Type 2 diabetes mellitus (T2DM) is a disease of multi-complications affecting more than 20 million US adults. Hyperglycemia is the classic clinical feature of diabetes, and uncontrolled hyperglycemia leads to deadly health complications. Thus, control of blood glucose represents a major goal for diabetics. Human and rodent studies revealed another clinical feature; diabetics have low tissue and plasma levels of polyunsaturated fatty acids (PUFAs), an effect often attributed by impaired endogenous PUFA synthesis. In this context, rodent studies have revealed a possible link between PUFA synthesis and high fat diet induced obesity and diabetes. These studies have shown that obese and diabetic mice have low hepatic expression and activity of fatty acid elongase-5 (Elovl5), a key enzyme involved in the PUFA synthesis pathway. Over-expression of Elovl5 in livers of chow fed C57BL/6J mice decreased fasting blood glucose and increased hepatic glycogen contents. Therefore, my hypothesis for the current work is that elevated hepatic Elovl5 activity or improved hepatic PUFA synthesis will improve systemic and hepatic carbohydrate metabolism in a mouse model of diet induced obesity and diabetes. Using a recombinant adenovirus approach, we over-expressed Elovl5 in livers of high fat diets (60% calories derived from fat as lard, Research Diets) induced obese-diabetic mice. Elevated hepatic Elovl5 activity increased hepatic and plasma C��������������� PUFA contents, reduced homeostatic model assessment for insulin resistance (HOMA-IR), improved glucose tolerance and lowered fasting blood glucose to euglycemic levels in obese-diabetic mice. The mechanism for insulin mimetic effect of Elovl5 on hepatic glucose metabolism was correlated with increased phosphorylation of Akt-S��������, FoxO1-S�������� and PP2Acat-Y��������, decreased nuclear content of FoxO1, and decreased expression of Pck1 and G6Pase; important enzymes involved in gluconeogenesis (GNG) and glucose production. Phospho-FoxO1 is excluded from nuclei, ubiquitinated and degraded by the proteasome. Loss of nuclear FoxO1, due to its increased phosphorylation, leads to the reduction in the expression of key genes involved in gluconeogenesis, i.e., Pck1 and G6Pase. Using obese-diabetic mice liver extracts and HepG2 cells, I established that Elovl5 uses two mechanisms to control hepatic GNG. The first mechanism involves Elovl5 mediated increased Akt2-S�������� and FoxO1-S�������� phosphorylation via mTORC2-rictor pathway. The second mechanism involves Elovl5 mediated attenuation of de-phosphorylation of FoxO1 via PP2A inhibition. Together, these mechanisms increase FoxO1 phosphorylation status in livers of fasted obese-diabetic mice, lower hepatic FoxO1 nuclear abundance and FoxO1 capacity to sustain transcription of GNG genes and inhibit GNG and restore blood glucose levels in fasted obese-diabetic mice. Results of these studies showed Elovl5 corrected high fat diet induced hyperglycemia in C57BL/6J mice, identified the molecular mechanism of Elovl5 control of GNG and explained how Elovl5 or PUFA synthesis controls GNG. Therefore, these findings will be eventually helpful in developing a therapeutic target to combat hyperglycemia. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from July 27, 2012 - July 27, 2013

Orientador: Gabriel Forato Anhê / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T11:52:59Z (GMT). No. of bitstreams: 1 Faria_JulianadeAlmeida_M.pdf: 3166901 bytes, checksum: d6395a9feadc0393429b22df87066881 (MD5) Previous issue date: 2012 / Resumo: O aumento da produção hepática de glicose (PHG) é o principal componente que contribui para os elevados valores da glicemia de jejum em indivíduos obesos com Diabetes Mellitus tipo 2 (DM2). ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The increase in hepatic glucose production (HGP) is the main component that contributes to high values of fasting glucose levels in obese individuals with diabetes mellitus type 2 (DM2). ...Note: The complete abstract is available with the full electronic document / Mestrado / Farmacologia / Mestra em Farmacologia

This thesis investigates the effects of alanine on rainbow trout’s glucose metabolism at the organismal and molecular levels. Rainbow trout is an important aquaculture species that belongs to the salmonid family. As a carnivorous fish, the requirement of protein/amino acids in trout’s diet is high. In contrast, rainbow trout are poor utilizers of carbohydrates. One prevalent hypothesis suggests that high levels of dietary amino acids could indeed contribute to the poor utilization of carbohydrates in this species. In mammals, there is evidence supporting the importance of alanine as a gluconeogenic precursor. However, a recent study found that alanine stimulates hepatic AMP-activated protein kinase (AMPK) to lower circulating glucose levels in mice. Alanine levels are high in all tissues in rainbow trout. The role of alanine in gluconeogenesis is less clear in trout and there is no evidence, to our knowledge, regarding its effects on glucose kinetics. Therefore, the main goal of the study was to investigate the impact of the continuous infusion of exogenous alanine for 4h on glucose fluxes and to identify potential mechanisms in tissues that could interpret the observed changes in glucose fluxes in vivo. Glucose turnover, appearance and disposal, Rt, Ra and Rd, respectively, were measured to determine the impact of alanine on glucose fluxes. The expression and/or activity of key genes in glucose transport, utilization and gluconeogenesis were assessed in liver and muscle. An additional goal was to assess whether alanine activates AMPK in trout. The levels of phosphorylated AMPK and other signaling proteins known to interact with the latter were quantified. Results show that alanine reduced plasma glucose levels and inhibited Ra and Rd glucose, consistent with previously observed effects of insulin in rainbow trout. The reduction in the expression of a paralogue of glut4, a key gene in glucose transport, and the activity of hexokinase (HK), a key enzyme in glucose utilization, in muscle can partially explain the observed reduction in Rd glucose. Together, these results suggest that glucose was not a preferred substrate under conditions of increased alanine availability and that alanine was probably oxidized to provide energy. Alanine failed to activate AMPK in trout, contrary to mammalian findings. However, it increased AKT (also known as protein kinase B) phosphorylation in muscle, similar to the effect of insulin in trout. In conclusion, my results suggest that alanine mediated at least some of the observed effects by stimulating insulin secretion given the similarities between the effects of exogenous alanine and insulin in rainbow trout as discussed above. Future studies are warranted to investigate the hypothesis that alanine is an insulin secretagogue in rainbow trout.

Hypoxia has become a subject of interest among the many environmental stressors as its role in biology is complex and diverse. Hypoxia is a significant low oxygen condition that causes many pathologies and adaptive responses in organisms. It can lead to a moderate or dangerous loss of respiration and can be an indication of tumorigenesis as many tumors lack adequate blood supply. Organisms possess adaptive responses to hypoxia that include hypoxia-inducible factors (HIFs) that activate several downstream pathways that are responsible for altering metabolism and maintaining homeostasis. Within aquatic organisms, hypoxia is an important ecological constraint as oxygen availability within bodies of water can vary greatly over time and space. Therefore, adaptation to hypoxia is likely pervasive, especially in genotypes originating from bodies of water that are prone to hypoxia. Here we report the transcriptional response to acute hypoxia in the clonal freshwater crustacean Daphnia magna. Daphnia were subjected to 1mg/O2 for 12 hours. Then, RNA was extracted, reverse transcribed, and sequenced using Oxford Nanopore MinION. We find that severe hypoxia significantly up-regulates key enzymes in the gluconeogenesis pathway. Additionally, we report genotype-by-environment interactions showing that Daphnia clones from habitats that are hypoxia prone survive better in hypoxia.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20568号 / 医博第4253号 / 新制||医||1022(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 原田 浩, 教授 武藤 学, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science

Diabetes mellitus is a growing public health concern, presently affecting 25.8 million or 8.3% of the American population. While the availability of novel drugs, techniques, and surgical intervention has improved the survival rate of individuals with diabetes, the prevalence of diabetes is still rising. Type 2 diabetes (T2D) is a result of chronic insulin resistance and loss of -cell mass and function, and it is is always associated with the impairment in energy metabolism, causing increased intracellular fat content in skeletal muscle (SkM), liver, fat, as well as pancreatic islets. As such, the search for novel agents that simultaneously promotes insulin sensitivity and 𝜷-cell survival may provide a more effective strategy to prevent the onset and progression of this disease. Kaempferol is a flavonol that has been identified in many plants and used in traditional medicine. It has been shown to elicit various pharmacological activities in epidemiological and preclinical studies. However, to date, the studies regarding its effect on the pathogenesis of diabetes are very limited. In this dissertation, I explored the anti-diabetic potential of the dietary intake of kaempferol in diet-induced obese mice and insulin-deficient diabetic mice. For the first animal study, kaempferol was supplemented in the diet to determine whether it can prevent insulin resistance and hyperglycemia in high fat (HF) diet-induced obese mice or STZ-induced obese diabetic mice. For the second animal study, kaempferol was administrated once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean STZ-induced diabetic mice to evaluate its efficacy for treating diabetes and further determining the underlying mechanism. The results demonstrated that dietary intake of kaempferol for 5 months (mo) improved insulin sensitivity and glucose tolerances, which were associated with increased Glut4 and AMPKα expression in muscle and adipose tissues in middle-aged mice fed a high-fat (HF) diet. In vitro, kaempferol increased lipolysis and restored chronic high fatty acid-impaired glucose uptake and glycogen synthesis in SkM cells, which were associated with improved AMPKα activity and Glut4 expression. In addition, dietary kaempferol treatment preserved functional pancreatic 𝜷-cell mass and prevented hyperglycemia and glucose intolerance in STZ-induced diabetic mice. Data from the second study show that oral administration of kaempferol significantly improved blood glucose control in obese mice, which was associated with reduced hepatic glucose production and improved whole body insulin sensitivity without altering body weight gain, food consumption, or the adiposity. In addition, kaempferol treatment increased Akt and hexokinase activity, but decreased pyruvate carboxylase and glucose-6 phosphatase activity in the liver homogenate without altering their protein expression. Consistently, kaempferol decreased pyruvate carboxylase activity and suppressed gluconeogenesis in HepG2 cells as well as primary hepatocytes isolated from the livers of obese mice. Kaempferol directly blunted the activity of purified pyruvate carboxylase. In the last study, we found that kaempferol stimulates basal glucose uptake in primary human SkM. In C2C12 mouse myotubes, kaempferol also increased insulin stimulated glycogen synthesis and preserved insulin dependent glycogen synthesis and glucose uptake in the presence of fatty acids. Kaempferol stimulated Akt phosphorylation in a similar time-dependent manner as insulin in human SkM cells. Consistent with this, kaempferol increased Akt and AMPK phosphorylation in isolated murine red SkM tissue. The effect of kaempferol on glucose uptake was blunted in the presence of chemical inhibitors of glucose transporter 4 (Glut4), phosphoinositide 3-kinase (PI3K), glucose transporter 1 (Glut1), and AMPK. The AMPK inhibitor also prevented kaempferol-stimulated Akt phosphorylation. Further, kaempferol improved the stability of insulin receptor substrate-1. Taken together, these studies suggest that the kaempferol is a naturally occurring compound that may be of use in the regulation of glucose homeostasis and diabetes by improving insulin sensitivity and glucose metabolism, as well as by preserving functional 𝜷-cell mass. / Ph. D.

Dans le cadre de l’évaluation de la néphrotoxicité métabolique du cadmium, nous avons étudié l'effet du chlorure de cadmium (CdCl2) sur la néoglucogenèse dans les tubules rénaux proximaux murins et humains. Les expériences de dose-effets in vitro montrent que le CdCl2 inhibe l’utilisation de lactate et la production de glucose de façon dose-dépendante. En outre, le CdCl2 induit une diminution importante des concentrations cellulaires de l'ATP et des principaux composés contenant des groupements thiols tels que les coenzymes A et le glutathion réduit. Les mesures enzymatiques et de spectroscopie RMN du carbone 13, montrent que le CdCl2 produit dans les tubules rénaux murins et humains respectivement aux concentrations de 10 et 100 μM, une inhibition des flux à travers la lactate déshydrogénase et l'ensemble de la voie de la néoglucogenèse. Nos résultats sont en faveur d’une action inhibitrice directe du cadmium sur les enzymes intervenant dans la voie de la néoglucogenèse. Toutefois, la baisse intracellulaire en ATP, coenzymes A et glutathion, aurait un effet potentialisateur de cette inhibition. Ainsi, cette étude fournit une base biochimique pour une meilleure compréhension des mécanismes cellulaires des tubulopathies proximales survenant chez l'homme suite à une exposition chronique au cadmium. / As part of the assessment of metabolism nephrotoxicity of cadmium, we have studied the effect of cadmium chloride (CdCl2) on gluconeogenesis in isolated mouse and human renal proximal tubules. The dose-response experiments in vitro have shown that CdCl2 inhibits the use of lactate and glucose production in a dose-dependent fashion. Besides, the CdCl2 induced a significant decrease in cellular concentrations of ATP and the main compounds containing thiol groups such as coenzyme A and reduced glutathione. The enzymatic steps and 13C-NMR spectroscopy showed that CdCl2 produced in mouse and human kidney tubules respectively at concentrations of 10 and 100 μM, an inhibition of fluxes through lactate dehydrogenase and the entire gluconeogenic pathway. Our results are in favor of a direct inhibitory action of cadmium on enzymes involved in the gluconeogenic pathway. However, the decrease in intracellular ATP, glutathione and coenzyme A, would have a potentiating effect of this inhibition. Therefore, this study provides a biochemical basis for better understanding the cellular mechanisms of proximal tubular nephropathy occurring in humans following chronic exposure to cadmium.

Master of Science / Department of Human Nutrition / Tonatiuh Melgarejo / Late-lactation Holstein cows (n=9/treatment) were used to evaluate effects of continuous adipose tissue TNFα administration on glucose and fatty acid (FA) metabolism. Cows were blocked by feed intake and milk yield and randomly assigned within block to control or TNFα treatments. Treatments (4 mL saline or 14 μg/kg TNFα in 4 mL saline) were infused continuously over 7 d via 2 osmotic pumps in the adipose layer in the tailhead region. Plasma, milk samples, milk yield, and dry matter intake (DMI) data were collected daily. On d 7, pumps were removed and liver and contralateral tailhead adipose biopsies were collected. Results were modeled with fixed effect of treatment and random effect of block; P values > 0.10 were considered non-significant. TNFα did not alter liver TNFα mRNA abundance, plasma TNFα, IL-4, IL-6, or interferon-γ concentrations, DMI, or rectal temperature. Milk fat and lactose concentrations decreased with TNFα (P < 0.05), but milk yield was unchanged and treatments did not alter the proportion of short vs. long-chain FA in milk on d 7. Treatments did not alter plasma NEFA concentration, liver triglyceride content, or adipose mRNA abundance for hormone-sensitive lipase or perilipin. Plasma glucose turnover rate, as measured by disappearance of U-13C-glucose bolus, was not altered by treatment, nor was liver mRNA abundance for phosphoenolpyruvate carboxykinase or pyruvate carboxylase. However, TNFα tended to decrease adipose TNFα mRNA abundance (P=0.09) and increase liver IL-10 mRNA abundance (P=0.05) compared to controls. Messenger RNA expression of IL-10 in adipose and IL-37 in liver tissue increased significantly in cows treated with TNFα (Figure 1; P = .02 adipose; P < 0.05 liver). This TNFα delivery protocol may have allowed for an adaptive anti-inflammatory response to suppress systemic inflammation, which may account for the lack of metabolic responses compared with previous responses to daily subcutaneous TNFα injections.