Adipose tissue-derived free fatty acids initiate myeloid cell accumulation in mouse liver in states of lipid oversupply.

Accumulation of myeloid cells in the liver, notably dendritic cells (DCs) and monocytes/macrophages (MCs), is a major component of the metainflammation of obesity. However, the mechanism(s) stimulating hepatic DC/MC infiltration remain ill defined. Herein, we addressed the hypothesis that adipose tissue (AT) free fatty acids (FFAs) play a central role in the initiation of hepatic DC/MC accumulation, using a number of mouse models of altered FFA supply to the liver. In two models of acute FFA elevation (lipid infusion and fasting) hepatic DC/MC and triglycerides (TGs) but not AT DC/MC were increased without altering plasma cytokines (PCs; TNFα and monocyte chemoattractant protein 1) and with variable effects on oxidative stress (OxS) markers. However, fasting in mice with profoundly reduced AT lipolysis (AT-specific deletion of adipose TG lipase; AAKO) failed to elevate liver DC/MC, TG, or PC, but liver OxS increased. Livers of obese AAKO mice that are known to be resistant to steatosis were similarly protected from inflammation. In high-fat feeding studies of 1, 3, 6, or 20-wk duration, liver DC/MC accumulation dissociated from PC and OxS but tracked with liver TGs. Furthermore, decreasing OxS by ~80% in obese mice failed to decrease liver DC/MC. Therefore, FFA and more specifically AT-derived FFA stimulate hepatic DC/MC accumulation, thus recapitulating the pathology of the obese liver. In a number of cases the effects of FFA can be dissociated from OxS and PC but match well with liver TG, a marker of FFA oversupply.

Role of Cardiorespiratory Fitness and Mitochondrial Oxidative Capacity in Reduced Walk Speed of Older Adults With Diabetes.

Cardiorespiratory fitness and mitochondrial oxidative capacity are associated with reduced walking speed in older adults, but their impact on walking speed in older adults with diabetes has not been clearly defined. We examined differences in cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity between older adults with and without diabetes, as well as determined their relative contribution to slower walking speed in older adults with diabetes. Participants with diabetes (n = 159) had lower cardiorespiratory fitness and mitochondrial respiration in permeabilized fiber bundles compared with those without diabetes (n = 717), following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. Four-meter and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walking speed between older adults with and without diabetes. Additional adjustments for BMI and comorbidities further explained the group differences in walking speed. Cardiorespiratory fitness and skeletal muscle mitochondrial oxidative capacity contribute to slower walking speeds in older adults with diabetes.

G-protein coupled receptor 19 (GPR19) knockout mice display sex-dependent metabolic dysfunction.

G-protein coupled receptors (GPCRs) mediate signal transduction from the cellular surface to intracellular metabolic pathways. While the function of many GPCRs has been delineated previously, a significant number require further characterization to elucidate their cellular function. G-protein coupled receptor 19 (GPR19) is a poorly characterized class A GPCR which has been implicated in the regulation of circadian rhythm, tumor metastasis, and mitochondrial homeostasis. In this report, we use a novel knockout (KO) mouse model to examine the role of GPR19 in whole-body metabolic regulation. We show that loss of GPR19 promotes increased energy expenditure and decreased activity in both male and female mice. However, only male GPR19 KO mice display glucose intolerance in response to a high fat diet. Loss of GPR19 expression in male mice, but not female mice, resulted in diet-induced hepatomegaly, which was associated with decreased expression of key fatty acid oxidation genes in male GPR19 KO livers. Overall, our data suggest that loss of GPR19 impacts whole-body energy metabolism in diet-induced obese mice in a sex-dependent manner.

Role of Cardiorespiratory Fitness and Mitochondrial Energetics in Reduced Walk Speed of Older Adults with Diabetes in the Study of Muscle, Mobility and Aging (SOMMA).

Rationale: Cardiorespiratory fitness and mitochondrial energetics are associated with reduced walking speed in older adults. The impact of cardiorespiratory fitness and mitochondrial energetics on walking speed in older adults with diabetes has not been clearly defined. Objective: To examine differences in cardiorespiratory fitness and skeletal muscle mitochondrial energetics between older adults with and without diabetes. We also assessed the contribution of cardiorespiratory fitness and skeletal muscle mitochondrial energetics to slower walking speed in older adults with diabetes. Findings: Participants with diabetes had lower cardiorespiratory fitness and mitochondrial energetics when compared to those without diabetes, following adjustments for covariates including BMI, chronic comorbid health conditions, and physical activity. 4-m and 400-m walking speeds were slower in those with diabetes. Mitochondrial oxidative capacity alone or combined with cardiorespiratory fitness mediated ∼20-70% of the difference in walk speed between older adults with and without diabetes. Further adjustments of BMI and co-morbidities further explained the group differences in walk speed. Conclusions: Skeletal muscle mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speeds in older adults with diabetes. Cardiorespiratory fitness and mitochondrial energetics may be therapeutic targets to maintain or improve mobility in older adults with diabetes. ARTICLE HIGHLIGHTS: Why did we undertake this study? To determine if mitochondrial energetics and cardiorespiratory fitness contribute to slower walking speed in older adults with diabetes. What is the specific question(s) we wanted to answer? Are mitochondrial energetics and cardiorespiratory fitness in older adults with diabetes lower than those without diabetes? How does mitochondrial energetics and cardiorespiratory fitness impact walking speed in older adults with diabetes? What did we find? Mitochondrial energetics and cardiorespiratory fitness were lower in older adults with diabetes compared to those without diabetes, and energetics, and cardiorespiratory fitness, contributed to slower walking speed in those with diabetes. What are the implications of our findings? Cardiorespiratory fitness and mitochondrial energetics may be key therapeutic targets to maintain or improve mobility in older adults with diabetes.

Intestinal HIF-2α Regulates GLP-1 Secretion via Lipid Sensing in L-Cells.

BACKGROUND & AIMS: Compelling evidence shows that glucagon-like peptide-1 (GLP-1) has a profound effect in restoring normoglycemia in type 2 diabetic patients by increasing pancreatic insulin secretion. Although L-cells are the primary source of circulating GLP-1, the current therapies do not target L-cells to increase GLP-1 levels. Our study aimed to determine the molecular underpinnings of GLP-1 secretion as an impetus to identify new interventions to target endogenous L-cells. METHODS: We used genetic mouse models of intestine-specific overexpression of hypoxia-inducible factor (HIF)-1α and HIF-2α (VhlΔIE), conditional overexpression of intestinal HIF-2α (Hif-2αLSL;Vilin-Cre/ERT2), and intestine-specific HIF-2α knockout mice (Hif-2αΔIE) to show that HIF signaling, especially HIF-2α, regulates GLP-1 secretion. RESULTS: Our data show that intestinal HIF signaling improved glucose homeostasis in a GLP-1-dependent manner. Intestinal HIF potentiated GLP-1 secretion via the lipid sensor G-protein-coupled receptor (GPR)40 enriched in L-cells. We show that HIF-2α regulates GPR40 in L-cells and potentiates fatty acid-induced GLP-1 secretion via extracellular regulated kinase (ERK). Using a genetic model of intestine-specific overexpression of HIF-2α, we show that HIF-2α is sufficient to increase GLP-1 levels and attenuate diet-induced metabolic perturbations such as visceral adiposity, glucose intolerance, and hepatic steatosis. Lastly, we show that intestinal HIF-2α signaling acts as a priming mechanism crucial for postprandial lipid-mediated GLP-1 secretion. Thus, disruption of intestinal HIF-2α decreases GLP-1 secretion. CONCLUSIONS: In summary, we show that intestinal HIF signaling, particularly HIF-2α, regulates the lipid sensor GPR40, which is crucial for the lipid-mediated GLP-1 secretion, and suggest that HIF-2α is a potential target to induce endogenous GLP-1 secretion.

Hepatocyte-Specific Ablation or Whole-Body Inhibition of Xanthine Oxidoreductase in Mice Corrects Obesity-Induced Systemic Hyperuricemia Without Improving Metabolic Abnormalities.

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of Xdh, the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte Xdh substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte Xdh is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative.

Elevated metabolic rate and skeletal muscle oxidative metabolism contribute to the reduced susceptibility of NF-κB p50 null mice to obesity.

Mice with a deletion of the p50 subunit of the proinflammatory nuclear factor kappa B pathway (NF-κB p50) have reduced weight compared to wild-type control mice. However, the physiological underpinning of this phenotype remains unknown. This study addressed this issue. Compared to littermate controls, lean male p50 null mice (p50-/- ) had an increased metabolic rate (~20%) that was associated with increased skeletal muscle (SkM, ~35%), but not liver, oxidative metabolism. These metabolic alterations were accompanied by decreases in adiposity, and tissue and plasma triglyceride levels (all ~30%). Notably, there was a marked decrease in skeletal muscle, but not liver, DGAT2 gene expression (~70%), but a surprising reduction in muscle PPARα and CPT1 (both ~20%) gene expression. Exposure to a high-fat diet accentuated the diminished adiposity of p50-/- mice despite elevated caloric intake, whereas plasma triglycerides and free fatty acids (both ~30%), and liver (~40%) and SkM (~50%) triglyceride accumulation were again reduced compared to WT. Although SkM cytokine expression (IL-6 and TNFα, each ~100%) were increased in p50-/- mice, neither cytokine acutely increased SkM oxidative metabolism. We conclude that the reduced susceptibility to diet-induced obesity and dyslipidemia in p50-/- mice results from an increase in metabolic rate, which is associated with elevated skeletal muscle oxidative metabolism and decreased DGAT2 expression.

The mammalian target of rapamycin regulates lipid metabolism in primary cultures of rat hepatocytes.

The mammalian target of rapamycin (mTOR) is a conserved serine-threonine kinase that regulates cell growth and metabolism in response to nutrient signals. However, the specific involvement of mTOR in regulation of energy metabolism is poorly understood. To determine if signaling via mTOR might be directly involved in regulation of fatty acid metabolism in hepatocytes, we performed studies with rapamycin, a specific inhibitor of mTOR. Rapamycin-mediated inhibition of mTOR (18-48 hours) increased oxidation of exogenous fatty acids (46%-100%, respectively). In addition, esterification of exogenous fatty acids and de novo lipid synthesis were reduced (40%-60%, respectively). Consistent with inhibition of lipogenic pathways, rapamycin decreased expression of genes encoding acetyl-coenzyme A carboxylase I and mitochondrial glycerol phosphate acyltransferase. Non-insulin-dependent glucose transport and glycogen synthesis were decreased by 20% to 30%, whereas glucose utilization was unaffected by rapamycin. The data suggest that the hyperlipidemia observed with the drug in vivo is likely not the result of enhanced hepatic synthesis, but rather of delayed peripheral clearance. However, these results are consistent with the idea that mTOR may play a significant role, not only in "energy sensing," but also in regulation of energy production through profound effects on hepatic fatty acid metabolism.

Rapamycin-mediated inhibition of mammalian target of rapamycin in skeletal muscle cells reduces glucose utilization and increases fatty acid oxidation.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an important role in cell growth and metabolism. mTOR has been postulated as a nutrient sensor, but its role in the regulation of fatty acid and glucose metabolism is poorly understood. For the first time, we show that mTOR inhibition in skeletal muscle cells has pronounced effects on intermediary metabolism. Rapamycin, a uniquely specific mTOR inhibitor with clinical applications, increased fatty acid oxidation by 60% accompanied by increased activities of carnitine palmitoyltransferases I and II, the former believed to be the primary intracellular regulatory enzyme of the fatty acid oxidation pathway. Furthermore, glucose transport capacity, glycogen synthesis, and glycolysis were reduced by approximately 40% under the same conditions. In addition, in the presence of rapamycin, hyperinsulinemic conditions (100 nmol/L insulin, 24 hours) were unable to suppress fatty acid oxidation in L6 myotubes. Rapamycin treatment also decreased baseline phosphorylation of mTOR residues S2448 and S2481 by 30% and almost completely abolished p70 S6 kinase phosphorylation. These results show that rapamycin causes a metabolic shift from glucose utilization to fatty acid oxidation in model muscle cells in the presence of nutrient abundance and underline the importance of mTOR as a key regulator in glucose and lipid metabolism.

Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes.

Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc-/- (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc-/- livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.

Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver.

Obesity-associated increases in adipose tissue (AT) CD11c(+) cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)-induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow-derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD.

Mice lacking NKT cells but with a complete complement of CD8+ T-cells are not protected against the metabolic abnormalities of diet-induced obesity.

The contribution of natural killer T (NKT) cells to the pathogenesis of metabolic abnormalities of obesity is controversial. While the combined genetic deletion of NKT and CD8(+) T-cells improves glucose tolerance and reduces inflammation, interpretation of these data have been complicated by the recent observation that the deletion of CD8(+) T-cells alone reduces obesity-induced inflammation and metabolic dysregulation, leaving the issue of the metabolic effects of NKT cell depletion unresolved. To address this question, CD1d null mice (CD1d(-/-)), which lack NKT cells but have a full complement of CD8(+) T-cells, and littermate wild type controls (WT) on a pure C57BL/6J background were exposed to a high fat diet, and glucose intolerance, insulin resistance, dyslipidemia, inflammation, and obesity were assessed. Food intake (15.5±4.3 vs 15.3±1.8 kcal/mouse/day), weight gain (21.8±1.8 vs 22.8±1.4 g) and fat mass (18.6±1.9 vs 19.5±2.1 g) were similar in CD1d(-/-) and WT, respectively. As would be expected from these data, metabolic rate (3.0±0.1 vs 2.9±0.2 ml O(2)/g/h) and activity (21.6±4.3 vs 18.5±2.6 beam breaks/min) were unchanged by NKT cell depletion. Furthermore, the degree of insulin resistance, glucose intolerance, liver steatosis, and adipose and liver inflammatory marker expression (TNFα, IL-6, IL-10, IFN-γ, MCP-1, MIP1α) induced by high fat feeding in CD1d(-/-) were not different from WT. We conclude that deletion of NKT cells, in the absence of alterations in the CD8(+) T-cell population, is insufficient to protect against the development of the metabolic abnormalities of diet-induced obesity.

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels.

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.