Dietary fat promotes pathological insulin resistance through chronic inflammation1–3. Macrophage inactivation of inflammatory proteins improves diet-induced diabetes4, but how nutrient-dense diets induce diabetes is unknown5. Membrane lipids affect the innate immune response6, which requires domains7 that influence high-fat diet (HFD)-induced chronic inflammation8,9 and alter cell function based on phospholipid composition10. Endogenous fatty acid synthesis, mediated by fatty acid synthase (FAS)11, affects membrane composition. Here we show that macrophage FAS is indispensable for dietinduced inflammation. Deleting FAS in macrophages prevents diet-induced insulin resistance, adipose macrophage recruitment, and chronic inflammation in mice. FAS deficiency alters membrane order and composition, impairing retention of plasma membrane cholesterol, as well as disrupting Rho GTPase trafficking required for cell adhesion, migration, and activation. Expressing a constitutively active Rho GTPase restored inflammatory signaling. Exogenous palmitate partitioned to different pools than endogenous lipids and did not rescue inflammatory signaling. However, exogenous cholesterol as well as other planar sterols rescued signaling, and exogenous cholesterol restored FAS-induced perturbations in membrane order. Endogenous fat production in macrophages is necessary for exogenous fat-induced insulin resistance by creating a receptive environment at the plasma membrane for assembly of cholesterol-dependent signaling networks.

Exogenous dietary fat can induce obesity and promote diabetes, but endogenous fat production is not thought to affect skeletal muscle insulin resistance, an antecedent of metabolic disease. Unexpectedly, the lipogenic enzyme fatty acid synthase (FAS) was increased in the skeletal muscle of mice with diet-induced obesity and insulin resistance. Skeletal muscle-specific inactivation of FAS protected mice from insulin resistance without altering adiposity, specific inflammatory mediators of insulin signaling, or skeletal muscle levels of diacylglycerol or ceramide. Increased insulin sensitivity despite high-fat feeding was driven by activation of AMPK without affecting AMP content or the AMP/ATP ratio in resting skeletal muscle. AMPK was induced by elevated cytosolic calcium caused by impaired sarco/endoplasmic reticulum calcium ATPase (SERCA) activity due to altered phospholipid composition of the sarcoplasmic reticulum (SR), but came at the expense of decreased muscle strength. Thus, inhibition of skeletal muscle FAS prevents obesity-associated diabetes in mice, but also causes muscle weakness, which suggests that mammals have retained the capacity for lipogenesis in muscle to preserve physical performance in the setting of disrupted metabolic homeostasis.

SUMMARY The intestinal mucus barrier prevents pathogen invasion and maintains host-microbiota homeostasis. We show that fatty acid synthase (FAS), an insulin-responsive enzyme essential for de novo lipogenesis, helps maintain the mucus barrier by regulating Mucin 2, the dominant mucin in the colon and a central component of mucus. Inducible Cre recombinase-directed inactivation of the FAS gene in the colonic epithelium of mice is associated with disruptions in the intestinal mucus barrier as well as increased intestinal permeability, colitis, systemic inflammation, and changes in gut microbial ecology. FAS deficiency blocked the generation of palmitoylated Mucin 2, which must be S-palmitoylated at its N-terminus for proper secretion and function. Furthermore, a diabetic mouse model exhibited lower FAS levels and a decreased mucus layer, which could be restored with insulin treatment. Thus, the role of FAS in maintaining intestinal barrier function may explain the pathogenesis of intestinal inflammation in diabetes and other disorders.

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De novo lipogenesis, the production of fats from simple precursors, is often dismissed as irrelevant to the pathobiology of obesity caused by positive energy balance due to typical high fat diets. However, emerging data implicate de novo lipogenesis in the generation of metabolic signals that alter disease risk. Exploiting this signaling pathway represents lipoexpediency. Lipoexpediency is the concept of directing fats toward benefit even in the setting of lipid overload, and represents a strategy to complement efforts aimed at improving energy balance. Optimizing lipid signals initiated by key lipogenic enzymes such as fatty acid synthase might limit morbidity in people unlikely to abandon the lifestyle of the sedentary gourmand. The reality of lipid excess An epidemic with limited novel treatment approachesOver 40% of Americans have diabetes or prediabetes [1] and as many as a quarter of American adults have the metabolic syndrome, which predisposes to diabetes and cardiovascular disease [2]. Tobacco use increases health risks that have been decreased by public health measures targeted at smoking, but the adverse effects of obesity will probably negate the beneficial effects of declining tobacco use in the United States by 2020 [3]. The metabolic mayhem promoted by a Western lifestyle has been exported worldwide, and diabetes is now a major and accelerating public health problem in China [4]. Diabetes, obesity, and the metabolic syndrome also increase the risk of nonalcoholic fatty liver disease [5]. Lifestyle changes are difficult to institute given the pressures of Western culture, and other therapies are limited. For example, insulin therapy lowers hemoglobin A1c but may increase adiposity. Statins and reninangiotensin system (RAS) inhibitors decrease but do not eliminate cardiovascular risk, i.e. individuals with dyslipidemia, hypertension, and diabetes are still at considerable risk for heart attacks even when treated with statins and RAS inhibitors. PPAR agonists have a mixed record that is still evolving, and it is unknown if GLP-1 modulators will decrease complications of diabetes and obesity.

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.Damage to blood vessels is inextricably linked with diabetes. Microvascular disease causes visual loss, renal failure, and neuropathy, and macrovascular disease (atherosclerotic coronary disease, stroke, and peripheral arterial disease) causes premature death. Macro-and microvascular complications characterize both type 1 (insulin-deficient) and type 2 (insulin-resistant with hyperinsulinemia) diabetes. Hyperglycemia is a biomarker of blood vessel disease (1), and glucose lowering delays microvascular complications (2, 3), but this therapy does not prevent progression of established microvascular (4) or macrovascular disease (5, 6). In addition to improving glycemia, optimal approaches to complications may require manipulating processes that are mostly obscure.One such process is endothelial lipid metabolism. The endothelium controls tissue access from the circulation and regulates vascular functions, including inflammation and angiogenesis. Endothelial dysfunction, largely due to defects in eNOS, 3 is characteristic of diabetes and probably promotes vascular disease (7,8). Circulating fatty acids interfere with eNOS and increase inflammation (9, 10), and insulin resistance appears to have similar effects by increasing fatty acid oxidation (11), but little is known about how fuels are partitioned inside endothelial cells. A critical pathway for fuel flow is de novo lipogenesis, the generation of fatty acids from simple sugars, which requires FAS (12, 13). One might predict that de novo lipogenesis would be irrelevant to endothelial cells, which are continually bathed in fatty acids, the product of the FAS reaction.Here, we report that endothelial...

Introduction: Bone loss adjacent to the implant is a major cause of joint arthroplasty failure. Although the cellular and molecular response to microscopic wear debris particles is recognized as causative, little is known concerning role of synovial fibroblasts in these events. Materials and Methods: Murine embryonic fibroblasts and knee synovial fibroblasts in culture stimulated with titanium particles were examined by FACS, real time RT-PCR, Northern blot, and Western blot for expressions of vascular cell adhesion molecule (VCAM)1, RANKL, cyclooxygenase (COX)-1, and COX-2, and the four prostaglandin E2 (PGE2) receptor isoforms. Experiments were performed in the presence and absence of COX inhibitors, protein kinase A (PKA) and protein kinase C (PKC) inhibitors, and various EP receptor agonists. Osteoclast formation was examined in co-cultures of pretreated glutaraldehyde-fixed fibroblasts and primary murine spleen cells treated with macrophage-colony stimulating factor (M-CSF) for 7-days. Results: TNF-␣ stimulated VCAM1 expression, consistent with a synovial fibroblast phenotype. Titanium particles stimulated RANKL gene and protein expressions in fibroblasts in a dose-dependent manner. Gene expression was increased 5-fold by 4 h, and protein levels reached a maximum after 48 h. Within 1 h, titanium particles also induced COX-2 mRNA and protein levels, whereas both indomethacin and celecoxib blocked the stimulation of RANKL, suggesting a COX-2-mediated event. Furthermore, PGE2 induced RANKL gene and protein expression and rescued RANKL expression in titanium-treated cultures containing COX-2 inhibitors. Fibroblast cultures pretreated with either PGE2 or titanium particles enhanced osteoclast formation, indicating the functional importance of RANKL induction. EP4 was the most abundant PGE2 receptor isoform, EP1 and EP2 were expressed at low levels, and EP3 was absent. The EP1 selective agonist iloprost and the EP2 selective agonist butaprost minimally stimulated RANKL. In contrast, the EP2 and EP4 agonist misoprostol induced RANKL to a magnitude similar to PGE2. Finally, PKA antagonism strongly repressed RANKL stimulation by PGE2. Conclusion: Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Thus, the synovial fibroblast is important mediator of osteolysis and target for therapeutic strategies.

SUMMARY Fatty acid synthase (FAS) is altered in metabolic disorders and cancer. Conventional FAS null mice die in utero so effects of whole body inhibition of lipogenesis following development are unknown. Inducible global knockout of FAS (iFASKO) in mice was lethal due to a disrupted intestinal barrier and leukopenia. Conditional loss of FAS was associated with the selective suppression of granulopoiesis without disrupting granulocytic differentiation. Transplantation of iFASKO bone marrow into wild type mice followed by Cre induction resulted in selective neutrophil depletion but not death. Impaired lipogenesis increased ER stress and apoptosis in neutrophils by preferentially decreasing peroxisome-derived membrane phospholipids containing ether bonds. Inducible global knockout of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, also produced neutropenia. FAS knockdown in neutrophil-like HL-60 cells caused cell loss that was partially rescued by ether lipids. Inhibiting ether lipid synthesis selectively constrains neutrophil development, revealing an unrecognized pathway in immunometabolism.