Student Number : 0101826W - M Med dissertation - School of Clinical Medicine - Faculty of Health Sciences / Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with hepatic histology that resembles alcoholic liver disease. It is a frequent cause of chronic liver disease and is attracting increasing scientific attention worldwide. I explored the possibility that increased gastrointestinal alcohol production may have a role as a “second hit” in the pathogenesis of NAFLD in study subjects with the metabolic syndrome. In an attempt to investigate this hypothesis, this study looked at blood, urine and breath levels of alcohol in patients with the metabolic syndrome versus matched age and ethnic group healthy controls. Of the twenty study subjects, 80% had dyslipidaemia, 60% had hypertension and 70% had type 2 diabetes mellitus. Their mean BMI was 35.1±8.2 kg/m² (mean ± SD, P < 0.0001 versus controls). The serum aminotransferases were significantly elevated in the study subjects, their ALT levels being 57.4±44.79 U/L versus 17.4±4.60 U/L in the controls (95% CI 18.02 – 61.42, P < 0.001), and their AST levels 52.5±36.21 U/L versus 23.4±4.86 U/L in the controls (95% CI 11.99 – 46.20, P < 0.01). Seventy five percent of the study group had sonar features suggestive of fatty liver disease. Two adipocytokines, adiponectin and leptin, mediators of insulin resistance, an important factor in the development and progression of NAFLD, were also measured. Adiponectin levels were significantly lower (6875 ng/L versus 15475 ng/L; median value, P < 0.01), and leptin concentration levels significantly higher (13.56 ng/L versus 3.05 ng/L; median value, P < 0.05) in the study subjects than in the control group. Alcohol was detected in 60% of the study subjects, of which 35% tested positive for ethanol, 55% tested positive for methanol, and 30% tested positive for both ethanol and methanol. This was a statistically significant result, as none of the control group tested positive for any of the alcohols. The ethanol concentration in the study subjects’ blood was 7.14±3.28 mg% (mean ± SD), in their urine 3.71± 12.87 mg% (mean ± SD) whilst none was detected in their breath. The methanol concentration in the study subjects’ blood was 16.17±17.95 mg% (mean ± SD), in their urine 6.8± 13.58 mg% (mean ± SD) while their breath level was 2.05±3.19 mg (mean ± SD). This study therefore suggests that endogenous alcohol production may be indeed be involved in the pathogenesis of NAFLD in subjects with the metabolic syndrome. Not only ethanol but also methanol was detected in the subjects tested. Endogenous alcohol may therefore be responsible for the ‘second hit’ theory in the pathogenesis of NAFLD, and it is likely that formaldehyde, the metabolite of methanol may be a more potent toxin of hepatocyte injury as opposed to acetaldehyde, the metabolite of ethanol. The most likely source of the alcohol is from intestinal bacterial flora. These findings provide further insight into the pathogenesis of NALFD, suggesting other therapeutic alternatives such as the use of antibiotics and probiotics as a potential treatment strategy for NAFLD.

Indiana University-Purdue University Indianapolis (IUPUI) / Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver. / 2024-05-22

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Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, and aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

The composition, size, and peptide structure of a fatty acid synthetase aggregate from etiolated Euglena gracilis was studied. The fatty acid synthetase was a lipoprotein containing about 40% lipid. Low-angle laser light scattering of the native fatty acid synthetase yielded a molecular weight of 6 · 106 up to concentrations of about 30 μg fatty acid synthetase/ml; at higher concentrations, the molecular weight increased to 11 · 106. Viscometry of the synthetase solutions yielded results that suggested that the asymmetric fatty acid synthetase aggregate formed a 'dimer' at concentrations above 30 μg fatty acid synthetase/ml by side-to-side interaction. The peptide structure of the fatty acid synthetase prepared in the presence of a variety of proteinase inhibitors included at least six peptides of Mr 150000 or less. More than 68% of the protein was in peptides of less than Mr 150000. N-terminal amino acid analysis gave eight different residues all present in integral amounts, seven at about 11% and one at 24% of the total α-N-dansyl amino acids. The Euglena-aggregated fatty acid synthetase appears to be a very large true multienzyme complex.

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Winter injury of bermudagrass (Cynodon spp.) continues to be a problem across the transition zone. In an attempt to delay or induce winter dormancy while maintaining cold hardiness, applications of seaweed extract (SWE) (0.54 kg ha-1), ethephon (16 L ha-1), Fe (1 kg ha-1), and N (49 kg ha-1) took place every three weeks during the fall of 2001 and 2002. Cultivars examined included 'Riviera', 'Midiron', 'Princess', and 'Tifway'. Tifway exhibited greatest fall color retention in both years of the study. Ethephon promoted early senescence and turfgrass quality during fall ratings in both years of the study while N, Fe, and SWE increased quality over the control in 2001 and only N showed better quality and color retention over the control in 2002. Samples removed from cold acclimated plots were artificially frozen as a measure of cold hardiness. Treatments did not have an effect on post freeze regrowth, however, cultivar was significant in both years. Midiron showed best regrowth followed by Riviera, Tifway, and Princess. In both years Riviera and Midiron displayed the quickest and greatest amount of spring greenup followed by Tifway and then Princess. Ethephon reduced greenup in both years and SWE, Fe, and N showed no differences from the control in 2001 and Fe showed significantly better greenup in 2002. Proline and Linolenic acid levels were highest in Midiron, followed by Riviera, Tifway, and Princess. Nitrogen, SWE, and Fe generally did not have an effect on linolenic acid and no consistent effects were noted on proline concentration. Ethephon treatments did not have an effect on linolenic acid levels, however, there was a negative effect on proline concentrations. The results of this study indicate that judicial N applications during the fall can promote color retention and do not have a negative effect on bermudagrass cold-tolerance. Linolenic acid and proline findings also help to explain differences in cold-tolerance between different bermudagrass cultivars. / Ph. D.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke. However, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. We have shown that conditions of hyperglycemia are associated with accumulation of intracellular glucosamine, a downstream metabolite of glucose. Our findings indicate that elevated levels of intracellular glucosamine can promote inflammation and lipid accumulation - the hallmark features of atherosclerosis - in vascular cells and HepG2 cells.</p> <p> Here I demonstrate that exposure of HepG2 cells to the branched chain fatty acid, valproic acid, increases cellular resistance to glucosamine-induced lipid accumulation and nuclear factor-KB activation. In vivo I show that hyperglycemic apolipoprotein E-deficient (ApoE-/-) mice fed a diet supplemented with 625 mg/kg valproic acid have significantly reduced lesion volumes relative to non-supplemented controls. Valproate supplementation has no apparent effect on the plasma levels of glucose, or lipids, nor does it affect the expression of ER chaperones. Significant reductions were observed in total hepatic lipids(> 50.4%) and hepatic glycogen synthase kinases (GSK)-3β activity (> 55.8%) in mice fed the valproate supplemented diet.</p> <p> In vitro I demonstrate that valproic acid directly inhibits GSK-3α/β. Also pretreatment with novel GSK-3 inhibitors protects primary mouse hepatocytes from glucosamine-induced unesterified cholesterol accumulation. I further establish the role of GSK-3 by showing that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol after glucosamine treatment. Dietary supplementation with 2-ethylbutyric acid, a novel and potent GSK-3 inhibitor in vitro, did not reduce lesion development in hyperglycemic ApoE-/- mice and significantly increased atherosclerosis in normoglycemic mice. This may be a side effect attributed to multiple cellular pathways controlled by GSK-3 .</p> <p> In conclusion, I have identified a pathway involving glucosamine-induced cellular dysfunction that leads to accelerated hyperglycemia-associated atherosclerosis. This pathway involves GSK-3, which regulates glucosamine-induced unesterified cholesterol accumulation.</p> / Thesis / Doctor of Philosophy (PhD)

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