The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive, inflammatory form of fatty liver disease. It is the most rapidly rising risk factor for the development of hepatocellular carcinoma (HCC), which can arise in NASH with or without cirrhosis. The inflammatory signals promoting the progression of NASH to HCC remain largely unknown. The propensity of neutrophils to expel decondensed chromatin embedded with inflammatory proteins, known as neutrophil extracellular traps (NETs), has been shown to be important in chronic inflammatory conditions and in cancer progression. In this study, we asked whether NET formation occurs in NASH and contributes to the progression of HCC. We found elevated levels of a NET marker in serum of patients with NASH. In livers from STAM mice (NASH induced by neonatal streptozotocin and high-fat diet), early neutrophil infiltration and NET formation were seen, followed by an influx of monocyte-derived macrophages, production of inflammatory cytokines, and progression of HCC. Inhibiting NET formation, through treatment with deoxyribonuclease (DNase) or using mice knocked out for peptidyl arginine deaminase type IV (PAD4-/- ), did not affect the development of a fatty liver but altered the consequent pattern of liver inflammation, which ultimately resulted in decreased tumor growth. Mechanistically, we found that commonly elevated free fatty acids stimulate NET formation in vitro. CONCLUSION: Our findings implicate NETs in the protumorigenic inflammatory environment in NASH, suggesting that their elimination may reduce the progression of liver cancer in NASH. (Hepatology 2018).

Nrf2 deletion from adipocytes, but not hepatocytes, potentiates systemic metabolic dysfunction after long-term high-fat diet-induced obesity in mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a canonical regulator of cytoprotective gene expression, but evidence of its cross talk with other pathways, including metabolic ones, is ever increasing. Pharmacologic or systemic genetic activation of the Nrf2 pathway partially protects from obesity in mice and ameliorates fasting hyperglycemia in mice and humans. However, systemic Nrf2 deletion also protects from diet-induced obesity and insulin resistance in mice. To further investigate the effect of the disruption of Nrf2 on obesity in a tissue-specific manner, we focused on adipocytes and hepatocytes with targeted deletion of Nrf2. To this end, mice with cell-specific deletion of Nrf2 in adipocytes (ANKO) or hepatocytes (HeNKO) were fed a high-fat diet (HFD) for 6 mo and showed similar increases in body weight and body fat content. ANKO mice showed a partially deteriorated glucose tolerance, higher fasting glucose levels, and higher levels of cholesterol and nonesterified fatty acids compared with their Control counterparts. The HeNKO mice, though, had lower insulin levels and trended toward improved insulin sensitivity without having any difference in liver triglyceride accumulation. This study compared for the first time two conditional Nrf2 knockout models in adipocytes and in hepatocytes during HFD-induced obesity. None of these models could completely recapitulate the unexpected protection against obesity observed in the whole body Nrf2 knockout mice, but this study points out the differential roles that Nrf2 may play, beyond cytoprotection, in different target tissues and rather suggests systemic activation of the Nrf2 pathway as an effective means of prevention and treatment of obesity and type 2 diabetes.

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.

Kupffer cells facilitate the acute effects of leptin on hepatic lipid metabolism.

Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120 min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on phosphoinositol-3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes. Notably, liver TG were elevated in both lean and obese myeloid cell ObR, but the degree of obesity and insulin resistance induced by a high-fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the coculture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually [granulocyte macrophage colony-stimulating factor, IL-1α, IL-1ß, IL-6, IL-10, and IL-18] or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1.NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice.

Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.

Hepatic overexpression of steroid sulfatase ameliorates mouse models of obesity and type 2 diabetes through sex-specific mechanisms.

The steroid sulfatase (STS)-mediated desulfation is a critical metabolic mechanism that regulates the chemical and functional homeostasis of endogenous and exogenous molecules. In this report, we first showed that the liver expression of Sts was induced in both the high fat diet (HFD) and ob/ob models of obesity and type 2 diabetes and during the fed to fasting transition. In defining the functional relevance of STS induction in metabolic disease, we showed that overexpression of STS in the liver of transgenic mice alleviated HFD and ob/ob models of obesity and type 2 diabetes, including reduced body weight, improved insulin sensitivity, and decreased hepatic steatosis and inflammation. Interestingly, STS exerted its metabolic benefit through sex-specific mechanisms. In female mice, STS may have increased hepatic estrogen activity by converting biologically inactive estrogen sulfates to active estrogens and consequently improved the metabolic functions, whereas ovariectomy abolished this protective effect. In contrast, the metabolic benefit of STS in males may have been accounted for by the male-specific decrease of inflammation in white adipose tissue and skeletal muscle as well as a pattern of skeletal muscle gene expression that favors energy expenditure. The metabolic benefit in male STS transgenic mice was retained after castration. Treatment with the STS substrate estrone sulfate also improved metabolic functions in both the HFD and ob/ob models. Our results have uncovered a novel function of STS in energy metabolism and type 2 diabetes. Liver-specific STS induction or estrogen/estrogen sulfate delivery may represent a novel approach to manage metabolic syndrome.

Impact of dietary fatty acids on metabolic activity and host intestinal microbiota composition in C57BL/6J mice.

Different dietary fat and energy subtypes have an impact on both the metabolic health and the intestinal microbiota population of the host. The present study assessed the impact of dietary fat quality, with a focus on dietary fatty acid compositions of varying saturation, on the metabolic health status and the intestinal microbiota composition of the host. C57BL/6J mice (n 9-10 mice per group) were fed high-fat (HF) diets containing either (1) palm oil, (2) olive oil, (3) safflower oil or (4) flaxseed/fish oil for 16 weeks and compared with mice fed low-fat (LF) diets supplemented with either high maize starch or high sucrose. Tissue fatty acid compositions were assessed by GLC, and the impact of the diet on host intestinal microbiota populations was investigated using high-throughput 16S rRNA sequencing. Compositional sequencing analysis revealed that dietary palm oil supplementation resulted in significantly lower populations of Bacteroidetes at the phylum level compared with dietary olive oil supplementation (P< 0·05). Dietary supplementation with olive oil was associated with an increase in the population of the family Bacteroidaceae compared with dietary supplementation of palm oil, flaxseed/fish oil and high sucrose (P< 0·05). Ingestion of the HF-flaxseed/fish oil diet for 16 weeks led to significantly increased tissue concentrations of EPA, docosapentaenoic acid and DHA compared with ingestion of all the other diets (P< 0·05); furthermore, the diet significantly increased the intestinal population of Bifidobacterium at the genus level compared with the LF-high-maize starch diet (P< 0·05). These data indicate that both the quantity and quality of fat have an impact on host physiology with further downstream alterations to the intestinal microbiota population, with a HF diet supplemented with flaxseed/fish oil positively shaping the host microbial ecosystem.

Divergent metabolic outcomes arising from targeted manipulation of the gut microbiota in diet-induced obesity.

OBJECTIVE: The gut microbiota is an environmental regulator of fat storage and adiposity. Whether the microbiota represents a realistic therapeutic target for improving metabolic health is unclear. This study explored two antimicrobial strategies for their impact on metabolic abnormalities in murine diet-induced obesity: oral vancomycin and a bacteriocin-producing probiotic (Lactobacillus salivarius UCC118 Bac(+)). DESIGN: Male (7-week-old) C57BL/J6 mice (9-10/group) were fed a low-fat (lean) or a high-fat diet for 20 weeks with/without vancomycin by gavage at 2 mg/day, or with L. salivarius UCC118Bac(+) or the bacteriocin-negative derivative L. salivarius UCC118Bac(-) (each at a dose of 1×10(9) cfu/day by gavage). Compositional analysis of the microbiota was by 16S rDNA amplicon pyrosequencing. RESULTS: Analysis of the gut microbiota showed that vancomycin treatment led to significant reductions in the proportions of Firmicutes and Bacteroidetes and a dramatic increase in Proteobacteria, with no change in Actinobacteria. Vancomycin-treated high-fat-fed mice gained less weight over the intervention period despite similar caloric intake, and had lower fasting blood glucose, plasma TNFα and triglyceride levels compared with diet-induced obese controls. The bacteriocin-producing probiotic had no significant impact on the proportions of Firmicutes but resulted in a relative increase in Bacteroidetes and Proteobacteria and a decrease in Actinobacteria compared with the non-bacteriocin-producing control. No improvement in metabolic profiles was observed in probiotic-fed diet-induced obese mice. CONCLUSION: Both vancomycin and the bacteriocin-producing probiotic altered the gut microbiota in diet-induced obese mice, but in distinct ways. Only vancomycin treatment resulted in an improvement in the metabolic abnormalities associated with obesity thereby establishing that while the gut microbiota is a realistic therapeutic target, the specificity of the antimicrobial agent employed is critical.

Leptin signalling reduces the severity of cardiac dysfunction and remodelling after chronic ischaemic injury.

AIMS: Leptin is elevated under conditions of both obesity and heart failure (HF), and activation of leptin receptor (ObR) signalling is known to increase in vivo cardiac contractility and to have anti-hypertrophic effects on the left ventricle (LV). However, it is unknown whether ObR signalling is altered in cardiomyocytes after myocardial infarction (MI) leading to HF, or if a deficiency in ObR signalling leads to worse HF. METHODS AND RESULTS: In separate experimental protocols, C57BL/6J and leptin-deficient (ob/ob) mice underwent open-chest surgery to induce permanent left coronary artery ligation (CAL) or had a sham operation. Subgroups of ob/ob mice examined were lean (food-restricted), obese (food-ad libitum), and leptin repleted. Four weeks post-surgery, cardiac structure and function was examined by echocardiography, and the activation of cardiac leptin signalling was characterized through quantitative PCR, western blotting, and DNA-binding activities. CAL produced echocardiographic evidence of HF in C57BL/6J mice, elevated circulating leptin, increased cardiomyocyte leptin and ObR expression, and activated myocardial signal transducer and activator of transcription-3 (STAT3). In leptin-deficient ob/ob mice, whether lean or obese, CAL caused increased hypertrophy and dilation, decreased contractility of the LV, and worsened survival relative to wildtype or leptin-repleted mice after CAL. In ob/ob mice, activation of cardiac STAT3 signalling after CAL is enhanced in the presence of leptin and parallels the induction of the STAT3-responsive genes, tissue-inhibitor of metalloproteinase-1 and heat shock protein-70. CONCLUSION: These data demonstrate that HF increases ObR signalling in cardiomyocytes and that activation of ObR signalling improves functional outcomes in chronic ischaemic injury leading to HF.

Adropin reduces blood glucose levels in mice by limiting hepatic glucose production.

Adropin is a liver- and brain-secreted peptide hormone with striking effects on fuel metabolism regulation in a number of tissues. Previous studies demonstrated that adropin secretion is decreased in obese mice subjected to a long-term high-fat diet (HFD), and that whole-body loss of adropin expression resulted in systemic insulin resistance. Treatment of obese mice with adropin improves glucose tolerance, which has been linked to increased glucose oxidation and inhibition of fatty acid utilization in isolated skeletal muscle homogenates. In this study, we used in vivo physiological measurements to determine how treatment of obese mice with adropin affects whole-body glucose metabolism. Treatment with adropin reduced fasting blood glucose and, as shown previously, increased glucose tolerance in HFD mice during standard glucose tolerance tests. Under hyperinsulinemic-euglycemic clamp conditions, adropin treatment led to a nonsignificant increase in whole-body insulin sensitivity, and a significant reduction in whole-body glucose uptake. Finally, we show that adropin treatment suppressed hepatic glucose production and improved hepatic insulin sensitivity. This correlated with reduced expression of fatty acid import proteins and gluconeogenic regulatory enzymes in the liver, suggesting that adropin treatment may impact the pathways that drive vital aspects of hepatic glucose metabolism.

Petite Integration Factor 1 (PIF1) helicase deficiency increases weight gain in Western diet-fed female mice without increased inflammatory markers or decreased glucose clearance.

Petite Integration Factor 1 (PIF1) is a multifunctional helicase present in nuclei and mitochondria. PIF1 knock out (KO) mice exhibit accelerated weight gain and decreased wheel running on a normal chow diet. In the current study, we investigated whether Pif1 ablation alters whole body metabolism in response to weight gain. PIF1 KO and wild type (WT) C57BL/6J mice were fed a Western diet (WD) rich in fat and carbohydrates before evaluation of their metabolic phenotype. Compared with weight gain-resistant WT female mice, WD-fed PIF1 KO females, but not males, showed accelerated adipose deposition, decreased locomotor activity, and reduced whole-body energy expenditure without increased dietary intake. Surprisingly, PIF1 KO females did not show obesity-induced alterations in fasting blood glucose and glucose clearance. WD-fed PIF1 KO females developed mild hepatic steatosis and associated changes in liver gene expression that were absent in weight-matched, WD-fed female controls, linking hepatic steatosis to Pif1 ablation rather than increased body weight. WD-fed PIF1 KO females also showed decreased expression of inflammation-associated genes in adipose tissue. Collectively, these data separated weight gain from inflammation and impaired glucose homeostasis. They also support a role for Pif1 in weight gain resistance and liver metabolic dysregulation during nutrient stress.

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.

Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic beta-cell replication in mice.

Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co-exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic beta-cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5-6% at 60 cycles h(-1) for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic beta-cells from 1.5 +/- 0.3 to 4.0 +/- 0.8% bromodeoxyuridine (BrdU)-positive) beta-cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in beta-cells, without any additive effects on beta-cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic beta-cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of beta-cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes.

Detailed molecular analysis of the induction of the L-PK gene by glucose.

Glucose has powerful effects on gene expression and participates in the fasted-to-fed transition of the liver. However, the molecular mechanism of glucose-regulated gene expression has not been completely described. In the present study, we performed a detailed analysis of the molecular events of the insulin-independent glucose response of the liver-type pyruvate kinase (L-PK) gene. L-PK mRNA was increased by glucose at the transcriptional level as determined by real-time RT-PCR, mRNA stability measurements, and nuclear run-on assays. LY294002 and LY303511 inhibited the glucose response of the L-PK gene at the transcriptional level. Histones H3 and H4 associated with the L-PK gene promoter were hyperacetylated and HNF4alpha was constitutively bound in low and high glucose. Treatment with 20mM glucose increased recruitment of ChREBP, additional HNF4alpha, and RNA polymerase II. Glucose-stimulated the phosphorylation of the C-terminal domain of RNA polymerase II, with increased Ser5 phosphorylation near the transcription start site and increased Ser2 phosphorylation near the termination signal. LY294002 and LY303511 blocked the recruitment of RNA polymerase II to the L-PK gene, reducing the rate of transcription. The results of these studies demonstrate fundamental details of the molecular mechanism of glucose activated gene expression.

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.

Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4.

Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here, we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (Csf1r, Spi1, Runx1) are enhanced, and lymphoid genes (Flt3, Tcf3, Ebf1) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid>lymphoid bias arises after 6 weeks of high-fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 to obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raises important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis, as fetal immune precursors are also sensitive to TLR4 signals.

Nrf2 prevents Notch-induced insulin resistance and tumorigenesis in mice.

Insulin resistance is associated with increased incidence and enhanced progression of cancers. However, little is known about strategies that can effectively ameliorate insulin resistance and consequently halt cancer progression. Herein, we propose that the transcription factor Nrf2 (also known as Nfe2l2) may be such a target, given its central role in disease prevention. To this end, we developed a mouse that overexpresses the Notch intracellular domain in adipocytes (AdNICD), leading to lipodystrophy-induced severe insulin resistance and subsequent development of sarcomas, as a model reflecting that Notch signaling is deregulated in cancers and shows positive associations with insulin resistance and fatty liver disease in humans. Nrf2 pathway activation was achieved by knocking down Keap1, a repressor of Nrf2, in the AdNICD background. Constitutively enhanced Nrf2 signaling in this setting led to prevention of hepatic steatosis, dyslipidemia, and insulin resistance by repressing hepatic lipogenic pathways and restoration of the hepatic fatty acid profile to control levels. This protective effect of Nrf2 against diabetes extended to significant reduction and delay in sarcoma incidence and latency. Our study highlights that the Nrf2 pathway, which has been induced by small molecules in clinical trials, is a potential therapeutic target against insulin resistance and subsequent risk of cancer.

Hepatic steatosis and plasma dyslipidemia induced by a high-sucrose diet are corrected by an acute leptin infusion.

High sucrose (HS) feeding in rats induces hepatic steatosis and plasma dyslipidemia. In previous reports (Huang W, Dedousis N, Bhatt BA, O'Doherty RM. J Biol Chem 279: 21695-21700, 2004; and Huang W, Dedousis N, Bandi A, Lopaschuk GD, O'Doherty RM. Endocrinology 147: 1480-1487, 2006), our laboratory demonstrated a rapid ( approximately 100 min) leptin-induced decrease in liver and plasma VLDL triglycerides (TG) in lean rats, effects that were abolished in obese rats fed a high-fat diet, a model that also presents with hepatic steatosis and plasma dyslipidemia. To further examine the capacity of acute leptin treatment to improve metabolic abnormalities induced by nutrient excess, hepatic leptin action was studied in rats after 5 wk of HS feeding. HS feeding induced hepatic steatosis (TG+80+/-8%; P=0.001), plasma hyperlipidemia (VLDL-TG+102+/-14%; P=0.001), hyperinsulinemia (plasma insulin +67+/-12%; P=0.04), and insulin resistance as measured by homeostasis model assessment (+125+/-20%; P=0.02), without increases in adiposity or plasma leptin concentration compared with standard chow-fed controls. A 120-min infusion of leptin (plasma leptin 13.6+/-0.7 ng/ml) corrected hepatic steatosis (liver TG-29+/-3%; P=0.003) and plasma hyperlipidemia in HS (VLDL-TG-42+/-4%; P=0.001) and increased plasma ketones (+45+/-3%; P=0.006), without altering plasma glucose, insulin, or homeostasis model assessment compared with saline-infused HS controls. In addition, leptin activated liver phosphatidylinositol 3-kinase (+70+/-18%; P=0.01) and protein kinase B (Akt; +90+/-29%; P=0.02), and inhibited acetyl-CoA carboxylase (40+/-7%; P=0.04) in HS, further demonstrating that hepatic leptin action was intact in these animals. We conclude that 1) leptin action on hepatic lipid metabolism remains intact in HS-fed rats, 2) leptin rapidly reverses hepatic steatosis and plasma dyslipidemia induced by sucrose, and 3) the preservation of hepatic leptin action after a HS diet is associated with the maintenance of low adiposity and plasma leptin concentrations.