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.

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.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate post-meal 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-LF, 15g; moderate-MF, 30g; high-HF, 60g). Meals were fed to 12 healthy men (mean±SD, age 31.3±9.2y, BMI 24.5±1.9 kg/m2) during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8h postprandially, 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) quantified by LC-MS. Total plasma TRL-TG concentrations were three-fold 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. Further, 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 pre-study 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.

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This dissertation is focused on factors that increase risk for nonalcoholic fatty liver disease (NALFD) and the more advanced form nonalcoholic steatohepatitis (NASH). These factors include postprandial lipid handling (chapter II); ceramide (CER) turnover (chapter III), hepatic and mitochondrial CER content (chapter III); glucose turnover (chapter IV), de novo lipogenesis (chapter IV), and hepatic fatty acid oxidation (chapter V). With regard to postprandial lipemia, healthy men consumed an oral isotope (2H11-oleate) in liquid meals of varying fat content and labeled triglycerides (TG) tracked into chylomicrons and triglyceride rich lipoproteins (TRL). This method may be applied to future studies of postprandial lipemia. Similar analytical methods (liquid chromatography mass spectrometry) were used to track the synthesis of CERs within hepatic tissues of mice consuming 13C3 15N L-serine dissolved in their water and a high fat (60 percent energy, HFD) or a control (low fat, 20 percentE, CD) diet for two weeks. The mice fed a HFD exhibited greater absolute CER turnover in both whole liver tissue and isolated mitochondria. Furthermore, while total liver concentrations did not differ between diet groups, the HFD elicited greater mitochondrial CER content which was related to total liver CER only in these animals. Plasma CER concentrations were measured in subjects with advanced NASH before and after a nine-month lifestyle program (and a standard care group) and the change in 16:0 CER was negatively related to improvements in liver fat. Similarly, an increase in glucose production and disposal were related negatively to histologic improvements. Together, these data support a hepatic benefit of routing substrates - glucose and lipotoxic lipids -- away from the liver. In addition to testing changes in glucose metabolism, these subjects had significant reductions in lipogenesis but no changes in a non-invasive breath test (13C4 Octanoate) to quantify total hepatic fatty acid oxidation using expelled breath. Correlations between baseline and the change in octanoate oxidation, glucose turnover, and steatosis support the utility of this method to investigate physiological processes that improve hepatic lipid burden. Currently, no drug therapies exist for the treatment of NAFLD or NASH and the results from the current studies support future investigations in identifying key factors for the regression of advanced liver disease through methodological innovation and novel findings with combined intensive lifestyle treatment.

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.

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