Epidemiology studies have found that type 2 diabetics treated with metformin are at a lower risk for developing cancer. It was speculated that the lowered risk might be attributed metformin's indirect physiological effect of lowering blood insulin levels, which is the opposite of many other antidiabetic drugs. However, further study of metformin's mechanism of action at the cellular level helped develop an understanding of its effect on the individual cell. This helped show why, mechanistically, it makes sense to use metformin for the treatment of cancer. As an activator of AMP-activated protein kinase (AMPK) via inhibition of complex 1 of the mitochondrial electron transport chain, metformin causes suppression of tumor growth and cell cycle arrest by acting on the mTOR pathway and cyclin/CDKs, respectively. Metformin has been most extensively studied in breast cancer, showing great efficacy in numerous breast cancer cell lines that include ER positive, HER2 positive, and triple negative breast cancer cell lines. This compilation of data and results of metformin's efficacy in various cancer subtypes will help push metformin forward as a new chemotherapeutic for breast cancer, and eventually for other cancer types as well.

Hexavalent Chromium (Cr(VI) induces malignant cell transformation in normal bronchial epithelial (BEAS-2B) cells. Cr(VI)-transformed cells exhibit increased level of antioxidants, are resistant to apoptosis, and are tumorigenic. RNAseq analysis in Cr(VI)-transformed cells showed that expression of transcripts associated with mitochondrial oxidative phosphorylation is reduced, and the expression of transcripts associated with pentose phosphate pathway, glycolysis, and glutaminolysis are increased. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. SIRT3 was upregulated and it positively regulated mitochondrial oxidative phosphorylation in Cr(VI)-transformed cells. Our results suggests that SIRT3 plays an important role in mitophagy deficiency of Cr(VI)-transformed cells. Furthermore, SIRT3 knockdown suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Nrf2 is a transcription factor that regulates oxidative stress response. This study investigated the role of Nrf2 in regulating metabolic reprogramming in Cr(VI)-transformed cells. We observed that in Cr(VI)-transformed cells p-AMPKthr172 was increased, when compared to normal BEAS-2B cells. Additionally, Nrf2 knockdown reduced p-AMPKthr172. Our results suggest that Nrf2 regulated glycolytic shift via AMPK regulation of PFK1/PFK2 pathway. Furthermore, our results showed that Nrf2 constitutive activation in Cr(VI-transformed cells increased cell proliferation and tumorigenesis. Overall this dissertation demonstrated that Cr(VI)-transformed cells undergo metabolic reprogramming. We demonstrated that Nrf2 constitutive activation plays decisive role on metabolic reprogramming induction, and SIRT3 activation contributing to increased cancer cell proliferation and tumorigenesis.

<p>The incidence of obesity and associated diseases such as type 2-diabetes and hypertension has reached epidemic portions worldwide and attracted increased interest to understand the mechanisms that are responsible for these diseases. Obesity can result from excessive energy intake, and increasing evidence has emphasized the role of the central nervous system, especially the hypothalamus, in regulating food intake. White adipose, as a direct target of obesity and an important endocrine organ, also has long been a subject of scientific inquiry. AMPK, a conserved energy sensor, has been shown to play important roles in both the hypothalamus and adipose. Recently, CaMKK2 was shown to function as an AMPK kinase. I used intracerebroventricular cannulation as a means to acutely inhibit hypothalamic CaMKK2 with STO-609 and characterize the appetite change associated with loss of CaMKK2 function. Infusion of STO-609 in wild-type mice, but not CaMKK2-null mice, inhibited appetite and promoted weight loss consistent with reduced NPY and AgRP mRNA. Furthermore, intraperitoneal injection of ghrelin increased food intake in wild-type but not CaMKK2-null mice, and 2-DG increased appetite in both types of mice, indicating that CaMKK2 functions downstream of ghrelin to activate AMPK and upregulate appetite. As CaMKK2-null mice were protected from high-fat diet-induced obesity and diabetes, I performed a pair feeding experiment using a high-fat diet and demonstrated that protection of CaMKK2-null mice did not require reduced food consumption. Analysis of brown adipose tissue and metabolic analysis indicated that CaMKK2-null mice did not expend more energy than WT mice. Interestingly, we were surprised to find that CaMKK2-null mice had more adipose than wild-type mice when fed standard chow (5001). By real-time PCR and immunoblot, I identified CaMKK2 expression in preadipocytes and showed that it decreased during adipogenesis. I used STO-609 or shRNA to block CaMKK2 activity in preadipocytes, which resulted in enhanced adipogenesis and increased mRNA of adipogenic genes. I also identified AMPK as the relevant downstream target of CaMKK2 involved in inhibiting adipogenesis via a pathway that maintained Pref-1 mRNA. Consistent with the in vitro data, we further demonstrated that CaMKK2-null mice have more adipocytes but fewer preadipocytes, which supports our hypothesis that loss of CaMKK2 enhances adipogenesis by depleting the preadipocyte pool. Together the data presented herein contribute to our understanding of distinct mechanisms by which CaMKK2 contributes to feeding behavior and adipogenesis.</p> / Dissertation

Nombroses evidències mostren l'important paper dels receptors nuclears PPAR en el control de l'homeòstasi energètica a través de les seves accions reguladores sobre el metabolisme lipídic i glucídic, a més de la seva implicació en processos inflamatoris, tumorals i en la reproducció i el desenvolupament embrionari. La falta de lligands específics de PPAR&#946;/&#948; fins fa relativament poc ha fet que els efectes fisiològics d'aquest subtipus hagin estat poc estudiats en relació als altres subtipus de PPAR, PPAR&#945; i PPAR&#947;. Actualment però, la disponibilitat d'agonistes selectius de PPAR&#946;/&#948; com GW501516, ha ajudat a conèixer i entendre l'important paper fisiològic que juga aquest receptor com regulador de múltiples funcions cel·lulars, especialment en la regulació del metabolisme i la resposta inflamatòria. Encara, però, resta per determinar els mecanismes moleculars responsables d'aquets efectes provocats per l'administració d'aquests fàrmacs, ja que només han estat parcialment estudiats. Els objectius d'aquesta tesi doctoral han estat:1. Establir els mecanismes responsables de l'efecte hipotrigliceremiant de l'agonista de PPAR&#946;/&#948; GW501516 en ratolins alimentats amb una dieta rica en greixos (HFD).2. Establir els mecanismes responsables pels quals l'activador de PPAR&#946;/&#948; GW501516 és capaç d'inhibir la resposta inflamatòria en queratinòcits humans estimulats amb TNF-&#945;.Respecte el primer objectiu els resultats obtinguts indiquen que l'agonista PPAR&#946;/&#948; GW501516 podria reduir els nivells de triglicèrids en plasma en un model animal de hipertrigliceridèmia evitant la reducció de l'AMPK i activant la via PGC1-&#945;-Lipin 1-PPAR&#945;. D'altra banda, respecte el segon objectiu els resultats també demostren que GW501516 pot evitar la inflamació induïda per TNF-&#945; en queratinòcits humans reduint l'activació del factor de transcripció pro-inflamatori NF-&#954;B per l'activació de l'AMPK i SIRT1. / The effects of PPAR&#946;/&#948; subtype and its role in the metabolic syndrome and the inflammatory diseases has been elucidated in the last years, in part, by the availability of PPAR&#946;/&#948; agonist GW501516, althought the mechanism involved is still unkonwn. In this study we examined the effects of PPAR&#946;/&#948; activator GW501516 on high-fat diet (HFD)-induced hypertriglyceridemia. Our results showed that the hipertrigliceridemia caused by the HFD was reduced by the drug treatment. Also the PPAR&#946;/&#948; activation prevented the reduction in the AMPK-Lipin1-PGC-1&#945; pathway caused by the HFD, leading to an increase in hepatic fatty acid oxidation. This effect, together with increased hepatic expression in VLDL-receptor might be responsible for the hypotriglyceridemic effect of GW501516.Moreover, the PPAR&#946;/&#948; activation by GW501516 also prevented TNF-&#945;-induced NF-&#954;B activation in human HaCaT cells by reducing p65 acetylation through AMPK and SIRT1.

Approximately two-thirds of US adults are overweight or obese, and obesity is also becoming more prevalent in children and adolescents. Similar to adults, obese children are at a higher risk of developing health problems due in part to dysfunctional immune surveillance. Obesity has been shown reduce the generation of new T-cells by accelerating thymic aging in an adult mouse. This study therefore aimed at determining whether similar diet induced obesity (DIO) changes can be induced in a young mouse. Comparisons made between lean and DIO C57Bl/6 mice showed a significant increase in thymic weight, decrease in thymic cellularity and thymic output, and impaired T-cell development at the double negative stage. We associate these alterations with changes in thymic architecture and accumulation of lipid droplets within the thymic cortex and medulla of the obese mice. The above observations indicate that DIO can induce fat accumulation and reduce thymic function at a young age. Resveratrol, a natural polyphenolic compound, was then used to regulate fat metabolism in an attempt to reduce these DIO changes we observed. Resveratrol induces fat oxidation via 5' adenosine monophosphate-activated protein kinase (AMPK), and its reciprocal regulation of glycerol-3-phosphate acyltransferase-1 (GPAT-1) and carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzymes required for glycerophospholipid biosynthesis and oxidation, respectively. Through resveratrol feeding, we were able to prevent the effects of DIO on thymic architecture and thymic T-cell proliferation. This was achieved by manipulating AMPK into inhibiting GPAT-1 and enhancing CPT-1 activity. Since the expression of GPAT-1 was upregulated in the obese mice, we investigated whether deleting GPAT-1 altogether might prevent the thymic involution, by inhibiting synthesis of glycerophospholipids and triacylglycerol. Instead, we found that GPAT-1 deletion slowed thymic growth and reduced cellularity in young mice, which we associated with impaired thymic T-cell function and development, suggesting that the deleterious effects of GPAT-1 deficiency may be due to perturbations in thymic T-cell activation and signaling. These data provide a novel link between lipid metabolism and T-cell development, and identify the use of the naturally-occurring resveratrol to reduce lipid accumulation within the involution-prone thymus, thus providing a useful approach to preventing a decline in thymic function in childhood. / text

Studies conducted for this dissertation utilized a rodent model exposed to single or multiple short duration heat loads in an effort to: 1) elucidate the changes in energy metabolism occurring at the tissue and whole-body level in response to hyperthermia, 2) characterize specific aspects of glucose utilization and hepatic glucose production following a heat load and 3) determine if aspects of mitochondrial function and/or dysfunction might play a role in the metabolic changes that occur in response to heat stress. Study 1 was conducted to determine if rodents exposed to heat stress shared similarities using a bovine heat stress model. Specifically, we were interested in identifying changes in blood metabolites and hormones, as well as gene expression and protein abundance of enzymes associated with energy metabolism in skeletal muscle (type I and type II), liver and adipose tissue. Previous bovine data indicates glucose may be preferentially utilized during heat stress, suggesting alterations in energy metabolism. This study provided evidence that tissue-specific changes occur in response to a heat load and that full glucose oxidation might be reduced, specifically in skeletal muscle where abundance of PDK4 mRNA was increased. Within skeletal muscle, glucose transporters (GLUTs 1 and 4) also tended to be increased in rats exposed to a heat load. Increases in skeletal muscle AMPK-α and PGC-1α as well as increased expression of energy substrate transporters suggests heat stress may impose a cellular energy deficit and/or increased energy demands which subsequently leads to changes in energy metabolism. Few changes were noted in either hepatic or adipose tissue in response to acute heat stress in this pilot study. Study aim of Chapter 3 was to further characterize the effects of heat stress on energy metabolism at the tissue and whole-body level in rats exposed to either 1 or 2 bouts of heat. Rats exposed to a 6 h heat load tended to have higher plasma glucose but reduced insulin levels, compared to thermal neutral controls, suggesting decreased glucose uptake or increased hepatic glucose output. Additionally, although heat stress likely increases whole-body energy demand, plasma NEFA levels were blunted in the early hours following onset of heat, suggesting increased adipocyte insulin sensitivity. Gene expression of enzymes associated with oxidative energy metabolism were increased in the TA (which is comprised primarily of glycolytic muscle fibers) following 2 bouts and in liver following a single bout of heat, while expression of oxidative enzymes were decreased within the soleus (a primarily oxidative muscle type). AMPK mRNA was increased following a single bout of heat in hepatic tissue and after 2 bouts of heat in type I skeletal muscle. AMPK mRNA abundance remained the same following 1 bout but was reduced following 2 bouts of heat within type II skeletal muscle. In the TA, phosphorylated AMPK protein abundance was reduced by HS. Abundance of PGC-1α mRNA was increased in types I and II skeletal muscle but was only numerically increased in liver following heat exposure. These data suggest differences at the transcription level in how heat effects energy metabolism within types I and II skeletal muscle as well as between muscle and hepatic tissue and also suggests a cellular attempt to increase energy production (by all mechanisms) in response to heat exposure. Study 3 (Chapter 4) focused on the effect of a heat load on glucose utilization in skeletal muscle and hepatic glucose production capacity. Similar to study 1, PDK4 expression was increased in types I and II skeletal muscle, while PDK2 expression was increased in hepatic tissue. Within skeletal muscle, increases in PDK expression paralled the increased protein abundance of PDHE1α following heat exposure, implying a decrease in oxidative glucose metabolism. Within the liver, protein abundance of PDH-E1α was reduced following a single heat load, but returned to TN levels after a 2nd heat exposure, suggesting that glucose oxidative metabolism is increased above normal levels after an initial heat exposure, but reduced following multiple heat bouts. Hepatic mRNA abundance for gluconeogenic enzymes were increased, implying an increase in hepatic glucose output capacity. The purpose of Study 4 (Chapter 5) was to determine if heat stress elicits changes on mitochondrial function/dysfunction (i.e. oxidative stress), that may account for changes observed in energy metabolism. Expression of genes associated with antioxidant defense were increased by heat stress, but differed between types I and II skeletal muscle as well as between muscle, hepatic tissue and WBCs. The abundance of mRNA for antioxidant enzymes was increased the greatest, and expression of DNA repair enzymes were also upregulated the most within hepatic tissue due to heat exposure, suggesting either increased damage at the level of hepatocytes or greater defensive capacity following an environmental insult. Taken together, this data provides evidence that heat alters energy metabolism, but these changes are tissue-specific and may be reflective of where damage is occurring, or which tissues are able to adapt and/or compensate for increased energy demands imposed by an environmental insult.

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Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.

Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.