Relationship between serum ß-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum ß-hydroxybutyrate (ß-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum ß-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum ß-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum ß-HB correlated with liver mitochondrial ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum ß-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum ß-hydroxybutyrate (ß-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating ß-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum ß-HB. Our work supports serum ß-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

Histological improvements following energy restriction and exercise: The role of insulin resistance in resolution of MASH.

BACKGROUND & AIMS: Metabolic-dysfunction associated steatohepatitis (MASH) is one of the most common liver diseases worldwide and is characterized by multi-tissue insulin resistance. The effects of a 10-month energy restriction and exercise intervention on liver histology, anthropometrics, plasma biochemistries, and insulin sensitivity were compared to standard of care (control) to understand mechanisms that support liver health improvements. METHODS: Following medical diagnosis of MASH, subjects were randomized to treatment (n=16) or control (n=8). Liver fat (MRS), 18-hour plasma biochemical measurements, and isotopically-labeled hyperinsulinemic-euglycemic clamps were completed pre- and post-intervention. Body composition and cardiorespiratory fitness (VO2peak) were also measured mid-intervention. Treatment subjects were counseled to reduce energy intake and completed supervised, high-intensity interval training (3x/week) for 10 months. Control subjects continued physician-directed care. RESULTS: Treatment induced significant (P<0.05) reductions in body weight, fat mass, and liver injury, while VO2peak (P<0.05) and fatty acid (NEFA) suppression (P=0.06) were improved. Both groups exhibited reductions in total energy intake, HbA1c, hepatic insulin resistance, and liver fat (P<0.05). Compared to control, treatment induced a two-fold increase in peripheral insulin sensitivity which was significantly related to higher VO2peak and resolution of liver disease, despite no group differences in peripheral insulin sensitivity. CONCLUSIONS: Exercise and energy-restriction elicited significant and clinically-meaningful treatment effects on liver health, potentially driven by a redistribution of excess nutrients to skeletal muscle, thereby reducing hepatic nutrient toxicity. Clinical guidelines should emphasize the addition of aerobic exercise in lifestyle treatments for the greatest histologic benefit in individuals with advanced MASH. CLINICAL TRIAL NUMBER: NCT03151798.

Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

BACKGROUND AND AIMS: NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant. APPROACH AND RESULTS: To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to CO2 and the rate-limiting enzyme in ß-oxidation (ß-hydroxyacyl-CoA dehydrogenase activity) were reduced by ~40%-50% with D-NASH compared with CTRL. This corresponded with increased hepatic mitochondrial reactive oxygen species production, as well as dramatic reductions in markers of mitochondrial biogenesis, autophagy, mitophagy, fission, and fusion in NAFL and NASH. CONCLUSIONS: These findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Isotope Labeling and Biochemical Assessment of Liver-Triacylglycerol in Patients with Different Levels of Histologically-Graded Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) prevalence is rapidly growing, and fatty liver has been found in a quarter of the US population. Increased liver lipids, particularly those derived from the pathway of de novo lipogenesis (DNL), have been identified as a hallmark feature in individuals with high liver fat. This has led to much activity in basic science and drug development in this area. No studies to date have investigated the contribution of DNL across a spectrum of disease, although it is clear that inhibition of DNL has been shown to reduce liver fat. OBJECTIVES: The purpose of this study was to determine whether liver lipid synthesis increases across the continuum of liver injury. METHODS: Individuals (n = 49) consumed deuterated water for 10 d before their scheduled bariatric surgeries to label DNL; blood and liver tissue samples were obtained on the day of the surgery. Liver lipid concentrations were quantitated, and levels of protein and gene expression assessed. RESULTS: Increased liver DNL, measured isotopically, was significantly associated with liver fatty acid synthase protein content (R = 0.470, P = 0.003), total steatosis assessed by histology (R = 0.526, P = 0.0008), and the fraction of DNL fatty acids in plasma very low-density lipoprotein-triacylglycerol (R = 0.747, P < 0.001). Regression analysis revealed a parabolic relationship between fractional liver DNL (percent) and NAFLD activity score (R = 0.538, P = 0.0004). CONCLUSION: These data demonstrate that higher DNL is associated with early to mid stages of liver disease, and this pathway may be an effective target for the treatment of NAFLD and nonalcoholic steatohepatitis. This study was registered at clinicaltrials.gov as NCT03683589.

Role of adropin in arterial stiffening associated with obesity and type 2 diabetes.

Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, we tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, we report that obesity and T2D are associated with reduced levels of adropin (in liver and plasma) and increased arterial stiffness in mice and humans. Establishing causation, we show that mesenteric arteries from adropin knockout mice are also stiffer, relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, we performed a set of follow-up experiments, in which we found that 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness, 2) adropin-induced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase, and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Lastly, we demonstrated that in vivo treatment of db/db mice with adropin for 4 wk reduces stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelium-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.NEW & NOTEWORTHY Arterial stiffening, a characteristic feature of obesity and type 2 diabetes (T2D), contributes to the development and progression of cardiovascular diseases. Herein we establish that adropin is decreased in obese and T2D models and furthermore provide evidence that reduced adropin may directly contribute to arterial stiffening. Collectively, findings from this work support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.

Mitochondrial Dysfunction and Acute Fatty Liver of Pregnancy.

The liver is one of the richest organs in mitochondria, serving as a hub for key metabolic pathways such as ß-oxidation, the tricarboxylic acid (TCA) cycle, ketogenesis, respiratory activity, and adenosine triphosphate (ATP) synthesis, all of which provide metabolic energy for the entire body. Mitochondrial dysfunction has been linked to subcellular organelle dysfunction in liver diseases, particularly fatty liver disease. Acute fatty liver of pregnancy (AFLP) is a life-threatening liver disorder unique to pregnancy, which can result in serious maternal and fetal complications, including death. Pregnant mothers with this disease require early detection, prompt delivery, and supportive maternal care. AFLP was considered a mysterious illness and though its pathogenesis has not been fully elucidated, molecular research over the past two decades has linked AFLP to mitochondrial dysfunction and defects in fetal fatty-acid oxidation (FAO). Due to deficient placental and fetal FAO, harmful 3-hydroxy fatty acid metabolites accumulate in the maternal circulation, causing oxidative stress and microvesicular fatty infiltration of the liver, resulting in AFLP. In this review, we provide an overview of AFLP and mitochondrial FAO followed by discussion of how altered mitochondrial function plays an important role in the pathogenesis of AFLP.

Mitochondrial Dysfunction Plays Central Role in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a global pandemic that affects one-quarter of the world's population. NAFLD includes a spectrum of progressive liver disease from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis and can be complicated by hepatocellular carcinoma. It is strongly associated with metabolic syndromes, obesity, and type 2 diabetes, and it has been shown that metabolic dysregulation is central to its pathogenesis. Recently, it has been suggested that metabolic- (dysfunction) associated fatty liver disease (MAFLD) is a more appropriate term to describe the disease than NAFLD, which puts increased emphasis on the important role of metabolic dysfunction in its pathogenesis. There is strong evidence that mitochondrial dysfunction plays a significant role in the development and progression of NAFLD. Impaired mitochondrial fatty acid oxidation and, more recently, a reduction in mitochondrial quality, have been suggested to play a major role in NAFLD development and progression. In this review, we provide an overview of our current understanding of NAFLD and highlight how mitochondrial dysfunction contributes to its pathogenesis in both animal models and human subjects. Further we discuss evidence that the modification of mitochondrial function modulates NAFLD and that targeting mitochondria is a promising new avenue for drug development to treat NAFLD/NASH.

Defects in long-chain 3-hydroxy acyl-CoA dehydrogenase lead to hepatocellular carcinoma: A novel etiology of hepatocellular carcinoma.

The incidence of both nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been increasing at an alarming rate. Little is known about NAFLD without cirrhosis as a risk for HCC. Here we report, for the first time, generation of a mouse model with a defect in long-chain 3-hydoxy acyl-CoA dehydrogenase (LCHAD). The LCHAD exon 15 deletion was embryonic lethal to the homozygous mice whereas heterozygous mice (HT) develop significant hepatic steatosis starting at young age (3 months old) and HCC at older age (>13 months old) without any evidence of fibrosis or cirrhosis. None of the wild-type (WT) mice developed steatosis and HCC (n = 39), whereas HT-LCHAD mice (n = 41) showed steatosis and ~20% (8/41) developed liver masses with histological features of HCC. Proteomic analysis of liver tissues from WT-mice and HT-mice with no signs of HCC was conducted. Proteins with significant changes in abundance were identified by mass spectrometry. Abundance of 24 proteins was significantly different (p < 0.01) between WT and HT-LCHAD mice. The proteins found to vary in abundance are associated with different cellular response processes ranging from intermediary metabolism of carbohydrate, protein and lipid to oxidative stress, signal transduction and the process of tumorigenesis. Protein expression pattern of the HT-LCHAD mouse liver indicates predisposition to HCC and suggests that impaired hepatic mitochondrial fatty acid oxidation plays an important role in the development and progression of HCC. To assess the implication of these studies in human disease, we demonstrated significant downregulation of HADHA transcripts in HCC patients.

eNOS deletion impairs mitochondrial quality control and exacerbates Western diet-induced NASH.

Dysregulated mitochondrial quality control leads to mitochondrial functional impairments that are central to the development and progression of hepatic steatosis to nonalcoholic steatohepatitis (NASH). Here, we identify hepatocellular localized endothelial nitric oxide synthase (eNOS) as a novel master regulator of mitochondrial quality control. Mice lacking eNOS were more susceptible to Western diet-induced hepatic inflammation and fibrosis in conjunction with decreased markers of mitochondrial biogenesis and turnover. The hepatocyte-specific influence was verified via magnetic activated cell sorting purified primary hepatocytes and in vitro siRNA-induced knockdown of eNOS. Hepatic mitochondria from eNOS knockout mice revealed decreased markers of mitochondrial biogenesis (PPARγ coactivator-1α, mitochondrial transcription factor A) and autophagy/mitophagy [BCL-2-interacting protein-3 (BNIP3), 1A/1B light chain 3B (LC3)], suggesting decreased mitochondrial turnover rate. eNOS knockout in primary hepatocytes exhibited reduced fatty acid oxidation capacity and were unable to mount a normal BNIP3 response to a mitophagic challenge compared with wild-type mice. Finally, we demonstrate that eNOS is required in primary hepatocytes to induce activation of the stress-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Thus, our data demonstrate that eNOS is an important regulator of hepatic mitochondrial content and function and NASH susceptibility.

Regulation of mitochondrial trifunctional protein modulates nonalcoholic fatty liver disease in mice.

Mitochondrial trifunctional protein (MTP) plays a critical role in the oxidation of long-chain fatty acids. We previously reported that aging mice (>9 months old) heterozygous for an MTP defect (MTP+/-) develop nonalcoholic fatty liver disease (NAFLD). We tested whether a high-fat diet (HFD) accelerates NAFLD in young MTP+/-mice, and whether overexpression of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 3 (SIRT3) deacetylates MTP and improves mitochondrial function and NAFLD. Three-month-old WT and MTP+/- mice were fed HFD (60% cal fat) for 16 weeks and livers were assessed for fatty acid oxidation (FAO) and NAFLD. Compared with WT, MTP+/- mice displayed reduced hepatic SIRT3 levels and reduced FAO, with increased hepatic steatosis and the inflammatory marker CD68. Hepatic overexpression of SIRT3 in HFD-fed MTP+/- mice increased hepatic MTP protein levels at the posttranscriptional level. Immunoprecipitation of MTP from liver mitochondria followed by Western blot with acetyl-lysine antibody showed higher acetylation of MTP in MTP+/- compared with WT mice. Overexpression of SIRT3 in MTP+/- mice significantly reduced the acetylation of MTP compared with ß-galactosidase controls, increased mitochondrial FAO, and reduced hepatic steatosis, CD68, and serum ALT levels. Taken together, our data indicate that deacetylation of MTP by SIRT3 improves mitochondrial function and rescues NAFLD in MTP+/- mice.

Role of 3-Hydroxy Fatty Acid-Induced Hepatic Lipotoxicity in Acute Fatty Liver of Pregnancy.

Acute fatty liver of pregnancy (AFLP), a catastrophic illness for both the mother and the unborn offspring, develops in the last trimester of pregnancy with significant maternal and perinatal mortality. AFLP is also recognized as an obstetric and medical emergency. Maternal AFLP is highly associated with a fetal homozygous mutation (1528G>C) in the gene that encodes for mitochondrial long-chain hydroxy acyl-CoA dehydrogenase (LCHAD). The mutation in LCHAD results in the accumulation of 3-hydroxy fatty acids, such as 3-hydroxy myristic acid, 3-hydroxy palmitic acid and 3-hydroxy dicarboxylic acid in the placenta, which are then shunted to the maternal circulation leading to the development of acute liver injury observed in patients with AFLP. In this review, we will discuss the mechanistic role of increased 3-hydroxy fatty acid in causing lipotoxicity to the liver and in inducing oxidative stress, mitochondrial dysfunction and hepatocyte lipoapoptosis. Further, we also review the role of 3-hydroxy fatty acids in causing placental damage, pancreatic islet ß-cell glucolipotoxicity, brain damage, and retinal epithelial cells lipoapoptosis in patients with LCHAD deficiency.

Corrigendum: 2016 ACG Abstracts- Outcomes Research.

This corrects the article DOI: 10.1038/ajg.2016.360.

Aerobic exercise training in the treatment of non-alcoholic fatty liver disease related fibrosis.

KEY POINTS: Physiologically relevant rodent models of non-alcoholic steatohepatitis (NASH) that resemble the human condition are limited. Exercise training and energy restriction are first-line recommendations for the treatment of NASH. Hyperphagic Otsuka Long-Evans Tokushima fatty rats fed a western diet high in fat, sucrose and cholesterol for 24 weeks developed a severe NASH with fibrosis phenotype. Moderate intensity exercise training and modest energy restriction provided some improvement in the histological features of NASH that coincided with alterations in markers of hepatic stellate cell activation and extracellular matrix remodelling. The present study highlights the importance of lifestyle modification, including exercise training and energy restriction, in the regulation of advanced liver disease. ABSTRACT: The incidence of non-alcoholic steatohepatitis (NASH) is rising but the efficacy of lifestyle modifications to improve NASH-related outcomes remain unclear. We hypothesized that a western diet (WD) would induce NASH in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat and that lifestyle modification would improve this condition. Eight-week-old Long-Evans Tokushima Otsuka (L) and OLETF (O) rats consumed a control diet (10% kcal fat, 3.5% sucrose) or a WD (45% kcal fat, 17% sucrose, 1% cholesterol) for 24 weeks. At 20 weeks of age, additional WD-fed OLETFs were randomized to sedentary (O-SED), food restriction (O-FR; ∼25% kcal reduction vs. O-SED) or exercise training (O-EX; treadmill running 20 m min(-1) with a 15% incline, 60 min day(-1) , 5 days week(-1) ) conditions for 12 weeks. WD induced a NASH phenotype in OLETFs characterized by hepatic fibrosis (collagen 1α1 mRNA and hydroxyproline content), as well as elevated inflammation and non-alcoholic fatty liver disease activity scores, and hepatic stellate cell activation (α-smooth muscle actin) compared to Long-Evans Tokushima Otsuka rats. FR and EX modestly improved NASH-related fibrosis markers (FR: hydroxyproline content, P < 0.01; EX: collagen 1α1 mRNA, P < 0.05; both: fibrosis score, P < 0.01) and inflammation (both: inflammation score; FR: interleukin-1ß and tumor necrosis factor α) vs. O-SED. FR reduced hepatic stellate cell activation markers (transforming growth factor-ß protein and α-smooth muscle actin mRNA), whereas EX increased the hepatic stellate cell senescence marker CCN1 (P < 0.01 vs. O-SED). Additionally, both FR and EX normalized extracellular matrix remodelling markers to levels similar to L-WD (P > 0.05). Although neither EX nor FR led to complete resolution of the WD-induced NASH phenotype, both independently benefitted liver fibrosis via altered hepatic stellate cell activation and extracellular matrix remodelling.

Aerobic capacity and hepatic mitochondrial lipid oxidation alters susceptibility for chronic high-fat diet-induced hepatic steatosis.

Rats selectively bred for high capacity running (HCR) or low capacity running (LCR) display divergence for intrinsic aerobic capacity and hepatic mitochondrial oxidative capacity, both factors associated with susceptibility for nonalcoholic fatty liver disease. Here, we tested if HCR and LCR rats display differences in susceptibility for hepatic steatosis after 16 wk of high-fat diets (HFD) with either 45% or 60% of kcals from fat. HCR rats were protected against HFD-induced hepatic steatosis, whereas only the 60% HFD induced steatosis in LCR rats, as marked by a doubling of liver triglycerides. Hepatic complete fatty acid oxidation (FAO) and mitochondrial respiratory capacity were all lower in LCR compared with HCR rats. LCR rats also displayed lower hepatic complete and incomplete FAO in the presence of etomoxir, suggesting a reduced role for noncarnitine palmitoyltransferase-1-mediated lipid catabolism in LCR versus HCR rats. Hepatic complete FAO and mitochondrial respiration were largely unaffected by either chronic HFD; however, 60% HFD feeding markedly reduced 2-pyruvate oxidation, a marker of tricarboxylic acid (TCA) cycle flux, and mitochondrial complete FAO only in LCR rats. LCR rats displayed lower levels of hepatic long-chain acylcarnitines than HCR rats but maintained similar levels of hepatic acetyl-carnitine levels, further supporting lower rates of ß-oxidation, and TCA cycle flux in LCR than HCR rats. Finally, only LCR rats displayed early reductions in TCA cycle genes after the acute initiation of a HFD. In conclusion, intrinsically high aerobic capacity confers protection against HFD-induced hepatic steatosis through elevated hepatic mitochondrial oxidative capacity.

Differential reduction in cardiac and liver monolysocardiolipin acyltransferase-1 and reduction in cardiac and liver tetralinoleoyl-cardiolipin in the α-subunit of trifunctional protein heterozygous knockout mice.

The contribution of α-subunit of trifunctional protein (αTFP) to cardiolipin (CL) (diphosphatidylglycerol) remodelling and mitochondrial supercomplex formation was examined in heart and liver mitochondria from wild-type (WT) and αTFP heterozygous knockout [Mtpa(+/-)] mice. Mtpa(+/-) mouse heart and liver exhibited an approximate 55% and 50% reduction in αTFP protein expression compared with WT respectively. Monolysocardiolipin (MLCL) acyltransferase (MLCL AT)-1 protein derived from αTFP was reduced by 30% in Mtpa(+/-) mouse heart but not in liver compared with WT. In vitro acylation of MLCL was significantly reduced in heart but not in liver mitochondria of Mtpa(+/-) mice compared with WT. CL mass was reduced and significant reductions in linoleate-containing CL species, in particular tetralinoleoyl-CL (L4-CL) and trilinoleoyl-CL (L3-MLCL) species, were observed in heart and liver mitochondria of Mtpa(+/-) mice compared with WT. Cardiac and liver mitochondrial supercomplex assembly and NADH dehydrogenase (complex I) activity within these supercomplexes were unaltered in both Mtpa(+/-) mouse heart and Mtpa(+/-) mouse liver compared with WT. The results indicate that αTFP may modulate CL molecular species composition in murine heart and liver. In addition, L4-CL might not be an essential requirement for mitochondrial supercomplex assembly.

Different mechanisms for histone acetylation by ethanol and its metabolite acetate in rat primary hepatocytes.

Ethanol and its major metabolite acetate both induced histone H3 acetylation in primary culture of rat hepatocytes. The acetylation by ethanol was dependent on the reactive oxygen species and mitogen-activated protein kinase pathway, whereas that by acetate was independent of both pathways. Ethanol increased CYP2E1 protein expression but acetate had negligible effect. The level of phospho-H2AX, an indicator of DNA breaks, was elevated by ethanol but not by acetate. Ethanol and acetate differentially activated mRNA expression for different genes, e.g., IL-6, PPARγ, c-Fos, Egr-1, and PNPLA3 in hepatocytes. The most striking increase (3-fold) was in PNPLA3 mRNA by ethanol with little change by acetate. It was further shown that acetate inhibited histone deacetylase activity. Taken together, these data establish for the first time that ethanol and acetate exhibit differences in their effects on hepatocytes in gene expression, P-H2AX levels, and the mechanism of histone H3 acetylation. The implications of these differences in the actions of ethanol in liver are discussed.

Unique transcriptomic signature of omental adipose tissue in Ossabaw swine: a model of childhood obesity.

To better understand the impact of childhood obesity on intra-abdominal adipose tissue phenotype, a complete transcriptomic analysis using deep RNA-sequencing (RNA-seq) was performed on omental adipose tissue (OMAT) obtained from lean and Western diet-induced obese juvenile Ossabaw swine. Obese animals had 88% greater body mass, 49% greater body fat content, and a 60% increase in OMAT adipocyte area (all P < 0.05) compared with lean pigs. RNA-seq revealed a 37% increase in the total transcript number in the OMAT of obese pigs. Ingenuity Pathway Analysis showed transcripts in obese OMAT were primarily enriched in the following categories: 1) development, 2) cellular function and maintenance, and 3) connective tissue development and function, while transcripts associated with RNA posttranslational modification, lipid metabolism, and small molecule biochemistry were reduced. DAVID and Gene Ontology analyses showed that many of the classically recognized gene pathways associated with adipose tissue dysfunction in obese adults including hypoxia, inflammation, angiogenesis were not altered in OMAT in our model. The current study indicates that obesity in juvenile Ossabaw swine is characterized by increases in overall OMAT transcript number and provides novel data describing early transcriptomic alterations that occur in response to excess caloric intake in visceral adipose tissue in a pig model of childhood obesity.

Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats.

Here, we sought to compare the efficacy of combining exercise and metformin for the treatment of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) in hyperphagic, obese, type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (age: 20 wk, hyperglycemic and hyperinsulinemic; n = 10/group) were randomly assigned to sedentary (O-SED), SED plus metformin (O-SED + M; 300 mg·kg(-1)·day(-1)), moderate-intensity exercise training (O-EndEx; 20 m/min, 60 min/day, 5 days/wk treadmill running), or O-EndEx + M groups for 12 wk. Long-Evans Tokushima Otsuka (L-SED) rats served as nonhyperphagic controls. O-SED + M, O-EndEx, and O-EndEx + M were effective in the management of type 2 diabetes, and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, O-EndEx lowered liver triglyceride content and fasting hyperglycemia more than O-SED + M. In addition, exercise elicited greater improvements compared with metformin alone on postchallenge glycemic control, liver diacylglycerol content, hepatic mitochondrial palmitate oxidation, citrate synthase, and ß-HAD activities and in the attenuation of markers of hepatic fatty acid uptake and de novo fatty acid synthesis. Surprisingly, combining metformin and aerobic exercise training offered little added benefit to these outcomes, and in fact, metformin actually blunted exercise-induced increases in complete mitochondrial palmitate oxidation and ß-HAD activity. In conclusion, aerobic exercise training was more effective than metformin administration in the management of type 2 diabetes and NAFLD outcomes in obese hyperphagic OLETF rats. Combining therapies offered little additional benefit beyond exercise alone, and findings suggest that metformin potentially impairs exercise-induced hepatic mitochondrial adaptations.

Intrinsic aerobic capacity impacts susceptibility to acute high-fat diet-induced hepatic steatosis.

Aerobic capacity/fitness significantly impacts susceptibility for fatty liver and diabetes, but the mechanisms remain unknown. Herein, we utilized rats selectively bred for high (HCR) and low (LCR) intrinsic aerobic capacity to examine the mechanisms by which aerobic capacity impacts metabolic vulnerability for fatty liver following a 3-day high-fat diet (HFD). Indirect calorimetry assessment of energy metabolism combined with radiolabeled dietary food was employed to examine systemic metabolism in combination with ex vivo measurements of hepatic lipid oxidation. The LCR, but not HCR, displayed increased hepatic lipid accumulation in response to the HFD despite both groups increasing energy intake. However, LCR rats had a greater increase in energy intake and demonstrated greater daily weight gain and percent body fat due to HFD compared with HCR. Additionally, total energy expenditure was higher in the larger LCR. However, controlling for the difference in body weight, the LCR has lower resting energy expenditure compared with HCR. Importantly, respiratory quotient was significantly higher during the HFD in the LCR compared with HCR, suggesting reduced whole body lipid utilization in the LCR. This was confirmed by the observed lower whole body dietary fatty acid oxidation in LCR compared with HCR. Furthermore, LCR liver homogenate and isolated mitochondria showed lower complete fatty acid oxidation compared with HCR. We conclude that rats bred for low intrinsic aerobic capacity show greater susceptibility for dietary-induced hepatic steatosis, which is associated with a lower energy expenditure and reduced whole body and hepatic mitochondrial lipid oxidation.

Selective hepatic insulin resistance in a murine model heterozygous for a mitochondrial trifunctional protein defect.

UNLABELLED: Earlier reports suggest a link between mitochondrial dysfunction and development of hepatic insulin resistance. Here we used a murine model heterozygous (HET) for a mitochondrial trifunctional protein (MTP) gene defect to determine if a primary defect in mitochondrial long-chain fatty acid oxidation disrupts hepatic insulin action. Hyperinsulinemic-euglycemic clamps and signaling studies were performed for assessment of whole-body and hepatic insulin resistance/signaling. In addition, hepatic fatty acid oxidation and hepatic insulin action were assessed in vitro using primary hepatocytes isolated from HET and wildtype (WT) mice. In both hepatic mitochondria and isolated primary hepatocytes, heterozygosity of MTP caused an ∼50% reduction in mitochondrial fatty acid oxidation, a significantly impaired glucose disposal during the insulin clamp, and a markedly lower insulin-stimulated suppression of hepatic glucose production. HET mice also exhibited impaired insulin signaling, with increased hepatic phosphorylation of IRS2 (ser731) and reduced Akt phosphorylation (ser473) in both hepatic tissue and isolated primary hepatocytes. Assessment of insulin-stimulated FOXO1/phospho-FOXO1 protein content and PEPCK/G6Pase messenger RNA (mRNA) expression did not reveal differences between HET and WT mice. However, insulin-induced phosphorylation of GSK3ß was significantly blunted in HET mice. Hepatic insulin resistance was associated with an increased methylation status of the catalytic subunit of protein phosphatase 2A (PP2A-C), but was not associated with differences in hepatic diacylglycerol content, activated protein kinase C-ϵ (PKC-ϵ), inhibitor κB kinase ß (IKK-ß), c-Jun N-terminal kinase (JNK), or phospho-JNK protein contents. Surprisingly, hepatic ceramides were significantly lower in the HET mice compared with WT. CONCLUSION: A primary defect in mitochondrial fatty acid ß-oxidation causes hepatic insulin resistance selective to hepatic glycogen metabolism that is associated with elevated methylated PP2A-C, but independent of other mechanisms commonly considered responsible for insulin resistance. (HEPATOLOGY 2013;).