Lifestyle treatment in the regression of NASH: insulin resistance, lipid synthesis, and methodological innovation

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.Includes bibliographical references

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