京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19367号 / 医博第4044号 / 新制||医||1011(附属図書館) / 32381 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 佐藤 俊哉, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) / Risk of coronary artery disease (CAD), the leading cause of death, greatly increases in metabolic syndrome. Metabolic syndrome (MetS; obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension) is increasing in prevalence with sedentary lifestyles and poor nutrition. Non-alcoholic steatohepatitis (NASH; i.e. MetS liver) is progressive and decreases life expectancy, with CAD as the leading cause of death. Pathogenic Ca2+ regulation transforms coronary artery smooth muscle from a healthy, quiescent state to a diseased, proliferative phenotype thus majorly contributing to the development of CAD. In particular, store-operated Ca2+ entry (SOCE) in vascular smooth muscle is associated with atherosclerosis. Genetic predisposition may render individuals more susceptible to Ca2+ dysregulation, CAD, NASH, and MetS. However, the metabolic and cellular mechanisms underlying these disease states are poorly understood. Accordingly, the goal of this dissertation was to investigate the role of dyslipidemia within MetS in the development of Ca2+ dysregulation, CAD, and NASH. The overarching hypothesis was that dyslipidemia within MetS would be necessary for induction of NASH and increased SOCE that would primarily mediate development of CAD. To test this hypothesis we utilized the Ossabaw miniature swine model of MetS. Swine were fed one of five diets for different lengths of time to induce varying severity of MetS. Lean swine were fed normal maintenance chow diet. F/MetS swine were fed high Fructose (20% kcal) diet that induced normolipidemic MetS. TMetS were fed excess high Trans-fat/cholesterol atherogenic diet that induced mildly dyslipidemic MetS and CAD. XMetS were TMetS swine with eXercise. DMetS (TMetS + high fructose) were moderately dyslipidemic and developed MetS and extensive CAD. sDMetS (Short-term DMetS) developed MetS with mild dyslipidemia, but no CAD. MMetS (Mixed-source-fat/cholesterol/fructose) were severely dyslipidemic, exhibited NASH, and developed severe CAD. Dyslipidemia in MetS predicted NASH severity (all groups < DMetS << MMetS), CAD severity (i.e. Lean, F/MetS, sDMetS < XMetS < TMetS < DMetS < MMetS), and was necessary for STIM1/TRPC1-mediated SOCE, which preceded CAD. Exercise ameliorated SOCE and CAD compared to TMetS. In conclusion, dyslipidemia elicits TRPC1/STIM1 SOCE that mediates CAD, is necessary for and predictive of NASH and CAD, and whose affects are attenuated by exercise.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24880号 / 医博第5014号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 江木 盛時, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Lifestyle modifications, including calorie deficit diets targeting weight loss, are the first line treatment and prevention measures for the development and progression of the emerging cardiometabolic disease, nonalcoholic fatty liver disease (NAFLD), particularly early in the disease course when interventions may alter the natural history. However, the optimal composition of eating patterns to protect liver health is still under debate. High-quality diets with anti-inflammatory, antioxidant, and anti-obesity effects have the potential to play an important role in NAFLD occurrence; however, evidence is still limited. Dietary Approaches to Stop Hypertension (DASH) and a Mediterranean-style diet are two major healthy dietary patterns that have been extensively reported to prevent cardiometabolic outcomes such as hypertension (HTN) and cardiovascular disease (CVD), major concomitant diseases of NAFLD. A few indexes have been developed to measure adherence to both of these dietary patterns in observational studies. However, none of the DASH indexes have been updated with current evidence in more than a decade. Research suggests that greater reductions in cardiometabolic disorders are possible with more updated guidance. In addition to the impact of overall diet quality, the effects of specific foods on liver fat are still unknown, and some, such as eggs, are controversial. Until recently, eggs have been considered unhealthy due to concerns about dietary cholesterol intake, but evidence suggests these concerns may have been influenced by confounding. Eggs are rich in many important nutrients and bioactive compounds, including dietary choline and carotenoids, that may benefit cardiometabolic outcomes, including NAFLD. Choline deficiency has been shown to cause liver steatosis in preclinical studies, but little is known about the relations between egg consumption, egg-rich nutrients, and liver fat in humans. The objectives of this dissertation are to evaluate the association between adherence to different healthy dietary patterns, including a Mediterranean-style diet and the DASH diet, and NAFLD risk. To account for updated evidence, we also developed and tested the reliability and validity of a newly modified DASH (mDASH) eating pattern and evaluated its association with incident NAFLD risk as well as changes in liver fat over a 6-year follow-up period. Lastly, we evaluated the associations between eggs and selected egg-rich nutrients (i.e., choline, lutein, and zeaxanthin) and NAFLD risk and prevalence alone and in combination with other dietary habits. We used data from several cohorts in the Framingham Heart Study, including the Offspring, Third Generation, Omni Generation Cohort 1, and Omni Generation Cohort 2. Liver fat was assessed using a computed tomography (CT) scan using the average liver fat attenuation relative to a control phantom to create the liver phantom ratio (LPR) at two sequential CT scans (2002–2005 and 2008–2011) in the Offspring and Third Generation cohorts; measures were available at a single CT scan (2008–2011) in Omni cohorts. NAFLD was defined as a LPR ≤0.33 in the absence of heavy alcohol use (>14 drinks per week for females and >21 drinks per week for males). Prevalent NAFLD was determined on the follow-up CT scan, which all cohorts had available. Among those with no NAFLD at the first CT scan, incident NAFLD was determined on the follow-up CT scan. To determine changes in liver fat, we calculated the difference in LPR from one exam to the next by subtracting the 1st CT scan LPR measure from the 2nd CT LPR measure. Next, we calculated annualized changes by dividing LPR change values by the year difference between the 1st and 2nd CT scan dates for each participant. For the assessment of incident NAFLD and liver fat change, dietary data were derived from food frequency questionnaires (FFQs) administered as close as possible in time to the first liver fat measurement. For the analysis of prevalent liver fat, we used the FFQ as close as possible in time to the second liver fat measurement. The first specific aim of this dissertation is to evaluate whether adherence to the Mediterranean diet (MeDiet Index) or a DASH eating pattern (Fung DASH index) was associated with NAFLD risk. Modified Poisson regression models were used to compute incident NAFLD risk ratios (RR) and 95% confidence intervals (CI) associated with three score categories (low, moderate, and high) on each index adjusting for confounding in 1413 Framingham Offspring and Third Generation participants. Multivariable linear regression models were used to compute adjusted annualized means of liver fat change over a median of 6 years of median follow-up in 1691 participants. In the second specific aim, we aimed to evaluate the adherence to a mDASH eating pattern on NAFLD risk and changes in liver fat using a newly developed mDASH index. Firstly, we developed and evaluated the psychometric properties (reliability and validity) of the new mDASH index. To develop the index, we started with the components included in the original DASH eating pattern (fruits, vegetables, and low-fat dairy), and then proceeded to add components, one at a time, that have been suggested in the literature to protect against known DASH-related outcomes. The selection of the components included in the final mDASH index (total vegetables (including potatoes), total fruit, total dairy (including full-fat dairy), red and processed meat, whole grains, legumes, nuts and seeds, sugar-sweetened beverages, and fish) was based on two criteria: 1) factors found in previous literature to be associated with blood pressure and cardiovascular outcomes, and 2) the ability of possible components in the new mDASH to predict the risk of known DASH-related outcomes (i.e., HTN and atherosclerotic CVD (ASCVD)). Test-retest reliability analyses of the proposed mDASH index were performed with Pearson correlation coefficients over four sequential examination visits in 1339 Offspring Cohort individuals. To assess construct validity, we computed cross-sectional linear regression and correlation analyses between the proposed mDASH index against key DASH nutrients such as calcium, potassium, magnesium, and fiber in 2763 Offspring Cohort individuals. Next, we used Cox regression models to evaluate the predictive validity of the mDASH index, with one modification at a time, for incident HTN (n=1714) and then for ASCVD (n=2700) risks over 11 and 25 medians years of follow-up, respectively. Once the mDASH index was finalized, we used Cox models to determine whether our final mDASH index represents an improvement over three previously established DASH indexes (Fung, Dixon, and Günther) for predicting HTN and ASCVD in two Framingham cohorts, the Offspring and the younger Third Generation cohorts. Lastly, we aimed to evaluate the association between mDASH (vs. earlier DASH indexes) and the risk of incident NAFLD. Multivariable modified Poisson regression and general linear models were used to compute incident NAFLD RR (n=1406) and adjusted means of annualized liver fat change (n=1692) associated with the mDASH index and other prior DASH indexes in a combined sample from the Offspring and Third Generation cohorts. In the third specific aim, we evaluated the impact of egg and egg-rich nutrients (choline, lutein, and zeaxanthin) on NAFLD risk. Egg intake was classified into three categories based on the food frequency questionnaire categories (<1, 1, and ≥2 per week). Dietary choline was body weight adjusted based on the residual method while lutein and zeaxanthin were classified into tertiles, respectively. Multivariable logistic regression models were used to compute prevalent NAFLD odds ratios (OR) associated with egg intakes in 2644 participants from the Offspring, Third Generation, Omni 1, and Omni 2 cohorts. Multivariable modified Poisson regression and general linear models were used to compute NAFLD incident RR (n=1414) and adjusted means of annualized liver fat change (n=1690) associated with egg, choline, and lutein and zeaxanthin intakes in a combined sample from the Offspring and Third Generation cohorts which had repeated measures of liver fat. In our analyses related to the comparison of the DASH eating pattern with a Mediterranean-style diet (Aim 1, Chapter 2), in a combined sample from the Offspring and Third Generation cohorts 19% of participants (n=1413, mean age 51 years, 53% female) developed new onset of NAFLD during follow-up and liver fat increased for most participants. High score category (>28 vs. ≤23 scores) on the Fung DASH index were associated with a 40% lower risk of incident NAFLD (95% CI: 0.42–0.84) after adjusting for age, sex, alcohol intake, education status, current smoking status, multivitamin use, moderate-to-vigorous physical activity, and annualized waist-to-height ratio changes. These findings were stronger in women than men (RR for higher (vs. lower) DASH scores: 0.46, 95% CI: 0.26–0.84 in women; 0.69, 95% CI: 0.45–1.05 in men). DASH scores were also associated with statistically significantly less acquisition of liver fat over study follow-up (p-value<0.05). Further, the DASH diet was protective against NAFLD risk even among individuals with prevalent HTN or an elevated triglyceride:high density lipoprotein cholesterol (TG:HDL-C) ratio. We observed no association between adherence to a MeDiet index and NAFLD risk or liver fat change. The development and testing of the mDASH index are shown in Chapter 3. We found in the analyses for this second specific aim that there was strong evidence of test-retest reliability for the newly-developed mDASH index as supported by moderate to high correlations (r=0.59–0.71) in total mDASH scores between four sequential exams. The construct validity of the mDASH index was supported by its associations with selected nutrients in expected directions. Total mDASH scores were strongly positively associated with intakes of calcium, potassium, magnesium, and fiber (p-values<0.05). These associations were similar to or stronger than those observed with previous DASH indexes. The predictive validity of the new mDASH index was supported by the statistically significant 31% and 29% lower risks of incident HTN and ASCVD associated with score quintile 5 (vs. quintile 1) on the new index in the older Offspring Cohort. Similarly, strong inverse associations between the mDASH index and both HTN and ASCVD were also observed in the younger Third Generation Cohort. These associations were of a similar magnitude to those observed with the Fung index for both HTN and ASCVD but stronger than those seen with the Dixon or Günther indexes. In Chapter 3, we also examined the association between the new mDASH index and risk of incident NAFLD. We found that score tertile 3 (vs. tertile 1) on the mDASH index was associated with a 29% lower NAFLD risk (95% CI: 0.53–0.95) after adjusting for age, sex, energy, sodium intake, current smoking status, education level, multivitamin use, and alcohol intake. In addition, the highest scores on all DASH indexes (mDASH, Fung, Dixon, and Günther) were associated with substantially smaller annualized increases in liver fat over the study follow-up (p-value <0.05). In Chapter 4 of this dissertation, we first examined the association between egg consumption and prevalent and incident NAFLD. NAFLD prevalence was 29% among participants (n=2644) in a combined sample from the Offspring, Third Generation, Omni 1, and Omni 2 cohorts. In a sample from the Offspring and Third Generation cohorts with two measures of liver fat (n=1414), the cumulative incidence of NAFLD was 19% and liver fat was found to increase for most participants during follow-up. After adjusting for confounding by age, sex, energy, red meat and alcohol intakes, prevalent HTN, and body mass index (BMI), we found no association between egg intake and prevalent NAFLD (OR for ≥2 vs. <1 eggs per week (referent): 1.15, 95% CI: 0.92–1.45). Similarly, we observed no associations between egg intake and incident NAFLD risk (RR for ≥2 vs. <1 eggs per week (referent): 1.00, 95% CI: 0.77–1.30) or annualized change in liver fat. Our final specific aim in Chapter 4 was to examine the associations between selected egg-rich nutrients and NAFLD risk. Here, we found that dietary choline intake was strongly associated with a 31% lower risk of incident NAFLD (RR for highest vs. lowest tertile: 0.69, 95% CI: 0.51-0.94) after adjusting for age, sex, education level, and waist-to-height ratio. No associations were found between lutein and zeaxanthin intakes and NAFLD. In summary, the findings of this dissertation suggested that adherence to a DASH eating pattern is beneficially associated with a lower risk of incident NAFLD. Our new mDASH index supports other evidence suggesting that the DASH eating pattern should no longer be limited to the consumption of low-fat dairy and that total vegetable intake does not need to exclude white or sweet potatoes from the diet. The new mDASH is associated with a lower risk of traditional DASH-related outcomes, including HTN and ASCVD, as well as with the emerging cardiometabolic risk factor, NAFLD. Lastly, higher egg intake alone or combined with other eating patterns was not associated with NAFLD, while dietary choline intakes were inversely associated with NAFLD risk.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder linked to obesity that is rapidly increasing in incidence worldwide. It is a disorder that ranges in severity; from a benign condition of hepatic steatosis to a potentially deadly one resulting in cirrhosis and hepatocellular carcinoma. It is currently known that NAFLD is strongly associated with various aspects of metabolic syndrome: insulin resistance, elevated triglyceride levels, obesity, and type two diabetes mellitus. The multifactorial pathogenesis of NAFLD is still uncertain and closer attention is needed on the effect of one’s diet on NAFLD. In this study, we directly compare a westernized diet containing high levels of fat and fructose to a diet high in fat and containing cholate using mouse models in order to determine the role of each dietary factor in the incidence and severity of the different stages of NAFLD. We will evaluate the severity of hepatic steatosis and hepatocellular damage via hematoxylin and eosin (H&E) stained liver tissue and the severity of hepatic fibrosis via trichrome-stained liver tissue. Our hypothesis is that mice on the fructose-based diet are expected to have higher levels of hepatic steatosis and hepatocellular damage relative to mice on the cholate-based diet while mice on the cholate-based diet are expected to have higher levels of hepatic fibrosis relative to the fructose-based diet. The results of this study will aid in elucidating and strengthening the connection between one’s diet and the prevalence and severity of NAFLD.

Using a novel, adipose-liver, two-organ, human-on-a-chip system, the metabolic disease non-alcoholic fatty liver disease was modeled. This model was then used to test the effects of the gut microbiome on NAFLD progression. Two products of the gut microbiome, Trimethylamine-n-oxide and butyrate, were selected as representatives of potentially harmful and potentially beneficial compounds. A dose response, adipocyte and hepatocyte monocultures controls, and HoaC systems were run for 14 days. Through this experimentation, it was found that a dysbiosis of the gut microbiome could be influencing NAFLD progression. Additionally, further development and discovery regarding adipose-liver systems was added to the ongoing conversation of HoaC systems and their usages.