Title: Inactivation of hypothalamic FAS protects mice from diet-induced obesity and inflammation
Abstract: 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. 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. Constant availability of food and sedentary living in contemporary wealthy cultures has dramatically limited the ability to utilize fat stores, resulting in an obesity epidemic. Worldwide, at least 1 in 10 adults is obese, and more than 25% are affected in many Western countries (1Ogden C.L. Carroll M.D. Curtin L.R. McDowell M.A. Tabak C.J. Flegal K.M. Prevalence of overweight and obesity in the United States, 1999–2004.JAMA. 2006; 295: 1549-1555Crossref PubMed Scopus (7400) Google Scholar), leading to striking increases in type 2 diabetes and heart disease (2Li Z. Bowerman S. Heber D. Health ramifications of the obesity epidemic.Surg. Clin. North Am. 2005; 85 (v.).: 681-701Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Over a quarter of adults in the United States have the metabolic syndrome (3Ford E.S. Giles W.H. Mokdad A.H. Increasing prevalence of the metabolic syndrome among US adults.Diabetes Care. 2004; 27: 2444-2449Crossref PubMed Scopus (1231) Google Scholar), a combination of central obesity, glucose intolerance/insulin resistance, dyslipidemia, and hypertension (4Alberti K.G. Zimmet P. Shaw J. Metabolic syndrome–a new world-wide definition. A consensus statement from the International Diabetes Federation.Diabet. Med. 2006; 23: 469-480Crossref PubMed Scopus (4429) Google Scholar), that confers a 2- to 3-fold increase risk for cardiovascular morbidity and mortality (5Isomaa B. Almgren P. Tuomi T. Forsen B. Lahti K. Nissen M. Taskinen M.R. Groop L. Cardiovascular morbidity and mortality associated with the metabolic syndrome.Diabetes Care. 2001; 24: 683-689Crossref PubMed Scopus (3849) Google Scholar). Given the reluctance of most modern adults to eat less and exercise more, many groups have altered lipid metabolism in mice in hopes of establishing proof of principle for new obesity therapies. Mice are resistant to diet-induced obesity after genetic manipulations that decrease lipid synthesis (acetyl-CoA carboxylase-2, stearoyl-CoA desaturase-1, diacylglycerol acyltransferase-1) and adipogenesis [peroxisome-proliferator-activated receptor (Ppar)γ], increase mitochondrial gene expression (Pparδ), increase respiratory uncoupling (uncoupling protein-1, liver X receptor), or perturb intracellular signaling (Ikkβ, S6k1) (6Abu-Elheiga L. Oh W. Kordari P. Wakil S.J. Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets.Proc. Natl. Acad. Sci. USA. 2003; 100: 10207-10212Crossref PubMed Scopus (327) Google Scholar, 7Chen H.C. Jensen D.R. Myers H.M. Eckel R.H. Farese Jr., R.V. Obesity resistance and enhanced glucose metabolism in mice transplanted with white adipose tissue lacking acyl CoA:diacylglycerol acyltransferase 1.J. Clin. Invest. 2003; 111: 1715-1722Crossref PubMed Scopus (81) Google Scholar, 8Jones J.R. Barrick C. Kim K.A. Lindner J. Blondeau B. Fujimoto Y. Shiota M. Kesterson R.A. Kahn B.B. Magnuson M.A. Deletion of PPARgamma in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance.Proc. Natl. Acad. Sci. USA. 2005; 102: 6207-6212Crossref PubMed Scopus (393) Google Scholar, 9Kalaany N.Y. Gauthier K.C. Zavacki A.M. Mammen P.P. Kitazume T. Peterson J.A. Horton J.D. Garry D.J. Bianco A.C. Mangelsdorf D.J. LXRs regulate the balance between fat storage and oxidation.Cell Metab. 2005; 1: 231-244Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 10Li B. Nolte L.A. Ju J.S. Han D.H. Coleman T. Holloszy J.O. Semenkovich C.F. Skeletal muscle respiratory uncoupling prevents diet-induced obesity and insulin resistance in mice.Nat. Med. 2000; 6: 1115-1120Crossref PubMed Scopus (265) Google Scholar, 11Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. Attie A.D. Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity.Proc. Natl. Acad. Sci. USA. 2002; 99: 11482-11486Crossref PubMed Scopus (880) Google Scholar, 12Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. et al.Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity.Nature. 2004; 431: 200-205Crossref PubMed Scopus (1367) Google Scholar, 13Wang Y.X. Lee C.H. Tiep S. Yu R.T. Ham J. Kang H. Evans R.M. Peroxisome-proliferator-activated receptor delta activates fat metabolism to prevent obesity.Cell. 2003; 113: 159-170Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar–14Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta.Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1627) Google Scholar). However, it is not clear that simply decreasing adiposity will universally improve the metabolic disease milieu. For instance, liver X receptor-deficient mice have increased metabolism, yet they are predisposed to the chronic inflammatory process of atherosclerosis (15Bradley M.N. Hong C. Chen M. Joseph S.B. Wilpitz D.C. Wang X. Lusis A.J. Collins A. Hseuh W.A. Collins J.L. et al.Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE.J. Clin. Invest. 2007; 117: 2337-2346Crossref PubMed Scopus (223) Google Scholar). Stearoyl-CoA desaturase-1 null mice are also hypermetabolic and thin but are predisposed to acute colitis (16Chen C. Shah Y.M. Morimura K. Krausz K.W. Miyazaki M. Richardson T.A. Morgan E.T. Ntambi J.M. Idle J.R. Gonzalez F.J. Metabolomics reveals that hepatic stearoyl-CoA desaturase 1 downregulation exacerbates inflammation and acute colitis.Cell Metab. 2008; 7: 135-147Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). We recently identified hypothalamic expression of another key protein in lipid metabolism, as a mediator of energy homeostasis (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). FAS is ubiquitously expressed and catalyzes the first committed step in fatty acid biosynthesis (18Semenkovich C.F. Regulation of fatty acid synthase (FAS).Prog. Lipid Res. 1997; 36: 43-53Crossref PubMed Scopus (204) Google Scholar). Pharmacologic inhibition of this enzyme with compounds such as C75 had previously implicated FAS in bioenergetics, but the lack of specificity of C75, notably its activation of the sympathetic nervous system (19Cha S.H. Hu Z. Chohnan S. Lane M.D. Inhibition of hypothalamic fatty acid synthase triggers rapid activation of fatty acid oxidation in skeletal muscle.Proc. Natl. Acad. Sci. USA. 2005; 102: 14557-14562Crossref PubMed Scopus (85) Google Scholar), raised the possibility that mechanisms independent of FAS could be involved. By mating FAS floxed mice (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) with RIPCre transgenic animals (known to express the Cre recombinase in pancreatic β cells and the hypothalamus), we generated animals with FAS deficiency limited to pancreatic β cells and the hypothalamus (FASKO). The lack of FAS, at least on a standard chow diet, did not affect β−cell function. However, hypothalamic FAS deficiency resulted in mice that were hypophagic, hypermetabolic, and lean on a standard chow diet (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). While these results provided unexpected insights into the role of hypothalamic FAS in feeding and behavior, they did not address effects of this manipulation on the risk for diet-induced obesity, insulin resistance, and chronic inflammation that are characteristic of human type 2 diabetes and metabolic syndrome. In addition, the high-fat feeding paradigm also allowed us to critically examine whether the beneficial metabolic effects in the FASKO mice are simply a consequence of decreased adiposity or due to a specific perturbation in central FAS signaling. Here we report the results of experiments testing the hypothesis that deficiency of hypothalamic FAS prevents diet-induced obesity and systemic inflammation, clinically relevant endpoints. Protocols were approved by the Washington University Animal Studies Committee. Generation of mice with FAS deletion in the hypothalamus and pancreatic β cells (FASKO), and wild-type (Cre negative with the FAS floxed allele) in a mixed (BL/6 and 129) background has been described (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). Starting at 6 weeks of age, FASKO and wild-type littermates were started on a Western-style diet (TD 88137, Harlan Teklad) [high-fat diet (HFD)] containing 21% (w/w) total lipid and 0.15% (w/w) total cholesterol for 10 weeks. Fatty acid composition of this diet is presented in supplementary Table I. Body weight was recorded weekly, and food intake [expressed as a function of lean body mass (g0.75)] was measured biweekly. On week 8, stools were collected to determine lipid content (21Gates A.C. Bernal-Mizrachi C. Chinault S.L. Feng C. Schneider J.G. Coleman T. Malone J.P. Townsend R.R. Chakravarthy M.V. Semenkovich C.F. Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease.Cell Metab. 2007; 6: 497-505Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and percentage of consumed lipid that was absorbed was calculated as (food intake × food lipid content) − (stool output × stool lipid content)/(food intake × food lipid content) × 100. After 10 weeks of high-fat feeding, another cohort of wild-type mice received an amount of food identical to that consumed by the FASKO mice (pair-fed) for an additional 15 days. These pair-fed wild-type mice were then subjected to the same experiments (see below) as freely fed wild-type and FASKO mice. Body composition, indirect calorimetry, locomotor activity, serum glucose, insulin, nonesterified fatty acids, triglycerides, cholesterol, β-hydroxybutyrate, leptin, adiponectin, glucose, and insulin tolerance tests were performed as described (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar, 20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Hepatic lipids were extracted in chloroform/methanol (2:1, v/v) followed by determination of triglyceride and cholesterol content (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Serum lipoprotein profiles were analyzed by size exclusion chromatography (21Gates A.C. Bernal-Mizrachi C. Chinault S.L. Feng C. Schneider J.G. Coleman T. Malone J.P. Townsend R.R. Chakravarthy M.V. Semenkovich C.F. Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease.Cell Metab. 2007; 6: 497-505Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Pancreatic islets isolated by collagenase digestion (22Pappan K.L. Pan Z. Kwon G. Marshall C.A. Coleman T. Goldberg I.J. McDaniel M.L. Semenkovich C.F. Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion.J. Biol. Chem. 2005; 280: 9023-9029Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) were cultured with 3 mM and 16.7 mM glucose, or 10 mM arginine, and insulin secreted in the media was assayed (22Pappan K.L. Pan Z. Kwon G. Marshall C.A. Coleman T. Goldberg I.J. McDaniel M.L. Semenkovich C.F. Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion.J. Biol. Chem. 2005; 280: 9023-9029Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Insulin content of islets and whole pancreas was determined after acid-ethanol extraction (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). Adipose and liver tissues were processed as described (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Adipose sections were stained with anti-F4/80 antibody (1:200, Abcam) and visualized using NovoRed (Vector) (23Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7473) Google Scholar). Livers were stained with Oil Red O (Sigma) to visualize neutral lipids (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Immunohistochemical and morphometric analyses for islet area, β cell, and non-β cell mass by point-counting morphometry utilized described methods (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). Internal jugular catheters were placed (24Bernal-Mizrachi C. Xiaozhong L. Yin L. Knutsen R.H. Howard M.J. Arends J.J. Desantis P. Coleman T. Semenkovich C.F. An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension.Cell Metab. 2007; 5: 91-102Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and after a 3-day recovery period, mice were subjected to an overnight fast. 3-[3H]glucose (PerkinElmer) was first infused (0.05 μCi/min) for 2 h to steady state. Human regular insulin was then bolused (50 μU/g) followed by a constant infusion at either 5 mU/kg/min (low-dose clamp) or 20 mU/kg/min (high-dose clamp) in separate cohorts of HFD fed animals. Infusion of 20% D-glucose was varied to maintain blood glucose at basal concentrations for at least 90 min. Rates of basal and clamped glucose production (Ra), and insulin-stimulated whole body glucose uptake (Rd) were determined as described (24Bernal-Mizrachi C. Xiaozhong L. Yin L. Knutsen R.H. Howard M.J. Arends J.J. Desantis P. Coleman T. Semenkovich C.F. An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension.Cell Metab. 2007; 5: 91-102Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Acute insulin stimulation was performed by intraportal injections (0.1 or 10 U/kg body weight) of regular insulin into anesthetized mice. Ten min later, liver and gastrocnemius were clamp-frozen and homogenized in buffer containing protease and phosphatase inhibitors (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Thirty μg of total protein was resolved by SDS-PAGE, electrotransferred onto PVDF membranes (Millipore), and immunoblotted with total and 473Ser-phosphoAkt (1:1,000, cell signaling), total and 9Ser-phospho glycogen synthase kinase-3beta (1:1.000, cell signaling), total and 256Ser-phosphoFOXO1 (1:1,000, cell signaling), TRB3 (1:2,500, Calbiochem), and actin (1:5,000, Sigma). Total and phospho-specific bands were detected by chemiluminescence (ECL kit, Amersham) (20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). RNA isolation, reverse transcription, and PCR (Applied BioSystems 7700) were performed using previously published primer-probe sequences (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar, 20Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Relative mRNA levels were calculated using the comparative CT and standard curve methods normalized to ribosomal protein L32, an invariant internal control. Urine was collected for 24 h in the presence of the antioxidant butylated hydroxytoluene (0.005%), and 15-isoprostane F2t and creatinine were analyzed by enzyme-linked immunosorbent assay (Oxford Biochemicals) and colorimetric assay (Cayman Chemical), respectively. In a separate cohort of HFD fed wild-type and FASKO mice, lipopolysaccharide (7 mg/kg, serotype 0111:B4, Sigma) was injected intraperitoneally, and serum was obtained before injection and at 1, 3, and 6 h after injection. Tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) were measured in serum by enzyme-linked immunosorbent assay (BD Biosciences). Data are expressed as mean ± SEM. Statistical comparisons were performed using an unpaired, two-tailed Student’s t-test or ANOVA (ANOVA). If the overall F was found to be significant (P < 0.05) for the latter, comparisons between means were made using appropriate posthoc tests. Wild-type mice gained weight (35% and 23% increase from baseline in males and females, respectively) after 10 weeks of HFD (Fig. 1A, B). Weight gain was considerably less in FASKO mice (Fig. 1A, B). FASKO mice after HFD feeding had ∼30% lower adiposity and an ∼8% increase in lean mass (Fig. 1C). There was no genotype effect on nasoanal lengths (not shown). Decreased adiposity in HFD fed FASKO animals was reflected by changes in white adipose tissue but not brown adipose tissue mass. Perigonadal fat pads weighed 3.17 ± 0.58 g in FASKO vs. 6.15 ± 1.12 g in wild-type, N = 10, P = 0.03). Intrascapular brown adipose tissue depots weighed 0.35 g in FASKO vs. 0.36 g in wild-type, P = NS. Adipocyte size was similar between genotypes (Fig. 1D). FASKO mice ate 17–22% less than controls at baseline and at 4, 7, and 10 weeks of eating HFD (Fig. 2A), exhibiting normal fat absorption at the end of the dietary study (Fig. 2B). FASKO animals demonstrated a 14–19% increase in VO2 compared with their wild-type littermates at baseline and at 4, 7, and 10 weeks of eating HFD (Fig. 2C). The respiratory quotient was similar between genotypes at the end of the dietary study, suggesting that although FASKO mice had increased oxygen consumption, their substrate utilization was the same as wild-type (Fig. 2D). There were no genotype-specific differences in serum β-hydroxybutyrate (FASKO 193 ± 26 vs. wild-type 215 ± 57 μmol/L, N = 7, P = NS), heat production (Fig. 2E), body temperature (Fig. 2F), or in the expression of genes controlling fat oxidation in liver (Pparα, acyl-CoA oxidase, carnitine palmitoyl transferase-1, medium-chain acyl-CoA dehydrogenase), skeletal muscle (lipoprotein lipase), and brown adipose tissue (uncoupling protein-1) (Fig. 2G), all of which suggested that elevated metabolism in peripheral tissues is unlikely to contribute to the increased whole-body energy expenditure in HFD fed FASKO mice. In contrast, HFD fed FASKO animals had increased locomotor activity assessed using an infrared sensing technique to measure home-cage activity over 3 days (Fig. 2H). Together, these data suggest that enhanced oxygen consumption in FASKO animals on a HFD appears to be mostly due to increased physical activity, which in combination with hypophagia protects them from diet-induced obesity. Serum chemistries and adipokines were unaffected by genotype at baseline (week 0, chow diet) (Fig. 3), consistent with our previous findings (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar). High-fat feeding increased fasting glucose and insulin in both genotypes, but levels of both were lower at 7 and 10 weeks in FASKO mice (Fig. 3A, B). Nonesterified fatty acids levels (Fig. 3C) were decreased (perhaps reflecting suppression of peripheral lipolysis by insulin) in FASKO animals, suggesting the presence of enhanced insulin sensitivity in these mice with high-fat feeding. Fasting serum triglyceride levels increased over time in both genotypes, but were 28% lower at 10 weeks in FASKO animals compared with controls (Fig. 3D). Lower triglycerides were reflected by differences in VLDL in lipoprotein analyses (not shown). Serum cholesterol levels were unaffected by genotype (not shown). Consistent with increasing adiposity in wild-type mice, serum leptin levels rose ∼8-fold over the course of the dietary intervention, while levels were lower in obesity-resistant FASKO animals (Fig. 3E). FASKO mice also resisted the HFD-induced reduction in serum adiponectin (Fig. 3F). Fatty acid profiling by mass spectrometry within the hypothalamus of HFD-fed animals showed no genotypic differences among several fatty acids, except for a significant reduction in palmitate (C16:0), the major product of the FAS reaction, in the FASKO mice (see supplementary Table II). These findings not only further validate the FASKO model, but also show that a diet rich in palmitate (see supplementary Table I) is unable to compensate for decreased de novo synthesis at this site. After 10 weeks of high-fat feeding, fed and 24 h-fasted serum glucose levels as well as serum insulin levels were lower in FASKO compared with control animals (Fig. 4A). There was no genotype effect on glucagon levels under these conditions (not shown). When given an acute bolus of D-glucose, HFD fed FASKO mice had lower glycemic excursions (Fig. 4B), indicating enhanced glucose tolerance. As FAS was also deleted in β cells (17Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (173) Google Scholar), increased glucose tolerance in FASKO mice could be caused by enhanced β−cell function. However, two lines of evidence suggest that β-cell function is unaffected by FAS deletion. First, lower glucose levels in FASKO mice (Fig. 4B) were matched with lower insulin levels (Fig. 4C), yielding lower insulin-to-glucose ratios in FASKO mice (Fig. 4D). Second, ex vivo insulin secretory responses of islets to glucose and arginine were nearly identical between genotypes under basal (3.3 mM glucose) and stimulated (16.7 mM glucose) conditions (Fig. 4E). These data suggest that enhanced glucose tolerance in HFD fed FASKO animals is not due to insulin hypersecretion. FASKO animals had lower β−cell mass (Fig. 4F) and a greater proportion of smaller-sized islets (19% in FASKO vs. 4% in WT had islet diameters of ∼75 μm or less), reflecting their insulin-sensitive metabolic and hormonal milieu (Fig. 3). Increased β-cell mass in WT mice (Fig. 4F) is an expected adaptive response to insulin resistance (25Weir G.C. Bonner-Weir S. Five stages of evolving beta-cell dysfunction during progression to diabetes.Diabetes. 2004; 53: S16-S21Crossref PubMed Scopus (802) Google Scholar). FASKO mice were more sensitive to insulin than their wild-type littermates with insulin tolerance testing (Fig. 4G). Hyperinsulinemic-euglycemic clamp experiments suggested that the liver was the major site affected in terms of glucose metabolism in mice with hypothalamic FAS inactivation. Hepatic glucose output was lower in HFD fed FASKO mice under basal and clamped (5 mU insulin/kg/min) conditions compared with littermate controls (Fig. 4H). No differences were seen between genotypes for insulin-dependent whole-body glucose disposal with either low-dose (5 mU/kg/min) (Fig. 4I), or high-dose (20 mU/kg/min) insulin (not shown). Consistent with the clamp data, mRNA levels for peroxisome-proliferator-activated receptor gamma coactivator-1α, Pepck, and glucose 6-phosphatase were decreased in FASKO compared with wild-type livers after 10 weeks of high-fat feeding (Fig. 5A). Glucokinase (GK) expression was increased in FASKO livers (Fig. 5A), reflecting enhanced hepatic insulin sensitivity. Key mediators of insulin signaling were assayed. Increased phosphorylation of Akt at serine 473, glycogen synthase kinase-3beta at serine 9, and Foxo1 at serine 256 were seen in FASKO compared with wild-type livers in response to 0.10 U/kg intraportal insulin (Fig. 5B, C). Protein levels for TRB3, an Akt inhibitor (26Du K. Herzig S. Kulkarni R.N. Montminy M. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver.Science. 2003; 300: 1574-1577Crossref PubMed Scopus (714) Google Scholar), were lower in FASKO livers (Fig. 5B, C). These differences were seen in the basal