Trey Coleman

Skeletal muscle respiratory uncoupling prevents diet-induced obesityand insulin resistance in mice

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.

Autophagy Links Inflammasomes to Atherosclerotic Progression

We 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.

PPARα deficiency reduces insulin resistance and atherosclerosis in apoE-null mice

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.

Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice

Vascular calcification is common in people with diabetes and its presence predicts premature mortality. To clarify the underlying mechanisms, we used low density lipoprotein receptor-deficient (LDLR −/−) mice to study vascular calcification in the ascending aorta. LDLR −/− mice on a chow diet did not develop obesity, diabetes, atheroma, or vascular calcification. In contrast, LDLR −/− mice on high fat diets containing cholesterol developed obesity, severe hyperlipidemia, hyperinsulinemic diabetes, and aortic atheroma. A high fat diet without cholesterol also induced obesity and diabetes, but caused only moderate hyperlipidemia and did not result in significant aortic atheroma formation. Regardless of cholesterol content, high fat diets induced mineralization of the proximal aorta (assessed by von Kossa staining) and promoted aortic expression ofMsx2 and Msx1, genes encoding homeodomain transcription factors that regulate mineralization and osseous differentiation programs in the developing skull. Osteopontin(Opn), an osteoblast matrix protein gene also expressed by activated macrophages, was up-regulated in the aorta by these high fat diets. In situ hybridization showed that peri-aortic adventitial cells in high fat-fed mice expressMsx2. Opn was also detected in this adventitial cell population, but in addition was expressed by aortic vascular smooth muscle cells and macrophages of the intimal atheroma. High fat diets associated with hyperinsulinemic diabetes activate an aortic osteoblast transcriptional regulatory program that is independent of intimal atheroma formation. The spatial pattern ofMsx2 and Opn gene expression strongly suggests that vascular calcification, thought to be limited to the media, is an active process that can originate from an osteoprogenitor cell population in the adventitia.

Dexamethasone induction of hypertension and diabetes is PPAR-α dependent in LDL receptor–null mice

Hypertension and diabetes are common side effects of glucocorticoid treatment. To determine whether peroxisome proliferator–activated receptor-α (PPAR-α) mediates these sequelae, mice deficient in low-density lipoprotein receptor (Ldlr−/−), with (Ppara+/+) or without (Ppara−/−) PPAR-α, were treated chronically with dexamethasone. Ppara+/+, but not Ppara−/−, mice developed hyperglycemia, hyperinsulinemia and hypertension. Similar effects on glucose metabolism were seen in a different model using C57BL/6 mice. Hepatic gluconeogenic gene expression was increased and insulin-mediated suppression of endogenous glucose production was less effective in dexamethasone-treated Ppara+/+ mice. Adenoviral reconstitution of PPAR-α in the livers of nondiabetic, normotensive, dexamethasone-treated Ppara−/− mice induced hyperglycemia, hyperinsulinemia and increased gluconeogenic gene expression. It also increased blood pressure, renin activity, sympathetic nervous activity and renal sodium retention. Human hepatocytes treated with dexamethasone and the PPAR-α agonist Wy14,643 induced PPARA and gluconeogenic gene expression. These results identify hepatic activation of PPAR-α as a mechanism underlying glucocorticoid-induced insulin resistance.

Brain fatty acid synthase activates PPARα to maintain energy homeostasis

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

Vascular respiratory uncoupling increases blood pressure and atherosclerosis

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.

Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking

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.

Respiratory Uncoupling Lowers Blood Pressure Through a Leptin-Dependent Mechanism in Genetically Obese Mice

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 (Ay/a) mice, genetically obese animals known to have elevated blood pressure. Despite increased food intake, UCP-Ay/a mice weighed less than their Ay/a littermates. The metabolic rate was higher in UCP-Ay/a mice than in Ay/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-Ay/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-Ay/a mice but not in their Ay/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.

Correction of Hypertriglyceridemia and Impaired Fat Tolerance in Lipoprotein Lipase–Deficient Mice by Adenovirus-Mediated Expression of Human Lipoprotein Lipase

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 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-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 < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.