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Microfibril-associated glycoprotein 1 (MAGP1) is a component of extracellular matrix microfibrils. Here we show that MAGP1 expression is significantly altered in obese humans, and inactivation of the MAGP1 gene (Mfap2−/−) in mice results in adipocyte hypertrophy and predisposition to metabolic dysfunction. Impaired thermoregulation was evident in Mfap2−/− mice prior to changes in adiposity, suggesting a causative role for MAGP1 in the increased adiposity and predisposition to diabetes. By 5 weeks of age, Mfap2−/− mice were maladaptive to cold challenge, uncoupling protein-1 expression was attenuated in the brown adipose tissue, and there was reduced browning of the subcutaneous white adipose tissue. Levels of transforming growth factor-β (TGF-β) activity were elevated in Mfap2−/− adipose tissue, and the treatment of Mfap2−/− mice with a TGF-β–neutralizing antibody improved their body temperature and prevented the increased adiposity phenotype. Together, these findings indicate that the regulation of TGF-β by MAGP1 is protective against the effects of metabolic stress, and its absence predisposes individuals to metabolic dysfunction.

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.

SummaryDe novo lipogenesis in adipocytes, especially with high fat feeding, is poorly understood. We demonstrate that an adipocyte lipogenic pathway encompassing fatty acid synthase (FAS) and PexRAP (peroxisomal reductase activating PPARγ) modulates endogenous PPARγ activation and adiposity. Mice lacking FAS in adult adipose tissue manifested increased energy expenditure, increased brown fat-like adipocytes in subcutaneous adipose tissue, and resistance to diet-induced obesity. FAS knockdown in embryonic fibroblasts decreased PPARγ transcriptional activity and adipogenesis. FAS-dependent alkyl ether phosphatidylcholine species were associated with PPARγ and treatment of 3T3-L1 cells with one such ether lipid increased PPARγ transcriptional activity. PexRAP, a protein required for alkyl ether lipid synthesis, was associated with peroxisomes and induced during adipogenesis. PexRAP knockdown in cells decreased PPARγ transcriptional activity and adipogenesis. PexRAP knockdown in mice decreased expression of PPARγ-dependent genes and reduced diet-induced adiposity. These findings suggest that inhibiting PexRAP or related lipogenic enzymes could treat obesity and diabetes.

Fatty acid synthase (FAS) promotes energy storage through de novo lipogenesis and participates in signaling by the nuclear receptor PPARα in noncardiac tissues. To determine if de novo lipogenesis is relevant to cardiac physiology, we generated and characterized FAS knockout in the myocardium (FASKard) mice. FASKard mice develop normally, manifest normal resting heart function, and have normal cardiac PPARα signaling as well as fatty acid oxidation. However, they decompensate with stress. Most die within 1 h of transverse aortic constriction, probably due to arrhythmia. Voltage clamp measurements of FASKard cardiomyocytes show hyperactivation of L-type calcium channel current that could not be reversed with palmitate supplementation. Of the classic regulators of this current, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not protein kinase A signaling is activated in FASKard hearts, and knockdown of FAS in cultured cells activates CaMKII. In addition to being intolerant of the stress of acute pressure, FASKard hearts were also intolerant of the stress of aging, reflected as persistent CaMKII hyperactivation, progression to dilatation, and premature death by ∼1 year of age. CaMKII signaling appears to be pathogenic in FASKard hearts because inhibition of its signaling in vivo rescues mice from early mortality after transverse aortic constriction. FAS was also increased in two mechanistically distinct mouse models of heart failure and in the hearts of humans with end stage cardiomyopathy. These data implicate a novel relationship between FAS and calcium signaling in the heart and suggest that FAS induction in stressed myocardium represents a compensatory response to protect cardiomyocytes from pathological calcium flux.

After loss of intestinal surface area, the remaining bowel undergoes a morphometric and functional adaptive response. Enterocytic expression of the transcriptional coregulator tetradecanoyl phorbol acetate induced sequence 7 (Tis7 )i s markedly increased in a murine model of intestinal adaptation. Mice overexpressing Tis7 in intestine have greater triglyceride absorption and weight gain when fed a high-fat diet (42% energy) than their wild-type (WT) littermates fed the same diet. These and other data suggest that Tis7 has a unique role in nutrient absorptive and metabolic adaptation. Herein, male Tis7 2/2 and WT mice were fed a high-fat diet (42% energy) for 8 wk. Weight was monitored and metabolic analyses and hepatic and intestinal lipid concentrations were compared after 8 wk. Intestinal lipid absorption and metabolism studies and intestinal resection surgeries were performed in separate groups of Tis7 2/2 and WT mice. At 8 wk, weight gain was less and jejunal mucosal and hepatic triglyceride and cholesterol concentrations were lower in Tis7 2/2 mice than in the WT controls. Following corn oil gavage, serum cholesterol, triglyceride, and FFA concentrations were lower in the Tis7 2/2 mice than in the WT mice. Incorporation of oral 3 [H] triolein into intestinal mucosal cholesterol ester and FFA was less in Tis7 2/2 compared with WT mice. Following resection, crypt cell proliferation rates and villus heights were lower in Tis7 2/2 than in WT mice, indicating a blunted adaptive response. Our results suggest a novel physiologic function for Tis7 in the gut as a global regulator of lipid absorption and metabolism and epithelial cell proliferation. J. Nutr. 140: 1907–1914, 2010.

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 beta cells protected them from diet-induced obesity. Though high-fat fed FASKO mice had no beta-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 (TNFalpha) 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.

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.

Endothelial Lipase (EL) is a newly identified member of the triacylglycerol lipase family. Recent studies suggested that EL may be an important determinant of HDL-metabolism and inflammation acting at the level of the vessel wall. The aim of this review is to summarize important facts derived from experimental approaches and from epidemiologic human studies to provide a comprehensive view on the role of EL in inflammation and atherogenesis as well as target for potential pharmaceutical interventions.

SummaryAge-related disease, not aging per se, causes most morbidity in older humans. Here we report that skeletal muscle respiratory uncoupling due to UCP1 expression diminishes age-related disease in three mouse models. In a longevity study, median survival was increased in UCP mice (animals with skeletal muscle-specific UCP1 expression), and lymphoma was detected less frequently in UCP female mice. In apoE null mice, a vascular disease model, diet-induced atherosclerosis was decreased in UCP animals. In agouti yellow mice, a genetic obesity model, diabetes and hypertension were reversed by induction of UCP1 in skeletal muscle. Uncoupled mice had decreased adiposity, increased temperature and metabolic rate, elevated muscle SIRT and AMP kinase, and serum characterized by increased adiponectin and decreased IGF-1 and fibrinogen. Accelerating metabolism in skeletal muscle does not appear to impact aging but may delay age-related disease.

Objective— High-fat, cholesterol-containing diets contribute to hyperlipidemia. Both high-fat diets and hyperlipidemia are associated with chronic inflammatory diseases like atherosclerosis. Integrins, heterodimeric mediators of inflammatory cell recruitment, are not generally thought to be affected by diet. However, high-fat feeding promotes inflammation, atherosclerosis, and death in hyperlipidemic mice with β3 integrin deficiency, and treatment of humans from Western populations with oral β3 integrin inhibitors increases mortality. The mechanisms responsible for these β3 integrin-associated events are unknown. Methods and Results— Here we show that diet-induced death in β3 integrin-deficient mice is a TNFα-dependent process mediated by bone marrow–derived cells. In 2 different hyperlipidemic models, apoE-null and LDL receptor–null mice, β3-replete animals transplanted with β3-deficient marrow died with Western-type high-fat feeding whereas β3-deficient animals transplanted with β3-replete marrow were rescued from diet-induced death. Transplantation with β3-deficient marrow also increased atherosclerosis. TNFα expression was increased in β3-deficient macrophages and normalized by either retroviral or adenoviral reconstitution of β3 integrin expression. Treatment with the anti-TNFα antibody infliximab rescued β3 integrin–deficient mice from Western diet–induced death, directly implicating TNFα in the pathophysiology triggered by diet-induced hyperlipidemia. Conclusions— These findings suggest that macrophage β3 integrin, acting through TNFα, suppresses inflammation caused by hyperlipidemia attributable to high-fat feeding.

Peroxisome proliferator-activated receptor-α is widely distributed in the vasculature where it is believed to exert pleiotropic antiatherogenic effects. Its role in the regulation of blood pressure is still unresolved; however, some evidence suggests that it may affect the renin-angiotensin system. We investigated its role in angiotensin II–induced hypertension in the Tsukuba hypertensive mouse (THM). This is a model of hypertension and atherosclerosis because of high angiotensin II and aldosterone levels as a result of the transgenic expression of the entire human renin-angiotensin system. Making the THM animals deficient in Peroxisome proliferator-activated receptor-α (THM/PPARKO) totally abolished hypertension and myocardial hypertrophy. This was accompanied by a reduction in plasma human active renin in THM/PPARKO mice compared with THM animals from 3525±128 mU/L to 1910±750 mU/L ( P P =0.003). In the THM/PPARKO mice, the extent of atherosclerosis at the aortic sinus after a 12-week period on an atherogenic diet was decreased by >80%. In addition, the spontaneous formation of foam cells from peritoneal macrophages, a blood pressure–independent event, was reduced by 92% in the THM/PPARKO mice, suggesting protection from the usual oxidative stress in these animals, possibly because of lower prevailing angiotensin II levels. Finally, chronic fenofibrate treatment further elevated blood pressure in THM animals but not in THM/PPARKO animals. Taken together, these data indicate that peroxisome proliferator-activated receptor-α may regulate the renin-angiotensin system. They raise the possibility that its activation may aggravate hypertension and hasten atherosclerosis in the context of an activated renin-angiotensin system.

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 beta cells and hypothalamus produced lean, hypophagic mice with increased physical activity and impaired hypothalamic PPARalpha signaling. Administration of a PPARalpha agonist into the hypothalamus increased PPARalpha target genes and normalized food intake. Inactivation of beta 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 PPARalpha. Thus, 2 diametrically opposed proteins, FAS (induced by feeding) and PPARalpha (induced by starvation), unexpectedly form an integrative sensory module in the central nervous system to orchestrate energy balance.

Objective— Grb2 is a ubiquitously expressed linker protein that couples growth factor receptor activation to downstream mitogen-activated protein kinase (MAPK) cascades. Macrophage proliferation and uptake of modified lipoproteins are critical components of atherogenesis which require MAPK activation. However, the precise role of upstream signaling factors and the interrelationship of various MAPK cascades in the pathogenesis of atherosclerosis remains uncertain. Complete deletion of Grb2 in mice results in early embryonic lethality. However, Grb2 heterozygous mice appear normal at birth. To test the role of the Grb2 adapter protein in atherosclerotic lesion formation, we generated Grb2 +/− mice in the apoE −/− genetic background. Methods and Results— Grb2 +/− apoE −/− and apoE −/− mice exhibited similar body weight and serum lipid profiles. However, Grb2 +/− apoE −/− mice on a Western diet had reduced lesion formation compared with apoE −/− mice by aortic sinus and en face assays. Transplantation of apoE −/− mice with Grb2 +/− apoE −/− or apoE −/− bone marrow indicated that Grb2 haploinsufficiency in blood-borne cells confers resistance to Western diet–induced atherosclerosis. Cell culture experiments with bone marrow–derived macrophages showed that Grb2 is required for oxidized low density lipoprotein (oxLDL)-induced MAPK activation and foam cell formation. Conclusions— Grb2 is required for atherosclerotic lesion formation and uptake of oxidized LDL by macrophages.

Following loss of functional small bowel surface area due to surgical resection, the remnant gut undergoes an adaptive response characterized by increased crypt cell proliferation and enhanced villus height and crypt depth, resulting in augmented intestinal nutrient absorptive capacity. Previous studies showed that expression of the immediate early gene tis7 is markedly up-regulated in intestinal enterocytes during the adaptive response. To study its role in the enterocyte, transgenic mice were generated that specifically overexpress TIS7 in the gut. Nucleotides -596 to +21 of the rat liver fatty acid-binding protein promoter were used to direct abundant overexpression of TIS7 into small intestinal upper crypt and villus enterocytes. TIS7 transgenic mice had increased total body adiposity and decreased lean muscle mass compared with normal littermates. Oxygen consumption levels, body weight, surface area, and small bowel weight were decreased. On a high fat diet, transgenic mice exhibited a more rapid and proportionately greater gain in body weight with persistently elevated total body adiposity and increased hepatic fat accumulation. Bolus fat feeding resulted in a greater increase in serum triglyceride levels and an accelerated appearance of enterocytic, lamina propria, and hepatic fat. Changes in fat homeostasis were linked to increased expression of genes involved in enterocytic triglyceride metabolism and changes in growth with decreased insulin-like growth factor-1 expression. Thus, TIS7 overexpression in the intestine altered growth, metabolic rate, adiposity, and intestinal triglyceride absorption. These results suggest that TIS7 is a unique mediator of nutrient absorptive and metabolic adaptation following gut resection.

De novo lipogenesis is an energy-expensive process whose role in adult mammals is poorly understood. We generated mice with liver-specific inactivation of fatty-acid synthase (FAS), a key lipogenic enzyme. On a zero-fat diet, FASKOL (FAS knockout in liver) mice developed hypoglycemia and fatty liver, which were reversed with dietary fat. These phenotypes were also observed after prolonged fasting, similarly to fasted PPARalpha-deficiency mice. Hypoglycemia, fatty liver, and defects in expression of PPARalpha target genes in FASKOL mice were corrected with a PPARalpha agonist. On either zero-fat or chow diet, FASKOL mice had low serum and hepatic cholesterol levels with elevated SREBP-2, decreased HMG-CoA reductase expression, and decreased cholesterol biosynthesis; these were also corrected with a PPARalpha agonist. These results suggest that products of the FAS reaction regulate glucose, lipid, and cholesterol metabolism by serving as endogenous activators of distinct physiological pools of PPARalpha in adult liver.

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.

Fatty acids may promote type 2 diabetes by altering insulin secretion from pancreatic beta cells, a process known as lipotoxicity. The underlying mechanisms are poorly understood. To test the hypothesis that peroxisome proliferator-activated receptor alpha (PPARalpha) has a direct effect on islet function, we treated INS-1 cells, an insulinoma cell line, with a PPARalpha adenovirus (AdPPARalpha) as well as the PPARalpha agonist clofibric acid. AdPPARalpha-infected INS-1 cells showed PPARalpha agonist- and fatty acid-dependent transactivation of a PPARalpha reporter gene. Treatment with either AdPPARalpha or clofibric acid increased both catalase activity (a marker of peroxisomal proliferation) and palmitate oxidation. AdPPARalpha induced carnitine-palmitoyl transferase-I (CPT-I) mRNA, but had no effect on insulin gene expression. AdPPARalpha treatment increased cellular triglyceride content but clofibric acid did not. Both AdPPARalpha and clofibric acid decreased basal and glucose-stimulated insulin secretion. Despite increasing fatty acid oxidation, AdPPARalpha did not increase cellular ATP content suggesting the stimulation of uncoupled respiration. Consistent with these observations, UCP2 expression doubled in PPARalpha-treated cells. Clofibric acid-induced suppression of glucose-simulated insulin secretion was prevented by the CPT-I inhibitor etomoxir. These data suggest that PPARalpha-stimulated fatty acid oxidation can impair beta cell function.

To determine whether uncoupling respiration from oxidative phosphorylation in skeletal muscle is a suitable treatment for obesity and type 2 diabetes, we generated transgenic mice expressing the mitochondrial uncoupling protein (Ucp) in skeletal muscle. Skeletal muscle oxygen consumption was 98% higher in Ucp-L mice (with low expression) and 246% higher in Ucp-H mice (with high expression) than in wild-type mice. Ucp mice fed a chow diet had the same food intake as wild-type mice, but weighed less and had lower levels of glucose and triglycerides and better glucose tolerance than did control mice. Ucp-L mice were resistant to obesity induced by two different high-fat diets. Ucp-L mice fed a high-fat diet had less adiposity, lower levels of glucose, insulin and cholesterol, and an increased metabolic rate at rest and with exercise. They were also more responsive to insulin, and had enhanced glucose transport in skeletal muscle in the setting of increased muscle triglyceride content. These data suggest that manipulating respiratory uncoupling in muscle is a viable treatment for obesity and its metabolic sequelae.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/−). LPL+/− mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/− mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/− islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/− and −/− islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-β-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.

Heterozygous lipoprotein lipase defi ciency (LPL+/-) is common and has been implicated in premature atherosclerosis in epidemiologic studies. However, in vitro data suggest that LPL deficiency in the vascular wall may be antiatherogenic. To address the role of LPL in atherosclerosis, LPL+/- mice in the C57BL/6J background were fed an atherogenic diet for 8 months. LPL+/- mice were more dyslipidemic than +/+ animals due to increased concentrations of non-HDL lipoproteins. There was no difference in aortic origin atherosclerosis between LPL+/- (n = 56) and +/+ (n = 55) mice. LPL+/- mice in the low density lipoprotein receptor knockout (LDLR-/-) background were fed the same atherogenic diet for 3 months. LPL+/-LDLR-/- mice were more dyslipidemic than LPL+/+LDLR-/- animals. There was no difference in atherosclerosis assayed for the entire aorta and no difference in aortic sterol content between LPL+/-LDLR-/- (n = 28) and LPL+/+LDLR-/- (n = 15) mice. LPL protein was detected in murine lesions in a consistent layered pattern. More luminal, lipid-laden macrophages generally did not stain for LPL, but deeper, lipid-poor macrophages as well as necrotic core regions contained immunoreactive LPL. LPL protein was more abundant in lesions from LPL+/+ LDLR-/- than LPL+/-LDLR-/- mice. After eating an atherogenic diet, LPL+/- as compared to LPL+/+ mice have more dyslipidemia, but no more atherosclerosis, and less LPL protein in atherosclerotic lesions. These data suggest that lipoprotein lipase deficiency in the vascular wall could prevent the retention of atherogenic lipoproteins.—Semenkovich, C. F., T. Coleman, and A. Daugherty. Effects of heterozygous lipoprotein lipase deficiency on diet-induced atherosclerosis in mice. J. Lipid Res. 1998. 39: 1141–1151.

Abstract Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5×10 9 plaque-forming units (pfu) of an E1/E3–deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial β-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n=57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834±133 vs 313±89 mU/mL, P P

To better understand the accelerated decay of fatty acid synthase (FAS) message that occurs after glucose deprivation (J. Biol. Chem. 1993. 268: 6961-6970), we characterized the 3‘ terminus of the human message and the kinetics of FAS mRNA decay in HepG2 cells. The FAS gene was localized to human chromosome 17q24-25 and to syntenic distal mouse chromosome 11. Expression of the FAS message in human tissues was ubiquitous with high levels in liver, lung, and intra-abdominal adipose tissue. The 806 nucleotide 3‘ untranslated region of the human mRNA contained two regions with the instability pentamer AUUUA. Unlike short-lived messages containing AUUUA motifs, FAS mRNA decay after glucose deprivation was not first order, and there were no detectable changes in the poly(A) tail. Glucose deprivation transiently caused FAS message to sediment more rapidly than control message in density gradients. In vivo treatment with different translational inhibitors showed that translation per se was not necessary for FAS mRNA decay; association of polysomes with FAS message protected it from decay. In cell-free decay experiments, FAS mRNA decay was more rapid using components from glucose-deprived than glucose-treated cells. These data suggest that glucose regulates cytoplasmic HepG2 FAS mRNA stability by partitioning the message between a translated pool not subject to degradation and a decay compartment, features reminiscent of regulated stability for other diet-responsive messages.

SummaryWe investigated the role of autophagy in atherosclerosis. During plaque formation in mice, autophagic markers colocalized predominantly with macrophages (mϕ). Atherosclerotic aortas had elevated levels of p62, suggesting that dysfunctional autophagy is characteristic of plaques. To determine whether autophagy directly influences atherogenesis, we characterized Beclin-1 heterozygous-null and mϕ-specific ATG5-null (ATG5-mϕKO) mice, commonly used models of autophagy haploinsufficiency and deficiency, respectively. Haploinsufficent Beclin-1 mice had no atherosclerotic phenotype, but ATG5-mϕKO mice had increased plaques, suggesting an essential role for basal levels of autophagy in atheroprotection. Defective autophagy is associated with proatherogenic inflammasome activation. Classic inflammasome markers were robustly induced in ATG5-null mϕ, especially when coincubated with cholesterol crystals. Moreover, cholesterol crystals appear to be increased in ATG5-mϕKO plaques, suggesting a potentially vicious cycle of crystal formation and inflammasome activation in autophagy-deficient plaques. These results show that autophagy becomes dysfunctional in atherosclerosis and its deficiency promotes atherosclerosis in part through inflammasome hyperactivation.

The mucopolysaccharidosis (MPS) type VII mouse was originally described as the adipose storage deficiency mouse because of its extreme lean phenotype of unknown etiology. Here, we show that adipose storage deficiency and lower leptin levels are common to five different lysosomal storage diseases (LSDs): MPSI, MPSIIIB, MPSVII, Niemann-Pick type A/B, and infantile neuronal ceroid lipofuscinosis. Elevated circulating pro-inflammatory proteins (VCAM1 and MCP1) were found in multiple LSDs. Multiple anti-inflammatory strategies (dexamethasone, MCP1 deficiency, M3 expression) failed to alter adiposity in LSD animals. All of the models had normal or greater caloric intake and lower to normal metabolic rate, fasting plasma glucose, non-esterified fatty acids, cholesterol, and triglycerides. Triglycerides were lower in the livers of MPSI mice, and the trend was lower in the muscle. Lipid absorption and processing in MPSI mice were indistinguishable from those in normal mice following oral gavage of olive oil. The increased lean mass of MPSI and MPSIIIB mice suggests a shift in adipose triglycerides to lysosomal storage. In agreement, MPSI livers had a similar total caloric content but reduced caloric density, indicating a shift in energy from lipids to proteins/carbohydrates (lysosomal storage). Enzyme replacement therapy normalized the caloric density within 48 h without reducing total caloric content. This was due to an increase in lipids. Recycling of stored material is likely reduced or nonexistent. Therefore, to maintain homeostasis, energy is likely diverted to synthesis at the expense of typical energy storage depots. Thus, these diseases will serve as important tools in studying the role of lysosome function in metabolism and obesity.

Niemann-Pick C1 (NPC1) is a key participant in cellular cholesterol trafficking. Loss of NPC1 function leads to defective suppression of SREBP-dependent gene expression and failure to appropriately activate liver X receptor-mediated (LXR-mediated) pathways, ultimately resulting in intracellular cholesterol accumulation. To determine whether NPC1 contributes to regulation of macrophage sterol homeostasis in vivo, we examined the effect of NPC1 deletion in BM-derived cells on atherosclerotic lesion development in the Ldlr-/- mouse model of atherosclerosis. High-fat diet-fed chimeric Npc1-/- mice reconstituted with Ldlr-/-Npc1-/- macrophages exhibited accelerated atherosclerosis despite lower serum cholesterol compared with mice reconstituted with wild-type macrophages. The discordance between the low serum lipoprotein levels and the presence of aortic atherosclerosis suggested that intrinsic alterations in macrophage sterol metabolism in the chimeric Npc1-/- mice played a greater role in atherosclerotic lesion formation than did serum lipoprotein levels. Macrophages from chimeric Npc1-/- mice showed decreased synthesis of 27-hydroxycholesterol (27-HC), an endogenous LXR ligand; decreased expression of LXR-regulated cholesterol transporters; and impaired cholesterol efflux. Lower 27-HC levels were associated with elevated cholesterol oxidation products in macrophages and plasma of chimeric Npc1-/- mice and with increased oxidative stress. Our results demonstrate that NPC1 serves an atheroprotective role in mice through regulation of LXR-dependent cholesterol efflux and mitigation of cholesterol-induced oxidative stress in macrophages.

Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.

The observations that atherosclerosis often occurs in non-smokers without elevated levels of low-density lipoprotein cholesterol, and that most atherosclerosis loci so far identified in mice do not affect systemic risk factors associated with atherosclerosis, suggest that as-yet-unidentified mechanisms must contribute to vascular disease. Arterial walls undergo regional disturbances of metabolism that include the uncoupling of respiration and oxidative phosphorylation, a process that occurs to some extent in all cells and may be characteristic of blood vessels being predisposed to the development of atherosclerosis. To test the hypothesis that inefficient metabolism in blood vessels promotes vascular disease, we generated mice with doxycycline-inducible expression of uncoupling protein-1 (UCP1) in the artery wall. Here we show that UCP1 expression in aortic smooth muscle cells causes hypertension and increases dietary atherosclerosis without affecting cholesterol levels. UCP1 expression also increases superoxide production and decreases the availability of nitric oxide, evidence of oxidative stress. These results provide proof of principle that inefficient metabolism in blood vessels can cause vascular disease.

The lysosomal storage disease MPS VII (mucopolysaccharidosis type VII) is caused by a deficiency in β-glucuronidase activity, and results in the accumulation of partially degraded glycosaminoglycans in many cell types. Although MPS VII is a simple monogenetic disorder, the clinical presentation is complex and incompletely understood. ERT (enzyme replacement therapy) is relatively effective at improving the clinical course of the disease; however, some pathologies persist. In order to clarify the molecular events contributing to the disease phenotype and how ERT might impact upon them, we analysed liver tissue from untreated and treated MPS VII mice at both 2 and 5 months of age using biochemical assays and microarray analysis. Overall, as the disease progresses, more genes have altered expression and, at either age, numerous transcriptional changes in multiple pathways appear to be refractory to therapy. With respect to the primary site of disease, both transcriptional and post-transcriptional mechanisms are involved in the regulation of lysosomal enzymes and other lysosome-associated proteins. Many of the changes observed in both lysosome-associated mRNAs and proteins are normalized by enzyme replacement. In addition, gene expression changes in seemingly unrelated pathways may account for the complex metabolic phenotype of the MPS VII mouse. In particular, β-glucuronidase deficiency appears to induce physiological malnutrition in MPS VII mice. Malnutrition may account for the pronounced adipose storage deficiency observed in this animal. Studying the molecular response to lysosomal storage, especially those changes recalcitrant to therapy, has revealed additional targets that may improve the efficacy of existing therapies.

Objectives— The α 2 β 1 integrin serves as a collagen or collagen/laminin receptor on many cell types, including endothelial cells and platelets. Many studies indicate that the α 2 β 1 integrin is a critical mediator of platelet adhesion to collagen. Epidemiologic studies suggest a direct correlation between the genetically determined platelet surface density of the α 2 β 1 integrin and the risk of thrombotic diseases, such as myocardial infarction and stroke, in the young, which are well-established complications of atherosclerosis. We have now used the α 2 β 1 integrin–deficient mouse to evaluate the contributions of the α 2 β 1 integrin to the development of atherosclerosis. Methods and Results— We generated wild-type (α 2 +/+ ) or α 2 β 1 integrin–deficient (α 2 −/− ) mice that were also deficient in the apolipoprotein E (ApoE) gene (ApoE −/− ) and compared atherosclerotic lesion development in α 2 +/+ ApoE −/− and α 2 −/− ApoE −/− mice that were fed a high-fat, cholesterol-containing diet for 6 or 15 weeks. Total lesional area did not differ significantly between the α 2 -null animals and the wild-type animals at either 6 or 15 weeks. Conclusions— Our results suggest that risk for arterial thrombotic disease associated with high-level α 2 β 1 integrin expression is not attributable to enhanced development of atherosclerosis per se but may rather be a consequence of thrombotic complications at the plaques.

Protease inhibitor-based highly active antiretroviral therapy (PI-HAART) has been implicated in dyslipidemia, peripheral insulin resistance, and abnormal adipose tissue deposition in human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome, or AIDS. In vitro evidence indicates that some PIs reduce adipocyte lipoprotein (LPL) and hepatic lipase (HL) expression and activities. We examined whether LPL and HL activities are reduced in HIV-infected patients with dyslipidemia. Fasting serum lipids, glucoregulatory hormones, and postheparin LPL and HL activities, as well as whole body and regional adiposity, were measured in 19 HIV-seronegative controls, 9 HIV+ patients naive to all anti-HIV medications, 9 HIV+ patients naive to PIs, 9 HIV+ patients with prior PI experience but not currently receiving PIs, and 47 HIV+ patients receiving PI-HAART. The PI-HAART group had low LPL and HL activities. However, multiple linear regression analysis indicated that low postheparin LPL activity contributed only partially to HIV-dyslipidemia. Central adiposity and high C-peptide levels (an indicator of high insulin secretion) were stronger predictors of HIV-dyslipidemia. Low LPL and HL activities, by themselves, were insufficient to explain HIV-dyslipidemia because the PI-naive group had low LPL and HL activities but had normal adiposity, C-peptide levels, and serum lipid and lipoprotein levels. HDL-cholesterol was lower in PI-HAART and PI-naive groups than seronegative controls and was directly associated with LPL activity. These findings suggest that HIV-dyslipidemia is mediated primarily by factors that influence triglyceride and lipoprotein synthesis (e.g., central adiposity and hyperinsulinemia) and mediated only partially by factors that influence triglyceride clearance (e.g., lipase activity).

Insulin resistance is commonly associated with hypertension, a condition that causes vascular disease in people with obesity and type 2 diabetes. The mechanisms linking hypertension and insulin resistance are poorly understood. To determine whether respiratory uncoupling can prevent insulin resistance-related hypertension, we crossed transgenic mice expressing uncoupling protein 1 (UCP1) in skeletal muscle with lethal yellow (A y /a) mice, genetically obese animals known to have elevated blood pressure. Despite increased food intake, UCP-A y /a mice weighed less than their A y /a littermates. The metabolic rate was higher in UCP-A y /a mice than in A y /a mice and did not impair their ability to alter oxygen consumption in response to temperature changes, an adaptation involving sympathetic nervous system activity. Compared with their nontransgenic littermates, UCP-A y /a mice had lower fasting insulin, glucose, triglyceride, and cholesterol levels and were more insulin sensitive. Blood pressure, serum leptin, and urinary catecholamine levels were also lower in uncoupled mice. Independent of sympathetic nervous system activity, low-dose peripheral leptin infusion increased blood pressure in UCP-A y /a mice but not in their A y /a littermates. These data indicate that skeletal muscle respiratory uncoupling reverses insulin resistance and lowers blood pressure in genetic obesity without affecting thermoregulation. The data also suggest that uncoupling could decrease the risk of atherosclerosis in type 2 diabetes.

The role of the enzyme lipoprotein lipase (LPL) in atherosclerosis is uncertain. To generate an animal model of LPL deficiency, we targeted the LPL gene in embryonic stem cells with a vector designed to disrupt the COOH terminus of the protein and used these cells to generate LPL-deficient mice. Germ line transmission of the disrupted LPL allele was achieved with two chimeric males, and offspring from each of these animals were phenotypically identical. Pups homozygous (-/-) for LPL deficiency died within 48 h of birth with extreme elevations of serum triglycerides (13,327 mg/dl) associated with essentially absent LPL enzyme activity in heart and carcass. Newborn heterozygous (+/-) LPL-deficient pups had lower LPL enzyme activity and higher triglycerides (370 versus 121 mg/dl) than wild type (+/+) littermates. Adult heterozygotes had higher triglycerides than wild type mice with ad libitum feeding (236 mg/dl for +/- versus 88 mg/dl for +/+) and after fasting for 4 h (98 mg/dl for +/- versus 51 for +/+) or 12 h (109 mg/dl for +/- versus 56 mg/dl for +/+). Triglycerides were present as very low density lipoprotein particles and chylomicrons, but high density lipoprotein cholesterol levels were not decreased in +/- animals. Plasma heparin-releasable LPL activity was 43% lower in +/- versus +/+ adult animals. LPL activity, mRNA, and protein were lower in the tissues of +/- versus +/+ mice. Homozygous LPL deficiency caused by disruption of the COOH terminus of the enzyme is lethal in mice. Heterozygous LPL deficiency caused by this mutation is associated with mild to moderate hypertriglyceridemia without affecting static HDL cholesterol levels. Heterozygous LPL-deficient mice could be useful for determining if hypertriglyceridemia, independently or in combination with other discrete defects, influences atherosclerosis.

Mucopolysaccharidosis (MPS) is due to deficiencies in enzymes that degrade glycosaminoglycans (GAGs). MPS I is due to deficient α-L-iduronidase activity (IDUA) and results in the accumulation of heparin sulfate (HS) and dermatan sulfate (DS). MPS VII is due to deficient β-glucuronidase activity and results in the accumulation of chondroitin sulfate (CS) in addition to HS and DS. Both disorders result in dysostosis multiplex in patients, which is characterized by short and thick bones. MPS can be treated with liver-directed gene therapy in which hepatocytes continuously secrete enzyme with mannose 6-phosphate, which can be taken up from blood. Although hepatic gene therapy reduced many manifestations of MPS VII, bone disease has not been completely corrected. The purpose of this study was to evaluate the effect of neonatal retroviral vector (RV)-mediated gene therapy on bone disease in MPS I mice. In contrast to untreated MPS VII mice in which long bones have only 81% to 87% of normal length, untreated MPS I mice had normal bone lengths at 6 weeks and 8 months after birth. Normal lengths were likely due to the fact that untreated MPS I mice had much less lysosomal storage material than did MPS VII mice in cartilage cells of the growth plate, which plays a pivotal role in bone elongation. This is likely due to the fact that CS is the major GAG in chondrocytes, and CS only accumulates in MPS VII. For untreated MPS I mice, bone mineral density (BMD) was normal at 6 weeks, but markedly increased at 0.069 gm/cm2 at 8 months [normal is 0.054 (p < 0.0001)]. The long bones of untreated MPS I mice had an increased diameter and were sclerotic, and there was substantial storage in osteocytes. The canine IDUA cDNA was cloned into a retroviral vector (RV) with a strong liver promoter, and the RV was injected into newborn MPS I mice at a high [1 × 109 transducing units (TU)/kg] or a low (1 × 108 TU/kg) dose. This resulted in transduction of liver cells and stable serum IDUA activity in most animals for 8 months at 1241 ± 147 and 110 ± 31 units (U)/ml for the high and low dose animals, respectively. At 8 months, mice that received a high dose of RV had normal long bone diameters without sclerosis, normal BMD [0.054 gm/cm2 (p < 0.0001 vs. values in untreated MPS I)], and marked reduction in lysosomal storage in the osteocytes. Mice that received a low dose of RV had some increase in diameter and sclerosis of the long bones, although they were not as severe as untreated MPS I mice. Similarly, the BMD (0.060 gm/cm2) was partially increased. We conclude that bone disease is much less severe in untreated MPS I mice than in untreated MPS VII mice. This is likely due to the fact that CS does not accumulate in MPS I, and CS is high in cartilage and accumulates in MPS VII. However, MPS I mice still had marked thickening and sclerosis of the bone, which were prevented with a high dose of RV at birth. However, the low dose of RV was less effective. These data help to define the serum activity necessary to prevent bone disease in MPS I.

The role of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in the development of insulin-resistant diabetes was evaluated using gain- and loss-of-function approaches. Transgenic mice overexpressing PPARalpha in muscle (MCK-PPARalpha mice) developed glucose intolerance despite being protected from diet-induced obesity. Conversely, PPARalpha null mice were protected from diet-induced insulin resistance in the context of obesity. In skeletal muscle, MCK-PPARalpha mice exhibited increased fatty acid oxidation rates, diminished AMP-activated protein kinase activity, and reduced insulin-stimulated glucose uptake without alterations in the phosphorylation status of key insulin-signaling proteins. These effects on muscle glucose uptake involved transcriptional repression of the GLUT4 gene. Pharmacologic inhibition of fatty acid oxidation or mitochondrial respiratory coupling prevented the effects of PPARalpha on GLUT4 expression and glucose homeostasis. These results identify PPARalpha-driven alterations in muscle fatty acid oxidation and energetics as a potential link between obesity and the development of glucose intolerance and insulin resistance.

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.

Carbohydrate feeding of animals results in striking increases in hepatic fatty acid synthesis but much of this induction is presumed to be hormone mediated. To clarify the mechanisms responsible for the specific effect of carbohydrate on fatty acid synthesis, the effects of D-glucose on the expression of human fatty acid synthase (FAS) in HepG2 cells cultured in serum-free medium were studied. Northern blots of total RNA from these cells showed a single FAS mRNA band of 9.3 kilobases that was increased 2.7-5.4-fold in the presence of D-glucose. Lactate and citrate but not L-glucose mimicked this effect. Glucose induction of FAS mRNA was time- and concentration-dependent and most of the increase in FAS message was detected within the range of physiologic glucose concentrations. Glucose induction of FAS mRNA, protein synthetic rate, and enzyme activity were similar suggesting that glucose regulates FAS expression at a pretranslational level in this system. Transcription run-off experiments showed that glucose feeding was associated with no change in the FAS transcription initiation rate despite a 5-fold increase in FAS mRNA. FAS mRNA stability as determined by actinomycin D chase was 7-fold greater in the presence of glucose. Differences between FAS mRNA levels in cells grown in the presence versus the absence of glucose were inhibited by cycloheximide but not puromycin, suggesting that glucose regulation of FAS mRNA stability is not dependent on translation. Glucose, at physiologic concentrations and in the absence of hormones, appears to regulate FAS gene expression in HepG2 cells predominantly by mediating FAS mRNA stability.

PPARalpha is a ligand-dependent transcription factor expressed at high levels in the liver. Its activation by the drug gemfibrozil reduces clinical events in humans with established atherosclerosis, but the underlying mechanisms are incompletely defined. To clarify the role of PPARalpha in vascular disease, we crossed PPARalpha-null mice with apoE-null mice to determine if the genetic absence of PPARalpha affects vascular disease in a robust atherosclerosis model. On a high-fat diet, concentrations of atherogenic lipoproteins were higher in PPARalpha(-/-)apoE(-/-) than in PPARalpha(+/+)apoE(-/-) mice, due to increased VLDL production. However, en face atherosclerotic lesion areas at the aortic arch, thoracic aorta, and abdominal aorta were less in PPARalpha-null animals of both sexes after 6 and 10 weeks of high-fat feeding. Despite gaining as much or more weight than their PPARalpha(+/+)apoE(-/-) littermates, PPARalpha(-/-)apoE(-/-) mice had lower fasting levels of glucose and insulin. PPARalpha-null animals had greater suppression of endogenous glucose production in hyperinsulinemic clamp experiments, reflecting less insulin resistance in the absence of PPARalpha. PPARalpha(-/-)apoE(-/-) mice also had lower blood pressures than their PPARalpha(+/+)apoE(-/-) littermates after high-fat feeding. These results suggest that PPARalpha may participate in the pathogenesis of diet-induced insulin resistance and atherosclerosis.