Angiogenesis is a key pathological feature of experimental and human steatohepatitis, a common chronic liver disease that is associated with obesity. We demonstrated that hepatocytes generated a type of membrane-bound vesicle, microparticles, in response to conditions that mimicked the lipid accumulation that occurs in the liver in some forms of steatohepatitis and that these microparticles promoted angiogenesis. When applied to an endothelial cell line, medium conditioned by murine hepatocytes or a human hepatocyte cell line exposed to saturated free fatty acids induced migration and tube formation, two processes required for angiogenesis. Medium from hepatocytes in which caspase 3 was inhibited or medium in which the microparticles were removed by ultracentrifugation lacked proangiogenic activity. Isolated hepatocyte-derived microparticles induced migration and tube formation of an endothelial cell line in vitro and angiogenesis in mice, processes that depended on internalization of microparticles. Microparticle internalization required the interaction of the ectoenzyme Vanin-1 (VNN1), an abundant surface protein on the microparticles, with lipid raft domains of endothelial cells. Large quantities of hepatocyte-derived microparticles were detected in the blood of mice with diet-induced steatohepatitis, and microparticle quantity correlated with disease severity. Genetic ablation of caspase 3 or RNA interference directed against VNN1 protected mice from steatohepatitis-induced pathological angiogenesis in the liver and resulted in a loss of the proangiogenic effects of microparticles. Our data identify hepatocyte-derived microparticles as critical signals that contribute to angiogenesis and liver damage in steatohepatitis and suggest a therapeutic target for this condition.

Androgen exposure during intrauterine life in nonhuman primates and in sheep results in a phenocopy of the reproductive and metabolic features of polycystic ovary syndrome (PCOS). Such exposure also results in reproductive features of PCOS in rodents. We investigated whether transient prenatal androgen treatment produced metabolic abnormalities in adult female rats and the mechanisms of these changes. Pregnant dams received free testosterone or vehicle injections during late gestation, and their female offspring were fed regular or high-fat diet (HFD). At 60 days of age, prenatally androgenized (PA) rats exhibited significantly increased body weight; parametrial and subcutaneous fat; serum insulin, cholesterol and triglyceride levels; and hepatic triglyceride content (all P < 0.0125). There were no significant differences in insulin sensitivity by intraperitoneal insulin tolerance test or insulin signaling in liver or skeletal muscle. HFD had similar effects to PA on body weight and composition as well as on circulating triglyceride levels. HFD further increased hepatic triglyceride content to a similar extent in both PA and control rats. In PA rats, HFD did not further increase circulating insulin, triglyceride, or cholesterol levels. In control rats, HFD increased insulin levels, but to a lesser extent than PA alone ( approximately 2.5- vs. approximately 12-fold, respectively). We conclude that transient prenatal androgen exposure produces features of the metabolic syndrome in adult female rats. Dyslipidemia and hepatic steatosis appear to be mediated by PA-induced increases in adiposity, whereas hyperinsulinemia appears to be a direct result of PA.

In addition to its role in reproduction, estradiol-17β is critical to the regulation of energy balance and body weight. Estrogen receptor α-null (Erα -/-) mutant mice develop an obese state characterized by decreased energy expenditure, decreased locomotion, increased adiposity, altered glucose homeostasis, and hyperleptinemia. Such features are reminiscent of the propensity of postmenopausal women to develop obesity and type 2 diabetes. The mechanisms by which ERα signaling maintains normal energy balance, however, have remained unclear. Here we used knockin mice that express mutant ERα that can only signal through the noncanonical pathway to assess the role of nonclassical ERα signaling in energy homeostasis. In these mice, we found that nonclassical ERα signaling restored metabolic parameters dysregulated in Erα -/-mutant mice to normal or near-normal values. The rescue of body weight and metabolic function by nonclassical ERα signaling was mediated by normalization of energy expenditure, including voluntary locomotor activity. These findings indicate that nonclassical ERα signaling mediates major effects of estradiol-17β on energy balance, raising the possibility that selective ERα agonists may be developed to reduce the risks of obesity and metabolic disturbances in postmenopausal women. IntroductionIn addition to its critical functions as a reproductive hormone, estradiol-17β (E 2 ) plays a vital role in the regulation of energy balance and body weight (1). Estrogen deficiency at menopause is associated with an increased probability of obesity as well as increased risk for the development of type 2 diabetes (2). In experimental animals, reduction of circulating estrogen levels by ovariectomy leads to the development of obesity, which can be reversed or prevented by E 2 treatment (1). The effects of E 2 on energy balance bear many similarities to the actions of leptin and insulin, key molecules involved in energy homeostasis (3, 4). Genetic and pharmacological studies have demonstrated that leptin and insulin act directly on neural networks to modulate energy homeostasis, where the net effect is to decrease food intake and increase energy expenditure (5-9). Both can activate STAT3 in various tissues, and hypothalamic leptin and insulin signaling are known to converge on the PI3K pathway (10-13). Similarly, E 2 is now also known to activate STAT3 as well as PI3K signaling cascades, suggestive of possible cross-talk among these molecules and possibly representing a common neuronal signaling mechanism that may help explain the similarities in their central effects on energy homeostasis (14-17).That these metabolic actions of E 2 are mediated by estrogen receptor α (ERα) has been demonstrated in Erα-null (Erα -/-) mutant mice, in which the ablation of ERα signaling results in a metabolic syndrome characterized by increased body weight, adiposity, altered glucose homeostasis, decreased energy expenditure, hyperinsulinemia, and hyperleptinemia (18)(19)(20). However, these metabolic character-

We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as “find me” signals to promote macrophage migration and activation. In this study we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with Metabolic Syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin and homeostatic model assessment of insulin resistance (HOMA-IR), in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35% (p<0.05) after a 3-month reduced calorie diet intervention. This translational study provides an extensive characterization of adipocyte-derived EVs. The findings identify perilipin A as a novel biomarker of circulating EVs of adipocyte origin and support the development of circulating perilipin A-positive EVs as indicators of adipose tissue health.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.

Obesity is associated with metabolic perturbations including liver and adipose tissue inflammation, insulin resistance, and type 2 diabetes. Omega-6 fatty acids (ω6) promote and omega-3 fatty acids (ω3) reduce inflammation as they can be metabolized to pro- and anti-inflammatory eicosanoids, respectively. 12/15-lipoxygenase (12/15-LO) enzymatically produces some of these metabolites and is induced by high fat (HF) diet. We investigated the effects of altering dietary ω6/ω3 ratio and 12/15-LO deficiency on HF diet-induced tissue inflammation and insulin resistance. We examined how these conditions affect circulating concentrations of oxidized metabolites of ω6 arachidonic and linoleic acids and innate and adaptive immune system activity in the liver. For 15 weeks, wild-type (WT) mice were fed either a soybean oil-enriched HF diet with high dietary ω6/ω3 ratio (11∶1, HFH), similar to Western-style diet, or a fat Kcal-matched, fish oil-enriched HF diet with a low dietary ω6/ω3 ratio of 2.7∶1 (HFL). Importantly, the total saturated, monounsaturated and polyunsaturated fat content was matched in the two HF diets, which is unlike most published fish oil studies in mice. Despite modestly increased food intake, WT mice fed HFL were protected from HFH-diet induced steatohepatitis, evidenced by decreased hepatic mRNA expression of pro-inflammatory genes and genes involved in lymphocyte homing, and reduced deposition of hepatic triglyceride. Furthermore, oxidized metabolites of ω6 arachidonic acid were decreased in the plasma of WT HFL compared to WT HFH-fed mice. 12/15-LO knockout (KO) mice were also protected from HFH-induced fatty liver and elevated mRNA markers of inflammation and lymphocyte homing. 12/15-LOKO mice were protected from HFH-induced insulin resistance but reducing dietary ω6/ω3 ratio in WT mice did not ameliorate insulin resistance or adipose tissue inflammation. In conclusion, lowering dietary ω6/ω3 ratio in HF diet significantly reduces steatohepatitis.

Aim: Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. Materials & methods: RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. Results: The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. Conclusion: These findings uncover RNP as a novel potential NASH therapy.

Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo­[5,1-b]­[1,3]­oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.