Manu V. Chakravarthy

Identification of a Physiologically Relevant Endogenous Ligand for PPARα in Liver

The nuclear receptor PPARalpha is activated by drugs to treat human disorders of lipid metabolism. Its endogenous ligand is unknown. PPARalpha-dependent gene expression is impaired with inactivation of fatty acid synthase (FAS), suggesting that FAS is involved in generation of a PPARalpha ligand. Here we demonstrate the FAS-dependent presence of a phospholipid bound to PPARalpha isolated from mouse liver. Binding was increased under conditions that induce FAS activity and displaced by systemic injection of a PPARalpha agonist. Mass spectrometry identified the species as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (16:0/18:1-GPC). Knockdown of Cept1, required for phosphatidylcholine synthesis, suppressed PPARalpha-dependent gene expression. Interaction of 16:0/18:1-GPC with the PPARalpha ligand-binding domain and coactivator peptide motifs was comparable to PPARalpha agonists, but interactions with PPARdelta were weak and none were detected with PPARgamma. Portal vein infusion of 16:0/18:1-GPC induced PPARalpha-dependent gene expression and decreased hepatic steatosis. These data suggest that 16:0/18:1-GPC is a physiologically relevant endogenous PPARalpha ligand.

Brain fatty acid synthase activates PPARα to maintain energy homeostasis

Central nervous system control of energy balance affects susceptibility to obesity and diabetes, but how fatty acids, malonyl-CoA, and other metabolites act at this site to alter metabolism is poorly understood. Pharmacological inhibition of fatty acid synthase (FAS), rate limiting for de novo lipogenesis, decreases appetite independently of leptin but also promotes weight loss through activities unrelated to FAS inhibition. Here we report that the conditional genetic inactivation of FAS in pancreatic β cells and hypothalamus produced lean, hypophagic mice with increased physical activity and impaired hypothalamic PPARα signaling. Administration of a PPARα agonist into the hypothalamus increased PPARα target genes and normalized food intake. Inactivation of β cell FAS enzyme activity had no effect on islet function in culture or in vivo. These results suggest a critical role for brain FAS in the regulation of not only feeding, but also physical activity, effects that appear to be mediated through the provision of ligands generated by FAS to PPARα. Thus, 2 diametrically opposed proteins, FAS (induced by feeding) and PPARα (induced by starvation), unexpectedly form an integrative sensory module in the central nervous system to orchestrate energy balance.

Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long‐Evans Tokushima Fatty (OLETF) rats

The purpose of this study was to delineate potential mechanisms initiating the onset of hepatic steatosis following the cessation of daily physical activity. Four‐week‐old, hyperphagic/obese Otsuka Long‐Evans Tokushima Fatty rats were given access to voluntary running wheels for 16 weeks to prevent the development of hepatic steatosis. The animals were then suddenly transitioned to a sedentary condition as wheels were locked (wheel lock; WL) for 5 h (WL5), 53 h (WL53) or 173 h (WL173). Importantly after the cessation of daily exercise (5–173 h), no changes occurred in body weight, fat pad mass (omental and retroperitoneal), food intake, serum insulin, hepatic triglycerides or in the exercise‐suppressed hepatic stearoyl‐CoA desaturase‐1 and peroxisome proliferator‐activated receptor‐γ protein content. However, complete hepatic fatty acid oxidation and mitochondrial enzyme activities were highest at WL5 and WL53 and dropped significantly to SED levels by WL173. In addition, cessation of daily exercise quickly increased the hepatic protein contents of fatty acid synthase and acetyl‐coenzyme A carboxylase (ACC), reduced ACC phosphorylation status, and dramatically increased hepatic malonyl‐CoA concentration. This study is the first to show that the sudden cessation of daily exercise in a hyperphagic/obese model activates a subgroup of precursors and processes known to initiate hepatic steatosis, including decreased hepatic mitochondrial oxidative capacity, increased hepatic expression of de novo lipogenesis proteins, and increased hepatic malonyl CoA levels; each probably increasing the susceptibility to non‐alcoholic fatty liver disease.

Insulin Resistance and Atherosclerosis

Insulin resistance characterizes type 2 diabetes and the metabolic syndrome, disorders associated with an increased risk of death due to macrovascular disease. In the past few decades, research from both the basic science and clinical arenas has enabled evidence-based use of therapeutic modalities such as statins and angiotensin-converting enzyme inhibitors to reduce cardiovascular (CV) mortality in insulin-resistant patients. Recently, promising drugs such as the thiazolidinediones have come under scrutiny for possible deleterious CV effects. Ongoing research has broadened our understanding of the pathophysiology of atherosclerosis, implicating detrimental effects of inflammation and the cellular stress response on the vasculature. In this review, we address current thinking that is shaping our molecular understanding of insulin resistance and atherosclerosis.

De Novo Lipogenesis Maintains Vascular Homeostasis through Endothelial Nitric-oxide Synthase (eNOS) Palmitoylation

Endothelial dysfunction leads to lethal vascular complications in diabetes and related metabolic disorders. Here, we demonstrate that de novo lipogenesis, an insulin-dependent process driven by the multifunctional enzyme fatty-acid synthase (FAS), maintains endothelial function by targeting endothelial nitric-oxide synthase (eNOS) to the plasma membrane. In mice with endothelial inactivation of FAS (FASTie mice), eNOS membrane content and activity were decreased. eNOS and FAS were physically associated; eNOS palmitoylation was decreased in FAS-deficient cells, and incorporation of labeled carbon into eNOS-associated palmitate was FAS-dependent. FASTie mice manifested a proinflammatory state reflected as increases in vascular permeability, endothelial inflammatory markers, leukocyte migration, and susceptibility to LPS-induced death that was reversed with an NO donor. FAS-deficient endothelial cells showed deficient migratory capacity, and angiogenesis was decreased in FASTie mice subjected to hindlimb ischemia. Insulin induced FAS in endothelial cells freshly isolated from humans, and eNOS palmitoylation was decreased in mice with insulin-deficient or insulin-resistant diabetes. Thus, disrupting eNOS bioavailability through impaired lipogenesis identifies a novel mechanism coordinating nutritional status and tissue repair that may contribute to diabetic vascular disease.

Inactivation of hypothalamic FAS protects mice from diet-induced obesity and inflammation

Obesity promotes insulin resistance and chronic inflammation. Disrupting any of several distinct steps in lipid synthesis decreases adiposity, but it is unclear if this approach coordinately corrects the environment that propagates metabolic disease. We tested the hypothesis that inactivation of FAS in the hypothalamus prevents diet-induced obesity and systemic inflammation. Ten weeks of high-fat feeding to mice with inactivation of FAS (FASKO) limited to the hypothalamus and pancreatic β cells protected them from diet-induced obesity. Though high-fat fed FASKO mice had no β-cell phenotype, they were hypophagic and hypermetabolic, and they had increased insulin sensitivity at the liver but not the periphery as demonstrated by hyperinsulinemic-euglycemic clamps, and biochemically by increased phosphorylated Akt, glycogen synthase kinase-3beta, and FOXO1 compared with wild-type mice. High-fat fed FASKO mice had decreased excretion of urinary isoprostanes, suggesting less oxidative stress and blunted tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) responses to endotoxin, suggesting less systemic inflammation. Pair-feeding studies demonstrated that these beneficial effects were dependent on central FAS disruption and not merely a consequence of decreased adiposity. Thus, inducing central FAS deficiency may be a valuable integrative strategy for treating several components of the metabolic syndrome, in part by correcting hepatic insulin resistance and suppressing inflammation.

Macrophage Fatty-acid Synthase Deficiency Decreases Diet-induced Atherosclerosis

Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. To determine whether fatty acid synthesis affects atherosclerosis, we inactivated fatty-acid synthase (FAS) in macrophages of apoE-deficient mice. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals. Atherosclerotic extent was decreased 20–40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor α (LXRα; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor α (PPARα) target gene expression was decreased in FASKOM macrophages. PPARα agonist treatment of FASKOM and wild type macrophages normalized PPARα target gene expression as well as Nr1h3 (LXRα). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRα-deficient/FAS-deficient bone marrow as compared with LXRα-replete/FAS-deficient marrow, consistent with anti-atherogenic effects of LXRα in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRα and suggest that FAS, which is induced by LXRα, may generate regulatory lipids that cause feedback inhibition of LXRα in macrophages.

Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion

Biliopancreatic diversion (BPD) improves insulin sensitivity and decreases serum cholesterol out of proportion with weight loss. Mechanisms of these effects are unknown. One set of proposed contributors to metabolic improvements after bariatric surgeries is bile acids (BAs). We investigated the early and late effects of BPD on plasma BA levels, composition, and markers of BA synthesis in 15 patients with type 2 diabetes (T2D). We compared these to the early and late effects of Roux-en-Y gastric bypass (RYGB) in 22 patients with T2D and 16 with normal glucose tolerance. Seven weeks after BPD, insulin sensitivity had doubled and serum cholesterol had halved. At this time, BA synthesis markers and total plasma BAs, particularly unconjugated BAs, had markedly risen; this effect could not be entirely explained by low FGF19. In contrast, after RYGB, insulin sensitivity improved gradually with weight loss and cholesterol levels declined marginally; BA synthesis markers were decreased at an early time point (2 weeks) after surgery and returned to the normal range 1 year later. These findings indicate that BA synthesis contributes to the decreased serum cholesterol after BPD. Moreover, they suggest a potential role for altered enterohepatic circulation of BAs in improving insulin sensitivity and cholesterol metabolism after BPD.

Decreased Fetal Size Is Associated With β-Cell Hyperfunction in Early Life and Failure With Age

OBJECTIVE—Low birth weight is associated with diabetes in adult life. Accelerated or “catch-up” postnatal growth in response to small birth size is thought to presage disease years later. Whether adult disease is caused by intrauterine β-cell–specific programming or by altered metabolism associated with catch-up growth is unknown. RESEARCH DESIGN AND METHODS—We generated a new model of intrauterine growth restriction due to fatty acid synthase (FAS) haploinsufficiency (FAS deletion [FASDEL]). Developmental programming of diabetes in these mice was assessed from in utero to 1 year of age. RESULTS—FASDEL mice did not manifest catch-up growth or insulin resistance. β-Cell mass and insulin secretion were strikingly increased in young FASDEL mice, but β-cell failure and diabetes occurred with age. FASDEL β-cells had altered proliferative and apoptotic responses to the common stress of a high-fat diet. This sequence appeared to be developmentally entrained because β-cell mass was increased in utero in FASDEL mice and in another model of intrauterine growth restriction caused by ectopic expression of uncoupling protein-1. Increasing intrauterine growth in FASDEL mice by supplementing caloric intake of pregnant dams normalized β-cell mass in utero. CONCLUSIONS—Decreased intrauterine body size, independent of postnatal growth and insulin resistance, appears to regulate β-cell mass, suggesting that developing body size might represent a physiological signal that is integrated through the pancreatic β-cell to establish a template for hyperfunction in early life and β-cell failure with age.

The ABCs of β-cell dysfunction in type 2 diabetes

Cholesterol toxicity may damage pancreatic β-cells, leading to decreased insulin secretion and impaired glucose tolerance ( pages 340–347 ).