with chronic inflammation, insulin resistance, β-cell dysfunction, and endoplasmic reticulum (ER) stress. The 12/15-lipoxygenase enzyme (12/15-LO) promotes inflammation and insulin resistance in adipose and peripheral tissues. Given that obesity is associated with ER stress and 12/15-LO is expressed in adipose tissue, we determined whether 12/15-LO could mediate ER stress signals. Addition of 12/15-LO lipid products 12(S)-HETE and 12(S)-HPETE to differentiated 3T3-L1 adipocytes induced expression and activation of ER stress markers, including BiP, XBP-1, p-PERK, and p-IRE1α. The ER stress inducer, tunicamycin, upregulated ER stress markers in adipocytes with concomitant 12/15-LO activation. Addition of a 12/15-LO inhibitor, CDC, to tunicamycin-treated adipocytes attenuated the ER stress response. Furthermore, 12/15-LO-deficient adipocytes exhibited significantly decreased tunicamycin-induced ER stress. 12/15-LO action involves upregulation of interleukin-12 (IL-12) expression. Tunicamycin significantly upregulated IL-12p40 expression in adipocytes, and IL-12 addition increased ER stress gene expression; conversely, LSF, an IL-12 signaling inhibitor, and an IL-12p40-neutralizing antibody attenuated tunicamycin-induced ER stress. Isolated adipocytes and liver from 12/15-LO-deficient mice fed a high-fat diet revealed a decrease in spliced XBP-1 expression compared with wild-type C57BL/6 mice on a high-fat diet. Furthermore, pancreatic islets from 12/15-LO-deficient mice showed reduced high-fat diet-induced ER stress genes compared with wild-type mice. These data suggest that 12/15-LO activity participates in ER stress in adipocytes, pancreatic islets, and liver. Therefore, reduction of 12/15-LO activity or expression could provide a new therapeutic target to reduce ER stress and downstream inflammation linked to obesity.

Type 2 diabetes is associated with obesity, insulin resistance, and inflammation in adipose tissue. 12/15-Lipoxygenase (12/15-LO) generates proinflammatory lipid mediators, which induce inflammation in adipose tissue. Therefore we investigated the role of 12/15-LO activity in mouse white adipose tissue in promoting obesity-induced local and systemic inflammatory consequences. We generated a mouse model for fat-specific deletion of 12/15-LO,aP2-Cre;12/15-LOloxP/loxP, which we call ad-12/15-LO mice, and placed wild-type controls and ad-12/15-LO mice on a high-fat diet for 16 weeks and examined obesity-induced inflammation and insulin resistance. High-fat diet-fed ad-12/15-LO exhibited improved fasting glucose levels and glucose metabolism, and epididymal adipose tissue from these mice exhibited reduced inflammation and macrophage infiltration compared to wild-type mice. Furthermore, fat-specific deletion of 12/15-LO led to decreased peripheral pancreatic islet inflammation with enlarged pancreatic islets when mice were fed the high-fat diet compared to wild-type mice. These results suggest an interesting crosstalk between 12/15-LO expression in adipose tissue and inflammation in pancreatic islets. Therefore, deletion of 12/15-LO in adipose tissue can offer local and systemic protection from obesity-induced consequences, and blocking 12/15-LO activity in adipose tissue may be a novel therapeutic target in the treatment of type 2 diabetes.

Adipose tissue inflammation in obesity is a major factor leading to cardiovascular disease and type 2 diabetes.12/15 lipoxygenases (ALOX) play an important role in the generation of inflammatory mediators, insulin resistance and downstream immune activation in animal models of obesity. However, the expression and roles of 12/15ALOX isoforms, and their cellular sources in human subcutaneous (sc) and omental (om) fat in obesity is unknown. The objective of this study was to examine the gene expression and localization of ALOX isoforms and relevant downstream cytokines in subcutaneous (sc) and omental (om) adipose tissue in obese humans. Paired biopsies of sc and om fat were obtained during bariatric surgeries from 24 morbidly obese patients. Gene and protein expression for ALOX15a, ALOX15b and ALOX 12 were measured by real-time PCR and western blotting in adipocytes and stromal vascular fractions (SVF) from om and sc adipose tissue along with the mRNA expression of the downstream cytokines IL-12a, IL-12b, IL-6, IFNγ and the chemokine CXCL10. In a paired analysis, all ALOX isoforms, IL-6, IL-12a and CXCL10 were significantly higher in om vs. sc fat. ALOX15a mRNA and protein expression was found exclusively in om fat. All of the ALOX isoforms were expressed solely in the SVF. Further fractionation of the SVF in CD34+ and CD34- cells indicated that ALOX15a is predominantly expressed in the CD34+ fraction including vascular and progenitor cells, while ALOX15B is mostly expressed in the CD34- cells containing various leucocytes and myeloid cells. This result was confirmed by immunohistochemistry showing exclusive localization of ALOX15a in the om fat and predominantly in the vasculature and non-adipocyte cells. Our finding is identifying selective expression of ALOX15a in human om but not sc fat. This is a study showing a major inflammatory gene exclusively expressed in visceral fat in humans.

Merlin, the product of the NF2 tumor suppressor gene, is closely related to the ERM (ezrin, radixin, moesin) proteins, which provide anchorage between membrane proteins and the underlying cortical cytoskeleton; all four proteins are members of the band 4.1 superfamily. Despite their similarity, the subcellular distributions and functional properties of merlin and the ERM proteins are largely distinct. Upon cell-cell contact merlin prevents internalization of and signaling from the epidermal growth factor receptor (EGFR) by sequestering it into an insoluble membrane compartment. Here we show that the extreme amino (N) terminus directs merlin biochemically to an insoluble membrane compartment and physically to the cortical actin network, with a marked concentration along cell-cell boundaries. This insoluble-membrane distribution is required for the growth-suppressing function of merlin and for the functional association of merlin with EGFR and other membrane receptors. Our data support a model whereby locally activated merlin sequesters membrane receptors such as EGFR at the cortical network, contributing to the long-held observation that the cortical actin cytoskeleton can control the lateral mobility of and signaling from certain membrane receptors.

The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a membrane/cytoskeleton-associated protein that mediates contact-dependent inhibition of proliferation. Here we show that upon cell–cell contact Merlin coordinates the processes of adherens junction stabilization and negative regulation of epidermal growth factor receptor (EGFR) signaling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized. In confluent Nf2−/− cells, EGFR activation persists, driving continued proliferation that is halted by specific EGFR inhibitors. These studies define a new mechanism of tumor suppression, provide mechanistic insight into the poorly understood phenomenon of contact-dependent inhibition of proliferation, and suggest a therapeutic strategy for NF2-mutant tumors.

Objectives— The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. Approach and Results— We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. Conclusions— The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Visceral adipose tissue (VAT) is a key contributor to chronic inflammation in obesity. The 12/15-lipoxygenase pathway (ALOX) is present in adipose tissue (AT) and leads to inflammatory cascades that are causal for the onset of insulin resistance in rodent models of obesity.The pathophysiology of the ALOX 12/15 pathway in human AT is unknown. We characterized the ALOX pathway in different AT depots in obese humans with or without type 2 diabetes (T2D).This study includes a cross-sectional cohort of 46 morbidly obese (body mass index39 kg/m(2)) nondiabetic (n = 25) and T2D (n = 21) subjects.This study was conducted at Eastern Virginia Medical School (Norfolk, Virginia) in collaboration with Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, Virginia).Twenty-five obese (body mass index 44.8 ± 4.4 kg/m(2)) nondiabetic (hemoglobin A1c 5.83% ± 0.27%) and 21 obese (43.4 ± 4.1 kg/m(2)) and T2D (hemoglobin A1c 7.66% ± 1.22%) subjects were included in the study. The subjects were age matched and both groups had a bias toward female gender.Expression of ALOX isoforms along with fatty acid substrates and downstream lipid metabolites were measured. Correlations with depot-specific inflammatory markers were also established.ALOX 12 expression and its metabolite 12(S)-hydroxyeicosatetraenoic acid were significantly increased in the VAT of T2D subjects. ALOX 15A was exclusively expressed in VAT in both groups. ALOX 12 expression positively correlated with expression of inflammatory genes IL-6, IL-12a, CXCL10, and lipocalin-2.ALOX 12 may have a critical role in regulation of inflammation in VAT in obesity and T2D. Selective ALOX 12 inhibitors may constitute a new approach to limit AT inflammation in human obesity.

AimsType 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D.Methods12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15(null) macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass.Results12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15(null) mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass.ConclusionsThese results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.

The lipoxygenases (LOs) are principal enzymes involved in the oxidative metabolism of polyunsaturated fatty acids, including arachidonic acid. 12- and 15-LO and their lipid metabolites have been implicated in the development of insulin resistance and diabetes. Adipose tissue, and in particular visceral adipose tissue, plays a primary role in the development of the inflammation seen in these conditions. 12- and 15-LO and their lipid metabolites act as upstream regulators of many of the cytokines involved in the inflammatory response in adipose tissue. While the role that 12- and 15-LO play in chronically inflamed adipose tissue is becoming clearer, there are still many questions that remain unanswered regarding their activation, signaling pathways, and roles in healthy fat. 12- and 15-LO also generate products with anti-inflammatory properties that are under investigation. Therefore, 12- and 15-LO have the potential to be very important targets for therapeutics aimed at reducing insulin resistance and the comorbid conditions associated with obesity.

Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.

Obesity, hypertension, cardiovascular disease, and inflammation are closely associated with the rising incidence of diabetes mellitus. One pharmacological target that may have significant potential to lower the risk of obesity-related diseases is the angiotensin type 1 receptor (AT1R). We examined the hypothesis that the AT1R blocker valsartan reduces the metabolic consequences and inflammatory effects of a high-fat (Western) diet in mice. C57BL/6J mice were treated by oral gavage with 10 mg/kg per day of valsartan or vehicle and placed on either a standard chow or Western diet for 12 weeks. Western diet-fed mice given valsartan had improved glucose tolerance, reduced fasting blood glucose levels, and reduced serum insulin levels compared with mice fed a Western diet alone. Valsartan treatment also blocked Western diet–induced increases in serum levels of the proinflammatory cytokines interferon-γ and monocyte chemotactic protein 1. In the pancreatic islets, valsartan enhanced mitochondrial function and prevented Western diet–induced decreases in glucose-stimulated insulin secretion. In adipose tissue, valsartan reduced Western diet–induced macrophage infiltration and expression of macrophage-derived monocyte chemotactic protein 1. In isolated adipocytes, valsartan treatment blocked or attenuated Western diet–induced changes in expression of several key inflammatory signals: interleukin 12p40, interleukin 12p35, tumor necrosis factor-α, interferon-γ, adiponectin, platelet 12-lipoxygenase, collagen 6, inducible NO synthase, and AT1R. Our findings indicate that AT1R blockade with valsartan attenuated several deleterious effects of the Western diet at the systemic and local levels in islets and adipose tissue. This study suggests that AT1R blockers provide additional therapeutic benefits in the metabolic syndrome and other obesity-related disorders beyond lowering blood pressure.

The antioxidant and anti-inflammatory compound AGI-1067 (succinobucol) has potential as an oral anti-diabetic agent. AGI-1067 reduces H b A1c, improves fasting plasma glucose, and reduces new-onset diabetes. We investigated AGI-1067 for possible effects on mouse pancreatic islets in vitro . Pretreatment with 10 μM AGI-1067 increased glucose-stimulated insulin secretion (11 mM) without affecting secretion in basal (3 mM) glucose. AGI-1067 enhanced the intracellular calcium response to glucose stimulation in 7 mM and 11 mM glucose, but had no effect in 28 mM or basal glucose. AGI-1067-pretreated islets also showed enhanced calcium responses to methyl pyruvate and alpha-ketoisocaproate at low doses, but not high doses. The AGI-1067-mediated effects on glucose-stimulated calcium were maintained during continuous diazoxide exposure, suggesting effects on the K ATP -channel-independent pathway. AGI-1067 also reduced cytokine-induced islet cell death and expression of iNOS, a key component in cytokine signaling. This is the first report of direct stimulatory and protective effects of a first-in-class potential anti-diabetic agent on pancreatic islets.

Inflammation and insulin resistance associated with visceral obesity are important risk factors for the development of type 2 diabetes, atherosclerosis, and the metabolic syndrome. The 12/15-lipoxygenase (12/15-LO) enzyme has been linked to inflammatory changes in blood vessels that precede the development of atherosclerosis. The expression and role of 12/15-LO in adipocytes have not been evaluated. We found that 12/15-LO mRNA was dramatically upregulated in white epididymal adipocytes of high-fat fed mice. 12/15-LO was poorly expressed in 3T3-L1 fibroblasts and was upregulated during differentiation into adipocytes. Interestingly, the saturated fatty acid palmitate, a major component of high fat diets, augmented expression of 12/15-LO in vitro. When 3T3-L1 adipocytes were treated with the 12/15-LO products, 12-hydroxyeicosatetranoic acid (12(S)-HETE) and 12-hydroperoxyeicosatetraenoic acid (12(S)-HPETE), expression of proinflammatory cytokine genes, including tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and IL-12p40, was upregulated whereas anti-inflammatory adiponectin gene expression was downregulated. 12/15-LO products also augmented c-Jun N-terminal kinase 1 (JNK-1) phosphorylation, a known negative regulator of insulin signaling. Consistent with impaired insulin signaling, we found that insulin-stimulated 3T3-L1 adipocytes exhibited decreased IRS-1(Tyr) phosphorylation, increased IRS-1(Ser) phosphorylation, and impaired Akt phosphorylation when treated with 12/15-LO product. Taken together, our data suggest that 12/15-LO products create a proinflammatory state and impair insulin signaling in 3T3-L1 adipocytes. Because 12/15-LO expression is upregulated in visceral adipocytes by high-fat feeding in vivo and also by addition of palmitic acid in vitro, we propose that 12/15-LO plays a role in promoting inflammation and insulin resistance associated with obesity.