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.

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis.

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered 13C3, 15N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.

High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

Understanding the variation in exercise responses to guide personalized physical activity prescriptions.

Understanding the factors that contribute to exercise response variation is the first step in achieving the goal of developing personalized exercise prescriptions. This review discusses the key molecular and other mechanistic factors, both extrinsic and intrinsic, that influence exercise responses and health outcomes. Extrinsic characteristics include the timing and dose of exercise, circadian rhythms, sleep habits, dietary interactions, and medication use, whereas intrinsic factors such as sex, age, hormonal status, race/ethnicity, and genetics are also integral. The molecular transducers of exercise (i.e., genomic/epigenomic, proteomic/post-translational, transcriptomic, metabolic/metabolomic, and lipidomic elements) are considered with respect to variability in physiological and health outcomes. Finally, this review highlights the current challenges that impede our ability to develop effective personalized exercise prescriptions. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) aims to fill significant gaps in the understanding of exercise response variability, yet further investigations are needed to address additional health outcomes across all populations.

Labeled breath tests in patients with NASH: Octanoate oxidation relates best to measures of glucose metabolism.

In vivo methods to estimate human liver mitochondrial activity are lacking and this project's goal was to use a non-invasive breath test to quantify complete mitochondrial fat oxidation and determine how test results changed when liver disease state was altered over time. Patients with suspected non-alcoholic fatty liver disease (NAFLD; 9 men, 16 women, 47 ± 10 years, 113 ± 23 kg) underwent a diagnostic liver biopsy and liver tissue was histologically scored by a pathologist using the NAFLD activity score (0-8). To assess liver oxidation activity, a labeled medium chain fatty acid was consumed orally (23.4 mg 13C4-octanoate) and breath samples collected over 135 min. Total CO2 production rates were measured using breath 13CO2 analysis by isotope ratio mass spectrometry. Fasting endogenous glucose production (EGP) was measured using an IV infusion of 13C6-glucose. At baseline, subjects oxidized 23.4 ± 3.9% (14.9%-31.5%) of the octanoate dose and octanoate oxidation (OctOx) was negatively correlated with fasting plasma glucose (r = -0.474, p = 0.017) and EGP (r = -0.441, p = 0.028). Twenty-two subjects returned for repeat tests 10.2 ± 1.0 months later, following lifestyle treatment or standardized care. OctOx (% dose/kg) was significantly greater across all subjects (p = 0.044), negatively related to reductions in EGP (r = -0.401, p = 0.064), and tended to correlate with reduced fasting glucose (r = -0.371, p = 0.090). Subjects exhibited reductions in steatosis (p = 0.007) which tended to correlate with increased OctOx (% of dose/kg, r = -0.411, p = 0.058). Based on our findings, the use of an 13C-octanoate breath test may be an indicator of hepatic steatosis and glucose metabolism, but these relationships require verification through larger studies in NAFLD populations.

The Tailgate Study: Differing metabolic effects of a bout of excessive eating and drinking.

INTRODUCTION: Excess energy intake by spectators at a sporting event (i.e., a tailgate) might cause acute negative health effects. However, limited data exist regarding the effects of overeating and alcohol consumption on lipid metabolism and the potential to gain intrahepatic triacylglycerols (IHTG). We tested the hypothesis that overconsumption of food and alcohol would significantly increase both hepatic de novo lipogenesis (DNL) and IHTG. METHODS: Eighteen males (mean ± SD, age: 31.4 ± 7.3 years, BMI: 32.1 ± 5.9 kg/m2) were given alcoholic drinks to elevate blood alcohol for 5 h, while highly palatable food was presented. Blood samples were collected and DNL in TG-rich lipoproteins (TRL) was measured by GC/MS, IHTG was measured via MRS (n = 15), and substrate oxidation was measured via indirect calorimetry. RESULTS: Subjects consumed 5087 ± 149 kcal (191 ± 25% excess of total daily energy needs including 171 ± 24 g alcohol), which increased plasma insulin, glucose, TG, and decreased NEFA (ANOVA p ≤ 0.003 for all). Both DNL and TRL-TG increased (p < 0.001), while IHTG did not change in the group as a whole (p = 0.229). Individual subject data revealed remarkably differing responses for IHTG (nine increased, five decreased, one did not change). Despite maintaining equal breath alcohol levels, subjects with IHTG elevations exhibited higher DNL, consumed 90% less alcohol (p = 0.048), tended to consume more carbohydrates, and exhibited lower whole-body fat oxidation (not significant) compared to those whose IHTG was reduced. DISCUSSION: This study demonstrates that acute excess energy intake may have differing effects on an individual's DNL and IHTG, and dietary carbohydrate may influence DNL more than alcohol.

Relationships between Very Low-Density Lipoproteins-Ceramides, -Diacylglycerols, and -Triacylglycerols in Insulin-Resistant Men.

This short report describes the relationships between concentrations of ceramides (CER), diacylglycerols (DAG), triacylglycerols (TAG) in very low-density lipoproteins (VLDL) particles, and hepatic lipid accumulation. VLDL particles were isolated from male subjects (n = 12, mean ± SD, age 42.1 ± 5.4 years, BMI 37.4 ± 4.1 kg/m2 , ALT 45 ± 21 U/L) and apolipoprotein B100 (apoB100), VLDL-TAG, -CER, and -DAG quantified. The contents of all three lipids were highly correlated with VLDL particle number (r ≥ 0.768, p ≤ 0.003). The molar quantity of VLDL-TAG was 3× that of DAG and 137× that of CER (14,053 ± 5714, 5004 ± 2714, and 105 ± 49 mol/mol apoB100, respectively). Reduced VLDL-CER concentrations were associated with both higher insulin levels (r = -0.645, p = 0.024) and intrahepatic-TAG (r = -0.670, p = 0.017). In fatty liver, the secretion of hepatic TAG, CER, and DAG may be suppressed and contribute to intrahepatic lipotoxicity. The mechanisms by which hepatic-CER and -DAG synthesis and assembly into VLDL is coordinately controlled with TAG will be important in understanding the emerging role of elevated CER contributing to cardiometabolic disease.

A Fad too Far? Dietary Strategies for the Prevention and Treatment of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem, and its prevalence has increased in recent years, concurrent with rising rates of obesity and other metabolic diseases. Currently, there are no FDA-approved pharmacological therapies for NAFLD, and lifestyle interventions, including weight loss and exercise, remain the cornerstones for treatment. Manipulating diet composition and eating patterns may be a sustainable approach to NAFLD treatment. Dietary strategies including Paleolithic, ketogenic, Mediterranean, high-protein, plant-based, low-carbohydrate, and intermittent fasting diets have become increasingly popular because of their purported benefits on metabolic disease. This review highlights what is currently known about these popular dietary approaches in the management of NAFLD in clinical populations with mechanistic insight from animal studies. It also identifies key knowledge gaps to better inform future preclinical and clinical studies aimed at the treatment of NAFLD.