Mitochondrial Fatty Acid Oxidation Research Articles

P-Coumaric Acid Enriched-Peanut Sprout Improves High Fat Diet-Induced Obesity and Insulin Resistance by Inhibiting Macrophage Recruitment

Abstract Objectives We have previously reported that Peanut Sprout Extract (PSE) inhibited adipogenesis through augmentation of mitochondrial fatty acid oxidation. However, it is unknown whether PS consumption reduced visceral obesity and obesity-mediated metabolic complications in vivo. Methods To test this question, obesity-prone C57BL/6 male mice were randomly assigned into three different group; 1) low-fat (LF) diets (11% calories from fat), 2) high-fat (HF) diets (60% calories from fat) or 2) HF diets plus PS (4% in diet, HF-P). Results HF diet for 8 weeks resulted in a significant increase in body weight, liver, and adipose tissue mass compared to LF alone. PS supplementation reduced 1) body weight gain, 2) lipid profile, 3) up-regulated glucose levels, and 4) recruitment of adipose tissue macrophage in HF diet-induced obese C57BL6 mice. In parallel, lipopolysaccharide (LPS)-mediated induction of inflammation was reversed by PSE treatment in macrophages and adipocytes with significant suppression of MAP kinase and NF-kB activation. PSE also reduced LPS-mediated M1 macrophage polarization in bone marrow stem cells. Additionally, we found that, among constitutes of PSE, p-coumaric acid has been identified as responsible polyphenol that exclusively showed a similar trends as PSE. Conclusions Collectively, these data suggest that p-coumaric acid enriched-Peanut Sprout attenuates visceral obesity and obesity-mediated metabolic complications by inhibiting adipose inflammation. Funding Sources This work has supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MSIT).

Association of Microbiome Metabolite Valerobetaine with Aging

Abstract Objectives The gut microbiome changes with age, and rapamycin treatment both delays aging and modulates the microbiome. This raises important questions concerning cause-effect relationships between microbiome, mTOR signaling and aging mechanisms. Microbiome metabolite valerobetaine (VB) disrupts mitochondrial fatty acid oxidation mechanism. Thus, we tested the hypothesis that increase in VB with aging activates mTORC1 and stimulates lung aging. Methods Plasma collected from 179 healthy humans (age; 21 to 75) without known disease (CHDWB, Center for Health Discover and Well Being), and lungs from mice (6 mo and 24 mo, n = 24 each) were analyzed for VB by high resolution metabolomics (HRM). Lung fibroblasts (LF) treated with VB were analyzed for activation of mechanistic target of rapamycin (mTOR) pathway by Western blot analysis. Results VB concentration in human plasma was significantly increased with age (P = 0.0004, r = 0.26). Consistently, lung VB level in old mice were significantly higher than young mice (P = 0.004). LF treated with VB stimulated phosphorylation of mTOR and ribosomal protein S6 as markers for mTORC1 activation, and rapamycin mTORC1 inhibitor substantially decreased VB-stimulated phosphorylation of S6 and mTOR. Conclusions The results show significant association of VB with aging in human and mouse lung studies, and VB-stimulated mTORC1. The finding suggests that microbiome metabolite VB could impact aging and indicates that studies are needed to understand potential dysfunctions related to age and age-associated diseases such as pulmonary fibrosis. Funding Sources NIH R01 ES023485 and S10 OD018006.

Empagliflozin Protects Cardiac Mitochondrial Fatty Acid Metabolism in a Mouse Model of Diet-Induced Lipid Overload

Sodium-glucose cotransporter 2 (SGLT2) inhibitors prevent heart failure and decrease cardiovascular mortality in patients with type 2 diabetes. Heart failure is associated with detrimental changes in energy metabolism, and the preservation of cardiac mitochondrial function is crucial for the failing heart. However, to date, there are no data to support the hypothesis that treatment with a SGLT2 inhibitor might alter mitochondrial bioenergetics in diabetic failing hearts. Thus, the aim of this study was to investigate the protective effects of empagliflozin on mitochondrial fatty acid metabolism. Mitochondrial dysfunction was induced by 18weeks of high-fat diet (HFD)-induced lipid overload. Empagliflozin was administered at a dose of 10mg/kg in a chow for 18weeks. Palmitate metabolism in vivo, cardiac mitochondrial functionality and biochemical parameters were measured. In HFD-fed mice, palmitate uptake was 1.7, 2.3, and 1.9 times lower in the heart, liver, and kidneys, respectively, compared with that of the normal chow control group. Treatment with empagliflozin increased palmitate uptake and decreased the accumulation of metabolites of incomplete fatty acid oxidation in cardiac tissues, but not other tissues, compared with those of the HFD control group. Moreover, empagliflozin treatment resulted in fully restored fatty acid oxidation pathway-dependent respiration in permeabilized cardiac fibers. Treatment with empagliflozin did not affect the biochemical parameters related to hyperglycemia or hyperlipidemia. Empagliflozin treatment preserves mitochondrial fatty acid oxidation in the heart under conditions of chronic lipid overload.

Hepatic steatosis induced in C57BL/6 mice by a non-\xdf oxidizable fatty acid analogue is associated with reduced plasma kynurenine metabolites and a modified hepatic NAD+/NADH ratio

BackgroundNon-alcoholic fatty liver disease is often associated with obesity, insulin resistance, dyslipidemia, and the metabolic syndrome in addition to mitochondrial dysfunction and nicotinamide adenine dinucleotide (NAD+) deficiency. The aim of this study was to investigate how inhibition of mitochondrial fatty acid oxidation using the compound tetradecylthiopropionic acid (TTP) would affect hepatic triacylglycerol level and plasma levels of kynurenine (Kyn) metabolites and nicotinamide.Methods12 C57BL/6 mice were fed a control diet, or an intervention diet supplemented with 0.9% (w/w) tetradecylthiopropionic acid for 14 days. Blood and liver samples were collected, enzyme activities and gene expression were analyzed in liver, in addition to fatty acid composition. Metabolites in the tryptophan/kynurenine pathway and total antioxidant status were measured in plasma.ResultsDietary treatment with tetradecylthiopropionic acid for 2 weeks induced fatty liver accompanied by decreased mitochondrial fatty acid oxidation. The liver content of the oxidized form of NAD+ was increased, as well as the ratio of NAD+/NADH, and these changes were associated by increased hepatic mRNA levels of NAD synthetase and nicotinamide mononucleotide adenyltransferase-3. The downstream metabolites of kynurenine were reduced in plasma whereas the plasma nicotinamide content was increased. Some effects on inflammation and oxidative stress was observed in the liver, while the plasma antioxidant capacity was increased. This was accompanied by a reduced plasma ratio of kynurenine/tryptophan. In addition, a significant decrease in the inflammation-related arachidonic fatty acid in liver was observed.ConclusionFatty liver induced by short-time treatment with tetradecylthiopropionic acid decreased the levels of kynurenine metabolites but increased the plasma levels of NAD+ and nicotinamide. These changes are most likely not associated with increased inflammation and oxidative stress. Most probably the increase of NAD+ and nicotinamide are generated through the Preiss Handler pathway and/or salvage pathway and not through the de novo pathway.The take home message is that non-alcoholic fatty liver disease is associated with the metabolic syndrome in addition to mitochondrial dysfunction and nicotinamide adenine dinucleotide (NAD+) deficiency. Inducing fatty liver in mice by inhibition of fatty acid oxidation resulted in a concomitant change in kynurenine metabolites increasing the plasma levels of nicotinamides and the hepatic NAD+/NADH ratio, probably without affecting the de novo pathway of kynurenines.

Improvement of steatotic rat liver function with a defatting cocktail during ex situ normothermic machine perfusion is not directly related to liver fat content.

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a “defatting” drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation.

HELLP Syndrome or Acute Fatty Liver of Pregnancy: A Differential Diagnostic Challenge: Common Features and Differences.

HELLP syndrome and the less common acute fatty liver of pregnancy (AFL) are unpredictable, life-threatening complications of pregnancy. The similarities in their clinical and laboratory presentations are often challenging for the obstetrician when making a differential diagnosis. Both diseases are characterised by microvesicular steatosis of varying degrees of severity. A specific risk profile does not exist for either of the entities. Genetic defects in mitochondrial fatty acid oxidation and multiple pregnancy are considered to be common predisposing factors. The diagnosis of AFL is based on a combination of clinical symptoms and laboratory findings. The Swansea criteria have been proposed as a diagnostic tool for orientation. HELLP syndrome is a laboratory diagnosis based on the triad of haemolysis, elevated aminotransferase levels and a platelet count < 100 G/l. Generalised malaise, nausea, vomiting and abdominal pain are common symptoms of both diseases, making early diagnosis difficult. Clinical differences include a lack of polydipsia/polyuria in HELLP syndrome, while jaundice is more common and more pronounced in AFL, there is a lower incidence of hypertension and proteinuria, and patients with AFL may develop encephalopathy with rapid progression to acute liver failure. In contrast, neurological symptoms such as severe headache and visual disturbances are more prominent in patients with HELLP syndrome. In terms of laboratory findings, AFL can be differentiated from HELLP syndrome by the presence of leucocytosis, hypoglycaemia, more pronounced hyperbilirubinemia, an initial lack of haemolysis and thrombocytopenia < 100 G/l, as well as lower antithrombin levels < 65% and prolonged prothrombin times. While HELLP syndrome has a fluctuating clinical course with rapid exacerbation within hours or transient remissions, AFL rapidly progresses to acute liver failure if the infant is not delivered immediately. The only causal treatment for both diseases is immediate delivery. Expectant management between 24 + 0 and 33 + 6 weeks of gestation is recommended for HELLP syndrome, but only in cases where the mother can be stabilised and there is no evidence of foetal compromise. The maternal mortality rate for HELLP syndrome in developed countries is approximately 1%, while the rate for AFL is 1.8 – 18%. Perinatal mortality rates are 7 – 20% and 15 – 20%, respectively. While data on the long-term impact of AFL on the health of mother and child is still insufficient, HELLP syndrome is associated with an increased risk of developing cardiovascular, metabolic and neurological diseases in later life.

Consumption of the fish oil high fat diet uncouples obesity and tumor growth through induction of reactive oxygen species in pro-tumor macrophages

Abstract It is clear that obesity is associated with increased risk of many types of cancer. However, obesity can be induced by various high fat diets (HFDs) from different fat sources. It remains unknown whether fatty acid composition in different HFDs influences obesity-associated tumor development. Herein we report that consumption of either a cocoa butter or fish oil HFD induced similar obesity in mouse models. While obesity induced by the cocoa butter HFD was associated with accelerated tumor growth, consumption of the fish oil HFD uncoupled obesity from increased tumor growth and exhibited a decrease in pro-tumor macrophages. Interestingly, compared to FA components in both HFDs, n-3 FAs rich in the fish oil HFD induced significant production of reactive oxygen species (ROS) and macrophage cell death. Moreover, we found that A-FABP expression in the pro-tumor macrophages facilitated n-3 FA intracellular transportation and mitochondrial FA oxidation. A-FABP deficiency diminished n-3 FA-mediated ROS production and macrophage cell death both in vitro and in vivo. Altogether, our results demonstrate a novel mechanism by which n-3 FAs induce ROS-mediated pro-tumor macrophage cell death in an A-FABP dependent manner. This study provides mechanistic insights into dietary supplementation with fish oil for cancer prevention and puts forwards a new concept that not all HFDs leading to obesity are tumorigenic.

Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids

Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway.

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.

Primrose syndrome: Characterization of the phenotype in 42 patients.

Primrose syndrome (PS; MIM# 259050) is characterized by intellectual disability (ID), macrocephaly, unusual facial features (frontal bossing, deeply set eyes, down‐slanting palpebral fissures), calcified external ears, sparse body hair and distal muscle wasting. The syndrome is caused by de novo heterozygous missense variants in ZBTB20. Most of the 29 published patients are adults as characteristics appear more recognizable with age. We present 13 hitherto unpublished individuals and summarize the clinical and molecular findings in all 42 patients. Several signs and symptoms of PS develop during childhood, but the cardinal features, such as calcification of the external ears, cystic bone lesions, muscle wasting, and contractures typically develop between 10 and 16 years of age. Biochemically, anemia and increased alpha‐fetoprotein levels are often present. Two adult males with PS developed a testicular tumor. Although PS should be regarded as a progressive entity, there are no indications that cognition becomes more impaired with age. No obvious genotype‐phenotype correlation is present. A subgroup of patients with ZBTB20 variants may be associated with mild, nonspecific ID. Metabolic investigations suggest a disturbed mitochondrial fatty acid oxidation. We suggest a regular surveillance in all adult males with PS until it is clear whether or not there is a truly elevated risk of testicular cancer.

The effect of SERCA2 activation on liver metabolism during acute lipotoxicity in vivo

Non‐alcoholic fatty liver disease (NAFLD) is the most common form of liver disease affecting about 30% of the US population. The prognosis for simple liver steatosis is relatively benign; however, chronic inflammation and progressive fibrosis may lead to nonalcoholic steatohepatitis (NASH) and later to cirrhosis and hepatocellular carcinoma. There is currently no established treatment to stop progression from NAFLD to NASH. It was previously shown (Egnatchik RA et al. Mol Metab. 2014; 3 (5): 544–53) that lipotoxic concentrations of fatty acids alter endoplasmic reticulum (ER) calcium stores and induce mitochondrial dysfunction, characterized by elevated citric acid cycle (CAC) flux and reactive oxygen species accumulation, leading to apoptosis. Furthermore, the function of SERCA2 (sarco/endoplasmic reticulum Ca2+‐ATPase), the main protein in hepatocytes that is responsible for transporting Ca2+ from cytoplasm to ER, is impaired in obese mice (Fu S et al. Nature. 2011; 473 (7348): 528–31). We thus hypothesize that restoration of SERCA2 activity might improve mitochondrial function in hepatocytes during acute lipotoxicity.To test this hypothesis, we developed a protocol to investigate the acute impact of fatty acids on liver energy metabolism. Conscious, unrestrained, overnight‐fasted, 12‐week‐old male C57Bl/6J mice were given continuous, intravenous infusions of either saline (control) or 20% lard oil (as described in Stein DT et al. J Clin Invest. 1997; 100 (2): 398–403) for 5 hrs to induce hepatic lipotoxicity. A third group of animals was given lard oil in combination with single intraperitoneal injection of small molecular allosteric activator CDN1163 (50 mg/kg) one week prior to study to activate SERCA2 (Kang S et al. J Biol Chem. 2016; 291 (10): 5185–98). Animals were simultaneously administered a primed, continuous infusion of [6,6‐2H2] glucose and sodium [13C3]lactate intravenously for 4 and 2 hrs, respectively, to assess in vivo liver metabolic fluxes. 13C and 2H‐enrichment of plasma glucose and liver alanine, glutamate, lactate and urea obtained at the end of the study were measured by GC‐MS and used to regress liver metabolic fluxes using a flexible modeling platform (INCA).Our analysis showed that by the end of the experiment both plasma and liver free fatty acids were significantly elevated after lard oil infusion, whereas liver triglycerides and plasma glucose were not affected, regardless of CDN1163 treatment. In contrast, CDN1163 significantly increased alanine aminotransferase and aspartate transaminase content in liver, and restored basal insulin level, which was significantly downregulated in the presence of lard oil alone. Analysis of liver metabolic fluxes revealed that lard oil increased fatty acid oxidation flux. On the other hand, lard oil coupled with CDN1163 led to further upregulation of fatty acid oxidation flux, but at the same time hepatocytes were characterized by increased endogenous glucose production, elevated pyruvate cycling and upregulated CAC activity. These data suggest that SERCA2 activation increases fatty acid oxidation in mitochondria, but at the same time proportionally increases other hepatic fluxes, which might restore energy balance and protect liver during acute lipotoxicity.Support or Funding InformationThis research was supported by NIH grant R01 DK106348.

Proximal tubular mitochondrial substrate overload is an independent factor in developing kidney disease

Alterations in mitochondrial function are linked to the development of chronic and diabetic kidney diseases (CKD/DKD). Proximal tubular epithelial cells (PTCs) are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. It is suggested, but not entirely clear whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage. Here we modeled mitochondrial substrate overload ‐ an important aspect of metabolic disease and diabetes ‐ by creating mice lacking the enzyme carnitine acetyl‐transferase (CrAT) in the PTC. CrAT is crucial in serving as a “relief valve”, linking excess acetyl‐CoA to carnitine and allowing transport from the mitochondria. Deletion of CrAT therefore models a scenario of substrate excess where the export of carbons is limited. Mice developed tubular disease and interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis and fibrosis markers, increased oxidative stress and abnormal profiles of acyl carnitines/organic acids including those derived from branch chain amino acid and glucose metabolism. These results indicate that during mitochondrial overload in the kidney cortex, all three major forms of metabolic pathways are impacted. When mice with CrAT deletion were placed on a high fat diet, kidney disease was more severe and developed faster. Primary PTCs isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology. Next generation sequencing analysis from cortices at different time points revealed that dysregulation of genes related to mitochondrial function precede development of pathology. Mitochondrial overload also led to the enrichment of genes that are deregulated in CKD/DKD. Importantly, mice with heterozygous CrAT deletion in the PTC also developed kidney disease, suggesting that even moderate impairments in substrate efflux impede PTC function. Our findings support the hypothesis that derailments of mitochondrial substrate metabolism in PTC alone may be causative to CKD. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell metabolism could be an important preventative strategy.Support or Funding InformationThis research is supported by NIH grants R01‐DK115749‐01A1 (to KS) and R01‐DK103860 (to RN)

Cyclophilin D Sequesters PPARα in Mitochondria to Impair Fatty Acid Oxidation in Cisplatin‐induced Acute Kidney Injury

ObjectiveKidney is a highly metabolic organ and ATP depletion is critical to acute kidney injury (AKI). Mitochondrial fatty acid oxidation (FAO) is the preferred energy source of the kidney and is inhibited during AKI. A pivotal role for the mitochondrial matrix protein cyclophilin D (CypD) in regulating overall energy metabolism is being unraveled. We hypothesize that mitochondrial interaction between CypD and PPARα in renal proximal tubule (PT) where is highly susceptible to acute stimuli modulates FAO in cisplatin‐induced AKI (cisplatin AKI).MethodsUsing genetic and pharmacological intervention and protein‐protein interaction studies, we investigated whether PT‐CypD modulates FAO in cisplatin AKI through mitochondrial CypD‐PPARα binding and its sequestration.ResultsCisplatin injury resulted in histological and functional damage in the kidney, along with downregulated mitochondrial FAO, increased intrarenal lipid accumulation and declined ATP level. However, PT‐specific deletion of CypD protected the malevolent effects. Immunoprecipitation and BioID methods demonstrated mitochondrial translocation of PPARα and its binding to CypD and sequestration. This led to inhibition of nuclear translocation of PPARα and reduced transcription of FAO genes during cisplatin AKI. Genetic or pharmacological inhibition of CypD suppressed mitochondrial CypD‐PPARα binding in cisplatin AKI, preventing the impairment of FAO and intracellular lipid accumulation.ConclusionsThese results uncover a novel mechanism by which mitochondrial interaction between CypD and PPARα impairs FAO in cisplatin AKI. Targeting their interaction may be a potential therapeutic strategy to prevent energy depletion and cell death in AKI.Support or Funding InformationThis work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).

Local and systemic actions of hepatic fatty acid oxidation

Mitochondrial long chain fatty acid beta‐oxidation is required for hepatic ketogenesis and gluconeogensis during fasting, starvation and times of low carbohydrate intake. The liver has a large capacity and dynamic range for fatty acid oxidation to facilitate ketogenesis and gluconeogenesis. To gain a better understanding of the roles of hepatic fatty acid oxidation on liver function, we generated mice with a hepatocyte‐specific knockout of Carnitine Palmitoyltransferase 2 (Cpt2L−/−), an obligate step in mitochondrial long‐chain fatty acid β‐oxidation. Mice with a loss in hepatic fatty acid oxidation were able to maintain systemic glucose homeostasis during a 24hr fast, but did not produce ketone bodies. Systemic energy homeostasis was largely maintained by adaptations in hepatic and systemic oxidative gene expression mediated in part by a robust hepatic transcriptional response. Open chromatin assessed by ATAC‐seq and histone H3K9 ChIP‐seq analysis revealed that many genes induced in fasted Cpt2L−/− mice exhibited Pparα regulatory sites. To understand the contribution of Pparα, we generated Pparα;Cpt2L−/− double knockout mice. The deletion of Pparα in Cpt2L−/− mice largely blunted this transcriptional response, demonstrating the requirement for Pparα. Triglyceride hydrolysis has been shown to be critical for Pparα‐mediated transcriptional control. To determine the requirement of hepatic triglyceride hydrolysis on the Cpt2L−/− transcriptional response, mice were generated with a liver‐specific defect in triglyceride hydrolysis (AtglL−/−) and fatty acid oxidation (AtglL−/−;Cpt2L−/− double knockout). The loss of Cpt2 or Atgl resulted in largely independent effects on hepatocyte morphology, intermediary metabolism and gene expression in response to fasting. However, high fat feeding revealed the interaction of Atgl and Cpt2 as the loss of only one of these genes results in steatosis but the loss of both components resulted in significant inflammation and fibrosis. These data suggest that defects in mitochondrial fatty acid oxidation induce an exaggerated fasting response that initiates lipid dependent transcription to mediate changes in local and systemic physiology.Support or Funding InformationThis work was supported in part by a National Institutes of Health grant R01DK116746 to M.J.W.

Cardiomyocyte‐Specific Deletion of GCN5L1 Limits Cardiac Contractile Decline in High Fat Diet Fed Mice

Healthy hearts use several fuel substrates for energy metabolism, with fatty acid oxidation (FAO) accounting for ~70% of total ATP production. Reduced substrate flexibility, and a shift towards increased fatty acid utilization, is a key feature of obesity and diabetes. We previously reported that increased acetylation of several mitochondrial FAO enzymes, regulated in part by increased abundance of the mitochondrial acetyltransferase GCN5L1, correlated with increased FAO enzyme activity in the heart. The focus of the current study was to investigate whether decreased acetylation, via cardiomyocyte‐specific deletion of GCN5L1 (GCN5L1 cKO), regulates cardiac energy metabolism following exposure to a high fat diet (HFD). Excess dietary fat led to similar cardiac hypertrophy in wildtype and GCN5L1 cKO mice, however animals lacking cardiac GCN5L1 expression were protected from a reduction in cardiac workload. We found that GCN5L1 deletion led to decreased acetylation and FAO activity following a HFD. Furthermore, we found that acetylation of pyruvate dehydrogenase (PDH) was significantly reduced in HFD GCN5L1 cKO animals, which resulted in its increased enzymatic activity relative to HFD wildtype controls. Finally, the acetylation of both electron transport chain Complex I protein NDUFB8 and manganese superoxide dismutase 2 (SOD2) was significantly reduced by cardiac GCN5L1 deletion in HFD animals, resulting in decreased lipid peroxidation. In summary, we demonstrate that GCN5L1 deletion in the heart limits cardiac contractile decline observed in HFD mice, through increased glucose oxidation potential and decreased reactive oxygen species damage.Support or Funding InformationThis work was supported by NIH T32HL110849 to D.T. and J.R.M., and by NIH K22HL116728, R56HL132917, R01HL132917 to I.S.

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.

Nitrosative Stress and Lipid Homeostasis as a Mechanism for Zileuton Hepatotoxicity and Resistance in Genetically Sensitive Mice.

Zileuton is an orally active inhibitor of leukotriene synthesis for maintenance treatment of asthma, for which clinical usage has been associated with idiosyncratic liver injury. Mechanistic understanding of zileuton toxicity is hampered by the rarity of the cases and lack of an animal model. A promising model for mechanistic study of rare liver injury is the Diversity Outbred (J:DO) mouse population, with genetic variation similar to that found in humans. In this study, female DO mice were administered zileuton or vehicle daily for 7 days (i.g.). Serum liver enzymes were elevated in the zileuton group, with marked interindividual variability in response. Zileuton exposure-induced findings in susceptible DO mice included microvesicular fatty change, hepatocellular mitosis, and hepatocellular necrosis. Inducible nitric oxide synthase and nitrotyrosine abundance were increased in livers of animals with necrosis and those with fatty change, implicating nitrosative stress as a possible injury mechanism. Conversely, DO mice lacking adverse liver pathology following zileuton exposure experienced decreased hepatic concentrations of resistin and increased concentrations of insulin and leptin, providing potential clues into mechanisms of toxicity resistance. Transcriptome pathway analysis highlighted mitochondrial dysfunction and altered fatty acid oxidation as key molecular perturbations associated with zileuton exposure, and suggested that interindividual differences in cytochrome P450 metabolism, glutathione-mediated detoxification, and farnesoid X receptor signaling may contribute to zileuton-induced liver injury (ZILI). Taken together, DO mice provided a platform for investigating mechanisms of toxicity and resistance in context of ZILI which may lead to targeted therapeutic interventions.

Maternal Fat-1 Transgene Protects Offspring from Excess Weight Gain, Oxidative Stress, and Reduced Fatty Acid Oxidation in Response to High-Fat Diet.

Overweight and obesity accompanies up to 70% of pregnancies and is a strong risk factor for offspring metabolic disease. Maternal obesity-associated inflammation and lipid profile are hypothesized as important contributors to excess offspring liver and skeletal muscle lipid deposition and oxidative stress. Here, we tested whether dams expressing the fat-1 transgene, which endogenously converts omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acid, could protect wild-type (WT) offspring against high-fat diet induced weight gain, oxidative stress, and disrupted mitochondrial fatty acid oxidation. Despite similar body mass at weaning, offspring from fat-1 high-fat-fed dams gained less weight compared with offspring from WT high-fat-fed dams. In particular, WT males from fat-1 high-fat-fed dams were protected from post-weaning high-fat diet induced weight gain, reduced fatty acid oxidation, or excess oxidative stress compared with offspring of WT high-fat-fed dams. Adult offspring of WT high-fat-fed dams exhibited greater skeletal muscle triglycerides and reduced skeletal muscle antioxidant defense and redox balance compared with offspring of WT dams on control diet. Fat-1 offspring were protected from the reduced fatty acid oxidation and excess oxidative stress observed in offspring of WT high-fat-fed dams. These results indicate that a maternal fat-1 transgene has protective effects against offspring liver and skeletal muscle lipotoxicity resulting from a maternal high-fat diet, particularly in males. Altering maternal fatty acid composition, without changing maternal dietary composition or weight gain with high-fat feeding, may highlight important strategies for n-3-based prevention of developmental programming of obesity and its complications.

Defective Mitochondrial Fatty Acid Oxidation and Lipotoxicity in Kidney Diseases

The kidney is a highly metabolic organ and uses high levels of ATP to maintain electrolyte and acid-base homeostasis and reabsorb nutrients. Energy depletion is a critical factor in development and progression of various kidney diseases including acute kidney injury (AKI), chronic kidney disease (CKD), and diabetic and glomerular nephropathy. Mitochondrial fatty acid β-oxidation (FAO) serves as the preferred source of ATP in the kidney and its dysfunction results in ATP depletion and lipotoxicity to elicit tubular injury and inflammation and subsequent fibrosis progression. This review explores the current state of knowledge on the role of mitochondrial FAO dysfunction in the pathophysiology of kidney diseases including AKI and CKD and prospective views on developing therapeutic interventions based on mitochondrial energy metabolism.

Acute Fatty Liver of Pregnancy

Abstract Acute fatty liver of pregnancy (AFLP) is a rare but life-threatening condition that develops in the third trimester of pregnancy. AFLP shares similar clinical features with other more common pregnancy-associated conditions. However, early correct diagnosis is important for maternal and fetal survival. Once the diagnosis has been established, immediate delivery and maternal intensive support should be undertaken. Both parents and the infant should be tested for deficiencies of the mitochondrial fatty acid oxidation, especially for long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, as many cases of AFLP are related to disruption of this physiological enzymatic pathway.