Hepatic malonyl-CoA synthesis restrains gluconeogenesis by suppressing fat oxidation, pyruvate carboxylation, and amino acid availability.

Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition.

Oral gavage delivery of Cornus officinalis extract delays type 1 diabetes onset and hyperglycemia in non-obese diabetic (NOD) mice.

Type 1 diabetes (T1D) is an autoimmune disease initiated by genetic predisposition and environmental influences, which result in the specific destruction of insulin-producing pancreatic ß-cells. Currently, there are over 1.6 million cases of T1D in the United States with a worldwide incidence rate that has been increasing since 1990. Here, we examined the effect of Cornus officinalis (CO), a well-known ethnopharmacological agent, on a T1D model of the non-obese diabetic (NOD) mouse. A measured dose of CO extract was delivered into 10-week-old NOD mice by oral gavage for 15 weeks. T1D incidence and hyperglycemia were significantly lower in the CO-treated group as compared to the water gavage (WT) and a no handling or treatment control group (NHT) following treatment. T1D onset per group was 30%, 60% and 86% for the CO, WT and NHT groups, respectively. Circulating C-peptide was higher, and pancreatic insulitis was decreased in non-T1D CO-treated mice. Our findings suggest that CO may have therapeutic potential as both a safe and effective interventional agent to slow early stage T1D progression.

Persistent fasting lipogenesis links impaired ketogenesis with citrate synthesis in humans with nonalcoholic fatty liver.

BACKGROUNDHepatic de novo lipogenesis (DNL) and ß-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of nonalcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic ß-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL.METHODSForty consecutive nondiabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24 hours, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry.RESULTSWithin a subset of subjects, DNL was detectable after a 24-hour fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic ß-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, non-esterified fatty acid, and ketones. Additionally, they sustained glycogenolysis and were spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis.CONCLUSIONMetabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL.TRIAL REGISTRATIONData were obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease, ClinicalTrials.gov NCT02690792.FUNDINGThis work was supported by the University of Texas Southwestern NORC Quantitative Metabolism Core (NIH P30DK127984), the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (R01DK078184, R01DK128168, R01DK087977, R01DK132254, and K01DK133630), the NIH/National Institute on Alcohol Abuse and Alcoholism (K01AA030327), and the Robert A. Welch Foundation (I-1804).

Proteomic examination of Cornus officinalis stimulated 1.1B4 human pancreatic cells reveals activation of autophagy and Keap1/Nrf2 pathway.

Type 1 diabetes (T1D) is an autoimmune disease initiated by genetic predisposition and environmental influences culminating in the immunologically mediated destruction of pancreatic ß-cells with eventual loss of insulin production. Although T1D can be accurately predicted via autoantibodies, therapies are lacking that can intercede autoimmunity and protect pancreatic ß-cells. There are no approved interventional modalities established for this purpose. One such potential source for clinical agents of this use is from the frequently utilized Cornus officinalis (CO) in the field of ethnopharmacology. Studies by our lab and others have demonstrated that CO has robust proliferative, metabolic, and cytokine protective effects on pancreatic ß-cells. To identify the molecular mechanism of the biological effects of CO, we performed a proteomic and phosphoproteomic analysis examining the cellular networks impacted by CO application on the 1.1B4 pancreatic ß-cell line. Our label-free mass spectrometry approach has demonstrated significant increased phosphorylation of the selective autophagy receptor of p62 (Sequestosome-1/SQSTM1/p62) and predicted activation of the antioxidant Kelch-like ECH-associated protein 1 (Keap1)/Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) pathway. Further validation by immunoblotting and immunofluorescence revealed markers of autophagy such as increased LC3-II and decreased total p62 along with nuclear localization of Nrf2. Both autophagy and the Keap1/Nrf2 pathways have been shown to be impaired in human and animal models of T1D and may serve as an excellent potential therapeutic target stimulated by CO.

Silencing alanine transaminase 2 in diabetic liver attenuates hyperglycemia by reducing gluconeogenesis from amino acids.

Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of 13C-alanine into newly synthesized glucose by hepatocytes. In vivo Gpt2 knockdown or knockout in liver has no effect on glucose concentrations in lean mice, but Gpt2 suppression alleviates hyperglycemia in db/db mice. These data suggest that ALT2 plays a significant role in hepatic gluconeogenesis from amino acids in diabetes.

Hepatic TM6SF2 Is Required for Lipidation of VLDL in a Pre-Golgi Compartment in Mice and Rats.

BACKGROUND & AIMS: Substitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2. METHODS: Two lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins. RESULTS: Tm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2. CONCLUSIONS: TM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B-containing lipoproteins, thus preventing fatty liver disease.

Measurement of lipogenic flux by deuterium resolved mass spectrometry.

De novo lipogenesis (DNL) is disrupted in a wide range of human disease. Thus, quantification of DNL may provide insight into mechanisms and guide interventions if it can be performed rapidly and noninvasively. DNL flux is commonly measured by 2H incorporation into fatty acids following deuterated water (2H2O) administration. However, the sensitivity of this approach is limited by the natural abundance of 13C, which masks detection of 2H by mass spectrometry. Here we report that high-resolution Orbitrap gas-chromatography mass-spectrometry resolves 2H and 13C fatty acid mass isotopomers, allowing DNL to be quantified using lower 2H2O doses and shorter experimental periods than previously possible. Serial measurements over 24-hrs in mice detects the nocturnal activation of DNL and matches a 3H-water method in mice with genetic activation of DNL. Most importantly, DNL is detected in overnight-fasted humans in less than an hour and is responsive to feeding during a 4-h study. Thus, 2H specific MS provides the ability to study DNL in settings that are currently impractical.

Simultaneous tracers and a unified model of positional and mass isotopomers for quantification of metabolic flux in liver.

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of 2H and 13C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from 2H and 13C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1-C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical by 2H or 13C NMR.

Pyruvate-Carboxylase-Mediated Anaplerosis Promotes Antioxidant Capacity by Sustaining TCA Cycle and Redox Metabolism in Liver.

The hepatic TCA cycle supports oxidative and biosynthetic metabolism. This dual responsibility requires anaplerotic pathways, such as pyruvate carboxylase (PC), to generate TCA cycle intermediates necessary for biosynthesis without disrupting oxidative metabolism. Liver-specific PC knockout (LPCKO) mice were created to test the role of anaplerotic flux in liver metabolism. LPCKO mice have impaired hepatic anaplerosis, diminution of TCA cycle intermediates, suppressed gluconeogenesis, reduced TCA cycle flux, and a compensatory increase in ketogenesis and renal gluconeogenesis. Loss of PC depleted aspartate and compromised urea cycle function, causing elevated urea cycle intermediates and hyperammonemia. Loss of PC prevented diet-induced hyperglycemia and insulin resistance but depleted NADPH and glutathione, which exacerbated oxidative stress and correlated with elevated liver inflammation. Thus, despite catalyzing the synthesis of intermediates also produced by other anaplerotic pathways, PC is specifically necessary for maintaining oxidation, biosynthesis, and pathways distal to the TCA cycle, such as antioxidant defenses.

Impaired ketogenesis and increased acetyl-CoA oxidation promote hyperglycemia in human fatty liver.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the U.S. population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, flux through pathways of hepatic oxidative metabolism and gluconeogenesis were studied using five simultaneous stable isotope tracers in ketotic (24-hour fast) individuals with a wide range of hepatic triglyceride contents (0-52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when ß-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is not a consequence of acetyl-CoA production per se, but how acetyl-CoA is further metabolized in liver.

Deep learning application engine (DLAE): Development and integration of deep learning algorithms in medical imaging.

Herein we introduce a deep learning (DL) application engine (DLAE) system concept, present potential uses of it, and describe pathways for its integration in clinical workflows. An open-source software application was developed to provide a code-free approach to DL for medical imaging applications. DLAE supports several DL techniques used in medical imaging, including convolutional neural networks, fully convolutional networks, generative adversarial networks, and bounding box detectors. Several example applications using clinical images were developed and tested to demonstrate the capabilities of DLAE. Additionally, a model deployment example was demonstrated in which DLAE was used to integrate two trained models into a commercial clinical software package.

Fibroblast growth factor 21 increases hepatic oxidative capacity but not physical activity or energy expenditure in hepatic peroxisome proliferator-activated receptor γ coactivator-1α-deficient mice.

NEW FINDINGS: What is the central question of this study? Does a reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been observed in an insulin-resistant obese state, impair the ability of fibroblast growth factor 21 (FGF21) to modulate metabolism? What is the main finding and its importance? A deficit in hepatic PGC-1α does not compromise the ability of FGF21 to increase hepatic fatty acid oxidation; however, the effects of FGF21 to regulate whole-body metabolism (i.e. total and resting energy expenditure), as well as ambulatory activity, were altered when hepatic PGC-1α was reduced. ABSTRACT: Fibroblast growth factor 21 (FGF21) treatment drives metabolic improvements, including increased metabolic flux and reduced hepatic steatosis, but the mechanisms responsible for these effects remain to be elucidated fully. We tested whether a targeted reduction in hepatic peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which has been shown to occur with obesity, had a negative impact on the metabolic effects of FGF21. We infused FGF21 (1 mg kg-1  day-1 ) or saline in chow-fed wild-type (WT) and liver-specific PGC-1α heterozygous (LPGC-1α) mice for 4 weeks. Administration of FGF21 lowered serum insulin and cholesterol (P ≤ 0.05) and tended to lower free fatty acids (P = 0.057). The LPGC-1α mice exhibited reduced complete hepatic fatty acid oxidation (FAO; LPGC-1α, 1788 ± 165 nmol g-1  h-1 compared with WT, 2572 ± 437 nmol g-1  h-1 ; P < 0.001), which was normalized by FGF21 treatment (2788 ± 519 nmol g-1  h-1 ; P < 0.001). FGF21 also increased hepatic incomplete FAO by 12% in both groups and extramitochondrial FAO by 89 and 56% in WT and LPGC-1α mice, respectfully (P = 0.001), and lowered hepatic triacylglycerol by 30-40% (P < 0.001). Chronic treatment with FGF21 lowered body weight and fat mass (P < 0.05), while increasing food consumption (P < 0.05), total energy expenditure [7.3 ± 0.60 versus 6.6 ± 0.39 kcal (12 h)-1 in WT mice; P = 0.009] and resting energy expenditure [5.4 ± 0.89 versus 4.6 ± 0.21 kcal (12 h)-1 in WT mice; P = 0.005]. Interestingly, FGF21 only increased ambulatory activity in the WT mice (P = 0.03), without a concomitant increase in non-resting energy expenditure. In conclusion, although reduced hepatic PGC-1α expression was not necessary for FGF21 to increase FAO, it does appear to mediate FGF21-induced changes in total and resting energy expenditure and ambulatory activity in lean mice.

How Stakeholder Assessment of E-Prescribing Can Help Determine Incentives to Facilitate Management of Care: A Delphi Study.

BACKGROUND: Little research has been conducted on the quality, benefits, costs, and financial considerations associated with health information technology (HIT), particularly informatics technologies such as e-prescribing, from the perspective of all of its stakeholders. OBJECTIVES: To (a) identify the stakeholders involved in e-prescribing and (b) identify and rank order the positives and negatives of e-prescribing from the perspective of stakeholders in order to create a framework for payers, integrated delivery systems, policymakers and legislators, and those who influence public policy to assist them in the development of incentives and payment mechanisms that result in the better management of care. METHODS: The Delphi method was used to enlist a panel of experts in e-prescribing, informatics, and/or HIT who have published in the field. This panel was presented with the results of initial research and an online survey of questions that sought to prioritize the quality, benefit, cost, and financial effects of e-prescribing from the perspective of each stakeholder. Eleven experts completed the first survey, which contained a list of stakeholders and positives and negatives associated with e-prescribing. Nine of the 11 experts completed the second survey, and 7 experts completed the final survey. From the results of these 3 surveys, a framework was presented to 5 framework experts, who were representatives from payers, integrated delivery systems, policymakers and legislators, and those who influence public policy. These framework experts were interviewed regarding the usefulness of the framework from their perspectives. RESULTS: The experts added stakeholders and many positives and negatives to the initial list and rank ordered the positives and negatives of e-prescribing from the perspective of each stakeholder. The aggregate results were summarized by stakeholder category. The positives and negatives were categorized as health, finance, effort, time, management, or data concerns. The framework experts evaluated the framework and found it useful. CONCLUSIONS: Positives and negatives associated with e-prescribing in the areas of quality, benefits, costs, and financial considerations can be rank ordered from the perspective of each stakeholder. The experts agreed on some points but disagreed on others. For example, they agreed that the main negative of e-prescribing from the perspective of pharmacists and pharmacies was its implementation costs but differed on the importance of providing faster information transfer. A framework was created that could be successfully used by payers, integrated delivery systems, policymakers and legislators, and those who influence public policy for the development of incentives and payment mechanisms. DISCLOSURES: This research was supported by the National Library of Medicine of the National Institutes of Health under Award Number T15LM007088. The authors declare no conflicts of interest in the research. Study concept and design were contributed by DeMuro, Ash, Middleton, and Fletcher. DeMuro took the lead in data collection, along with Ash, and data interpretation was performed by DeMuro, Ash, Madison, Middleton, and Fletcher. The manuscript was written primarily by DeMuro, along with Ash and Middleton, and revised by DeMuro, Madison, and Ash, along with Middleton and Fletcher.

Anti-inflammatory effects of exercise training in adipose tissue do not require FGF21.

Exercise enhances insulin sensitivity; it also improves adipocyte metabolism and reduces adipose tissue inflammation through poorly defined mechanisms. Fibroblast growth factor 21 (FGF21) is a pleiotropic hormone-like protein whose insulin-sensitizing properties are predominantly mediated via receptor signaling in adipose tissue (AT). Recently, FGF21 has also been demonstrated to have anti-inflammatory properties. Meanwhile, an association between exercise and increased circulating FGF21 levels has been reported in some, but not all studies. Thus, the role that FGF21 plays in mediating the positive metabolic effects of exercise in AT are unclear. In this study, FGF21-knockout (KO) mice were used to directly assess the role of FGF21 in mediating the metabolic and anti-inflammatory effects of exercise on white AT (WAT) and brown AT (BAT). Male FGF21KO and wild-type mice were provided running wheels or remained sedentary for 8 weeks (n = 9-15/group) and compared for adiposity, insulin sensitivity (i.e., HOMA-IR, Adipo-IR) and AT inflammation and metabolic function (e.g., mitochondrial enzyme activity, subunit content). Adiposity and Adipo-IR were increased in FGF21KO mice and decreased by EX. The BAT of FGF21KO animals had reduced mitochondrial content and decreased relative mass, both normalized by EX. WAT and BAT inflammation was elevated in FGF21KO mice, reduced in both genotypes by EX. EX increased WAT Pgc1alpha gene expression, citrate synthase activity, COX I content and total AMPK content in WT but not FGF21KO mice. Collectively, these findings reveal a previously unappreciated anti-inflammatory role for FGF21 in WAT and BAT, but do not support that FGF21 is necessary for EX-mediated anti-inflammatory effects.

A Quality, Benefit, Cost, and Financial Framework for Health Information Technology, E-Prescribing: A Delphi Study.

BACKGROUND: Little research has been conducted about the quality, benefits, costs, and financial considerations associated with health information technology (HIT), particularly informatics technologies, such as e-prescribing, from the perspective of all its stakeholders. OBJECTIVES: This research effort sought to identify the stakeholders involved in e-prescribing and to identify and rank-order the positives and the negatives from the perspective of the stakeholders to create a framework to assist in the development of incentives and payment mechanisms which result in better managed care. METHODS: The Delphi method was employed by enlisting a panel of experts. They were presented with the results of initial research in an online survey of questions which sought to prioritize the quality, benefit, cost, and financial effects of e-prescribing from the perspective of each stakeholder. From the results of this study, a framework was presented to framework experts. RESULTS: The experts added stakeholders and positives and negatives to the initial lists and rank-ordered the positives and negatives of e-prescribing from the perspective of each stakeholder. The aggregate results were summarized by category of stakeholder. The framework experts evaluated the framework. CONCLUSIONS: Positives and negatives can be rank-ordered from the perspective of each stakeholder. A useful framework was created.

Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis.

KEY POINTS: Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. ABSTRACT: Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1ß) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

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.

A return to ad libitum feeding following caloric restriction promotes hepatic steatosis in hyperphagic OLETF rats.

Hyperphagic Otsuka Long-Evans Tokushima fatty (OLETF) rats develop obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD), but lifestyle modifications, such as caloric restriction (CR), can prevent these conditions. We sought to determine if prior CR had protective effects on metabolic health and NAFLD development following a 4-wk return to ad libitum (AL) feeding. Four-week-old male OLETF rats (n = 8-10/group) were fed AL for 16 wk (O-AL), CR for 16 wk (O-CR; ∼70% kcal of O-AL), or CR for 12 wk followed by 4 wk of AL feeding (O-AL4wk). CR-induced benefit in prevention of NAFLD, including reduced hepatic steatosis, inflammation, and markers of Kupffer cell activation/number, was largely lost in AL4wk rats. These findings occurred in conjunction with a partial loss of CR-induced beneficial effects on obesity and serum triglycerides in O-AL4wk rats, but in the absence of changes in serum glucose or insulin. CR-induced increases in hepatic mitochondrial respiration remained significantly elevated (P < 0.01) in O-AL4wk compared with O-AL rats, while mitochondrial [1-(14)C]palmitate oxidation, citrate synthase activity, and ß-hydroxyacyl-CoA dehydrogenase activity did not differ among OLETF groups. NAFLD development in O-AL4wk rats was accompanied by increases in the protein content of the de novo lipogenesis markers fatty acid synthase and stearoyl-CoA desaturase-1 and decreases in phosphorylated acetyl-CoA carboxylase (pACC)/ACC compared with O-CR rats (P < 0.05 for each). The beneficial effects of chronic CR on NAFLD development were largely lost with 4 wk of AL feeding in the hyperphagic OLETF rat, highlighting the importance of maintaining energy balance in the prevention of NAFLD.