Antecedentes. El olivo constituye una fuente de compuestos bioactivos, tanto en su fruto, como en sus subproductos. Los extractos de aceitunas son ricos en polifenoles y no son tóxicos. Algunos de sus compuestos han mostrado beneficios para la salud en modelos celulares y animales. Así, los polifenoles de la oliva presentan actividad antiinflamatoria, previenen la apoptosis por estrés oxidativo, favorecen el neurodesarrollo y neuroprotección y tienen efecto antiadipogénico en diferentes modelos celulares, protegiendo contra el aumento de peso y la acumulación de grasa. Objetivos. Se han planteado los siguientes objetivos: Evaluar la bioseguridad in vitro e in vivo de extractos de huesos de aceituna ricos en polifenoles. Analizar el posible efecto antiadipogénico de los polifenoles del olivo en pez cebra midiendo la ganancia de peso, colesterol, triglicéridos y ácidos grasos. Evaluar la influencia de los extractos de aceituna en los mecanismos de digestión y absorción de polisacáridos y grasas mediante la cuantificación de amilasa, glucosa, fosfolipasa y colesterol en el modelo de pez medaka. Comprobar la actividad antiadipogénica de dichos extractos, utilizando la diferenciación a adipocitos de la línea celular 3T3-L 1 de fibroblastos de ratón. Evaluar el posible efecto de los extractos de oliva ricos en polifenoles sobre el metabolismo de los lípidos en pez medaka cuantificando la expresión de genes lipogénicos y lipolíticos. Material y métodos. Para el primer objetivo, se ha evaluado la citotoxicidad mediante adición de extracto de hueso de olivas disuelto en PBS (0-400 mg/1) a un cultivo de la línea celular THP1-XBlue-CD14 y evaluación de la viabilidad celular mediante la reacción de reducción de la resazurina por las células vivas. La bioseguridad se ha evaluado en pez cebra, incubando huevos fecundados en extracto de O a 100 mg/I durante 24 a 72 horas y midiendo los parámetros: a) letales (embriones muertos, huevos coagulados), b) subletales (movimientos espontáneos, pigmentación, edemas) y c) teratogénicos (malformaciones, retraso desarrollo). Para el segundo objetivo, Se han utilizado huevos fecundados e incubados en placas de pocillos a 26±1 º C durante 72 horas: Grupo control en agua con DMSO al O, 1 %. Grupo intervención, añadiendo extracto polifenólico a 1 00mg/1. Al finalizar la incubación se procedió a la cuantificación en ambos grupos de la masa corporal de las larvas secas, proteínas, colesterol total, triglicéridos y ácidos grasos. Para el cuarto objetivo, se cultivan y diferencian las células 3T3-L 1 de fibroblastos de ratón (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 1 O y 50 mg/I de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con Oil-Red y medida de la absorbancia a 490 nm y la expression de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. Por último, para el quinto objetivo, los peces medaka adultos se mantuvieron en tanques durante cinco días con cinco extractos al 0.01 % en agua, causando obesidad a través de una dieta rica en carbohidratos, con un grupo de control mantenido en agua con una dieta normal. Los extractos contenían polifenoles que oscilaban entre 7 y 116 mg/g (oleuropeína, hidroxitirosol) con un poder antioxidante de 2-13 mmol de 2,4,6-tri (2-piridil) -1,3,5-triazina/100 g. Después de cinco días, los peces fueron sacrificados y el ARNm hepático y su ADN complementario se obtuvó mediante transcripción inversa. Los ADN complementarios se cuantificaron para tres genes lipolíticos y tres lipogénicos por PCR en tiempo real. La expresión genética relativa se calculó a partir de la amplificación utilizando curvas en referencia al grupo de control. Resultados. En primer lugar, se analizó la citotoxicidad (efecto tóxico cuando viabilidad inferior al 75%) del extracto de huesos de oliva en la línea celular THP1-XBlue-CD14, está en concentraciones superiores a 50 mg/I de extracto (viabilidad 77,5%), calculando una LD50 (dosis de letalidad 50%) superior a 800 mg/1. La bioseguridad in vivo con los embriones de pez cebra expuestos a concentraciones de extracto de 0-100 mg/I mostró total viabilidad a 24, 48 y 72 horas postfecundación (hpf), no observándose mortalidad ni se apreciaron embriones con efectos subletales, teratógenos, ni adelanto o retraso en la eclosión. Los valores de peso de los peces analizados no muestran diferencias entre control e intervención, igualmente todos los demás parámetros analizados, proteínas, cholesterol total, triglicéridos y ácidos grasos, no han presentado diferencias significativas con la introducción del extracto durante la incubación. Estos resultados no son concluyentes debido posiblemente a que no se ha tenido en cuenta que el tejido adiposo aparece en el pez cebra a las 120 horas postfertilización. Se deben realizar nuevos ensayos con el fin de confirmar o descartar el efecto sobre diferentes parámetros relacionados con el metabolismo lipídico, ensayos que tengan en cuenta las condiciones fisiológicas normales del modelo animal escogido. Los extractos de polifenoles no fueron tóxicos en los peces medaka a una concentración de 0.1 %, diez veces mayor que la concentración utilizada. Se observó una disminución general en los niveles de glucosa en peces sobrealimentados con carbohidratos con la adición de extractos, pero sin volver a los niveles del grupo control con alimentación estándar (disminución entre 15-40%). No hubo impacto sobre la amilasa en adultos o alevines, se observó una disminución general pero no significativa del colesterol y un aumento general de la fosfolipasa en los alevines. En la línea cellular 3T3-L 1 de fibroblastos de ratón, la adición de 50 mg/I de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 1 O mg/I de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/1. La expresión de genes implicados en la lipólisis, incluyendo el receptor activado para la proliferación de peroxisomas, acil-CoA oxidasa 1 y carnitina palmitoiltransferasa 1, se redujeron claramente en los peces sometidos a una dieta obesogénica, y esta situación no se invierte con la adición de los extractos ricos en polifenoles. En contraste, los genes implicados en la lipogenesis, genes de la ácido graso sintasa, acetil-CoA carboxilasa 1 y de la proteína 1 de unión a elementos reguladores de esteroles aumentaron considerablemente con la dieta obesogénica y volvió al estado normal con los extractos de aceituna. Este efecto no fue dependiente del contenido total de polifenoles, la concentración específica de oleuropeína o hidroxitirosol, o el poder antioxidante, lo que sugiere un efecto sinérgico. Conclusiones. Se puede concluir que el extracto de huesos de olivas es altamente bioseguro hasta al menos concentraciones de 100 mg/1. Los extractos de aceitunas ricos en polifenoles reducen los niveles de glucosa en peces sobrealimentados. La formación de los cuerpos grasos característicos de la adipogénesis en la diferenciación de los fibroblastos 3T3-L 1 queda inhibida previa adición de 50 mg/I de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina. Por último, los polifenoles del olivo actúan como agentes antilipogénicos, tienen un efecto positivo en el metabolismo de los lípidos, pero su mecanismo en la expression de cada gen es diferente en relación a cada extracto utilizado, lo que apoya la presencia de mecanismos sinérgicos respecto a las diferentes proporciones de polifenoles y fitoquímicos acompañantes en cada extracto.

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Growth hormone (GH) is a poly-peptide hormone produced by the anterior pituitary. Growth hormone not only stimulates body and muscle growth but also inhibits adipose tissue growth. The overall objective of this study was to determine the mechanisms by which GH inhibits adipose tissue growth. Three studies were conducted to achieve this objective. The first study was conducted to determine if GH inhibits fat tissue growth by stimulating lipolysis. In this study, adipose tissue weight and adipocyte size were compared between GH-deficient growth hormone releasing hormone receptor (Ghrhr) homozygous mutant mice (i.e., lit/lit mice), lit/+ mice, and lit/lit mice injected with GH. lit/lit mice had less body weight but more subcutaneous fat and larger adipocytes compared to lit/+ mice at the same ages. GH treatment to lit/lit mice for four weeks partially reversed these differences. These data suggest that GH inhibits adipose tissue growth in mice at least in part by stimulating lipolysis. Additional data from this study suggest that GH indirectly stimulates lipolysis in vivo and this indirect mechanism is independent of " adrenergic receptors in the adipose tissue. The second study was conducted to investigate if GH inhibits fat tissue growth also by inhibiting adipogenesis. In this study, stromal vascular fraction (SVF) cells were isolated from subcutaneous fat of lit/+ and lit/lit mice and were induced to differentiate into adipocytes in vitro. Oil Red O staining and gene expression analysis revealed that the SVF cells from lit/lit mice had greater adipogenic potential than from lit/+ mice. This suggests that GH inhibits adipose tissue growth also through inhibition of adipogenesis. Additional data from this study suggest that GH may inhibit adipogenesis by inhibiting the formation of adipogenic precursor cells in adipose tissue in mice. The third study was conducted to determine the role of the central component of GH receptor signaling, STAT5, in GH inhibition of differentiation of bovine preadipocytes. In this study, preadipocytes were isolated from subcutaneous fat of adult cattle and were induced to differentiate with or without GH. Based on Oil Red O staining, gene expression, glycerol-3-phosphate dehydrogenase (G3PDH) activity and acetate incorporation assays, GH inhibited differentiation of bovine preadipocytes into adipocytes. GH induced phosphorylation of STAT5 in differentiating bovine preadipocytes. Overexpression of constitutively active STAT5 through adenovirus mimicked the effect of GH on differentiation of bovine preadipocytes. These data support a role of STAT5 in mediating the inhibitory effect of GH on differentiation of bovine preadipocytes into adipocytes. Overall, GH inhibits adipose tissue by both stimulating lipolysis and inhibiting adipogenesis; GH stimulates lipolysis through an indirect mechanism that is independent of the " adrenergic receptors; GH inhibits adipogenesis through a direct mechanism that may involve the transcription factor STAT5. / Ph. D.

The objective of this dissertation research was to investigate the effect of dietary macronutrient composition on neuropeptide Y (NPY)-mediated regulation of adipose tissue physiology in broiler chickens during the early post-hatch period. A high-carbohydrate (HC), high-fat (HF) or high-protein (HP) diet was fed to broiler chicks in all experiments and various facets of physiology were evaluated at day 4 post-hatch, including diet-, fasting-, and neuropeptide Y-induced effects on gene expression, cellular morphology, and lipid metabolism. Experiment 1 was designed to study the effects of diet on molecular changes in different adipose tissue depots (subcutaneous, clavicular and abdominal) after 3 hours of fasting and 1 hour of refeeding. Adipose tissue weights were decreased in chicks that consumed the HP diet, whereas adipocyte diameter was increased in response to the HF diet. There was greater expression of mRNAs encoding fatty acid binding protein 4 (FABP4) and monoglyceride lipase in chicks fed the HC and HF diets than the HP diet in all three adipose tissue depots. Fasting increased plasma non-esterified fatty acid concentrations in chicks fed the HC and HP diets. Results suggest that the heavier fat depots and larger adipocytes in chicks fed the HF diet are explained by greater rates of hypertrophy, whereas the HP diet led to a decrease in adipose tissue deposition, likely as a result of decreased rates of adipogenesis. Experiments 2 and 3 were designed to investigate how dietary macronutrient composition affects the effect of centrally or peripherally administered NPY, respectively, on lipid metabolism-associated factor mRNAs in adipose tissue. In experiment 2, vehicle or 0.2 nmol of NPY was injected intracerebroventricularly (ICV) and abdominal and subcutaneous fat samples were collected at 1 hour post-injection. In the subcutaneous fat, ICV NPY injection decreased peroxisome proliferator-activated receptor gamma (PPAR gamma) and sterol regulatory element-binding transcription factor 1 (SREBP1) mRNAs in chicks fed the HF diet, whereas there was an increase in SREBP1 expression in chicks fed the HF diet after NPY injection. Expression of PPAR gamma and FABP4 mRNAs increased in the abdominal fat of HF diet-fed chicks after NPY injection. Thus, HF diet consumption may have enhanced the sensitivity of chick adipose tissue to the effect of centrally-injected NPY on gene expression of adipogenesis-associated factors. In experiment 3, vehicle, 60, or 120 micrograms/kg BW of NPY was injected intraperitoneally (IP), and subcutaneous, clavicular, and abdominal fat was collected at 1 and 3 hours post-injection. Food intake and plasma NEFA concentrations were not different among chicks fed the HC, HF or HP diet after IP NPY injection, indicating that the effects of NPY on adipogenesis were independent of secondary effects due to altered energy intake. In response to the lower dose of NPY, the expression of NPY receptor sub-type 2 mRNA was increased at 1 hour post-injection in the subcutaneous fat of chicks fed the HP diet, whereas there was less 1-acylglycerol-3-phosphate O-acyltransferase 2 mRNA in the subcutaneous fat of chicks fed the HC diet. The higher dose of NPY was associated with greater AGPAT2 mRNA in the clavicular fat of chicks that consumed the HP diet and less CCAAT/enhancer-binding protein alpha in the abdominal fat of chicks that were provided the HF diet. However, there was also a decrease in the expression of some of these factors, although mechanisms are unclear. In conclusion, dietary macronutrient composition influenced the response of adipose tissue to the adipogenic effects of NPY and metabolic effects of short-term fasting and refeeding during the first week post-hatch. Collectively, this research may provide insights on understanding NPY's effects on the development of adipose tissue during the early life period and mechanisms underlying diet-dependent and depot-dependent differences in adipose tissue physiology across species. / Ph. D.

Les apolipoprotéines L (APOL) forment une famille de protéines conservées chez les mammifères. L’APOL6 murine est principalement exprimée par les adipocytes présents dans le tissu adipeux. Dans un modèle de culture d’adipocytes, l’adipogénèse a causé l’induction de l’expression de l’APOL6. Celle-ci a pu encore être modulée à la hausse par de l’IFNγ, et à la baisse par du TGFβ. Des facteurs élevant la concentration en AMP cyclique ont aussi permis de diminuer l’expression d’APOL6. In vivo, lorsque des souris APOL6 KO ont été nourries par un régime riche en graisses, elles ont pris moins de poids que les souris WT correspondantes. De plus, les adipocytes des souris APOL6 KO obèses étaient plus petits que ceux des contrôles WT. Finalement, la recherche de protéines interagissant avec l’APOL6 par immunoprécipitation a permis de mettre en évidence une majorité de protéines associées au cytosquelette d’actine. En conclusion, l’APOL6 semble être associée au cytosquelette d’actine des adipocytes et permettrait la régulation de la taille de leurs gouttelettes lipidiques. / Apolipoproteins L (APOL) are a family of conserved proteins among mammals. Murine APOL6 is mainly expressed by adipocytes in the adipose tissue. In a model of in vitro adipocyte cell culture, adipogenesis induced the expression of APOL6. This expression increased with IFNγ and decreased with TGFβ. Cyclic-AMP elevating agents also decreased the expression of APOL6. In vivo, APOL6 KO mice that were fed with a high fat diet gained less weight than their wild type (WT) counterparts. Furthermore, adipocytes from obese APOL6 KO mice were smaller than those from WT controls. Finally, immunoprecipitation experiments showed that APOL6 probably interacted with actin cytoskeleton proteins within adipocytes. In conclusion, APOL6 is likely associated with the actin cytoskeleton in adipocytes and could be involved in the regulation of the size of lipid droplets. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Polychlorinated biphenyls (PCBs) are persistent, man-made toxicants that are linked to adverse health effects and diseases such as endocrine disruption, diabetes, obesity, cardiovascular effects, and cancer. Since their manufacturing began in 1929 for industrial use, and was banned in 1979, they have bioaccumulated in water, sediment, food, animals, humans and more. PCBs are also found in indoor air of older buildings and as inadvertent byproducts in the manufacture of paints and pigments. The lower chlorinated PCBs, those with fewer than 5 chlorine atoms, are readily metabolized to form hydroxylated PCBs (OH-PCBs) that are further converted to PCB-sulfates in reactions catalyzed by cytosolic sulfotransferases (SULTs). Steroid sulfotransferases SULT1E1 and SULT2A1 participate in regulating the homeostasis of estrogens and androgens, respectively, through the deactivation of active hormones. The estrogen sulfotransferase (SULT1E1) is also a potential key player in adipogenesis. Recent literature has shown that downregulating expression of SULT1E1 in cells derived from humans and mice caused opposite effects, where adipogenesis was inhibited or stimulated, respectively. Adipogenesis is the maturation of preadipocytes into mature adipocytes, which is regulated by peroxisome proliferating-activator γ (PPARγ). Adipocytes are a main component of adipose tissue, which is important for energy homeostasis, organ protection, and thermoregulation. Adipose tissue also secretes various cytokines such as adiponectin. Adipose tissue dysfunction can result from adipocyte dysfunction, which can be caused by alterations in cell signaling. The objective of this dissertation research was to determine if OH-PCBs and PCB-sulfates are inhibitors of SULT1E1 and SULT2A1 and if inhibition of SULT1E1 by OH-PCBs could potentially affect adipogenesis. We hypothesized that PCB metabolites would inhibit SULT1E1 and SULT2A1 and potentially affect adipogenesis in both human and murine cell models. Using purified recombinant human SULT1E1 and SULT2A1, I found that 4’-OH-PCB 3, 4-OH-PCB 8, 4-OH-PCB 11, 4’-OH-PCB 25, and 4-OH-PCB 52 were potent inhibitors of the sulfation of representative substrates (7.0 nM estradiol for SULT1E1 and 1.0 µM dehydroepiandrosterone for SULT2A1, Figures 3-3 and 3-4, respectively). Moreover, 4-OH-PCB 11 and 4-OH-PCB 52 were the most potent inhibitors of SULT1E1 and SULT2A1 with IC50 values of 7.2 nM and 1.5 μM, tables 3-1 and 3-2, respectively. The least potent inhibitor of SULT1E1 was 4’-OH-PCB 3, with an IC50 of 1300 nM. The PCB-sulfates were not potent inhibitors for either enzyme. 4-OH-PCB 11 inhibited the sulfation of estradiol in the cytosol of both pre-adipocytes and fully differentiated adipocytes (Figure 4-9). Immortalized human adipocytes were treated with 10 µM of triclosan (a known inhibitor of SULT1E1), 4’-OH-PCB 3 and 4-OH-PCB 11. Experiments included exposure to these toxicants for 1) 72 hours to preadipocytes, 2) 72 hours to preadipocytes followed by 11-day differentiation, 3) to differentiating adipocytes and for 48 hours post-differentiation. The lipid accumulation levels remained unaffected, as determined by microscopic imaging and quantification using AdipoRed. The mRNA expression levels of prominent adipogenic markers SULT1E1, PPARγ, and AdipoQ were measured using RT-Q-PCR. Changes in SULT1E1 and PPARγ expression were unaffected upon treatment before, during and after adipogenesis when compared to controls. However, the increase in AdipoQ expression was reduced upon treatment with 4-OH-PCB 11 in differentiated adipocytes and in preadipocytes exposed for 72 hours followed by 11-day differentiation (Figure 4-14). This could be an indicator of adipocyte dysfunction that was not manifested by a change in lipid accumulation. Murine 3T3-L1 cells were also treated with 10 µM of triclosan, 4’-OH-PCB 3 and 4-OH-PCB 11 for 48 hours to preadipocytes, during 8-day differentiation and for 48 hours after differentiation. The mRNA expression levels of prominent markers of cardiovascular and adipogenesis functions, ACE2, PPARγ, FABP4, and AdipoQ were measured using RT-PCR. Compared to controls, the increase in AdipoQ expression was reduced following treatment of preadipocytes with triclosan and 4-OH-PCB 11 and subsequent differentiation (Figure 5-11). The increase in PPARγ expression remained either unchanged from controls or slightly stimulated in differentiating and differentiated adipocytes (Figures 5-11 and 5-13). Angiotensin-converting enzyme 2 (ACE2) expression was decreased compared to control values, upon treatment with 4’-OH-PCB 3 (Figure 5-12), while fatty acid binding protein 4 (FABP4) expression was stimulated to the same extent across all treatment groups in differentiating adipocytes (Figure 5-12). The results, overall, show that these OH-PCBs did not affect lipid accumulation in human adipocytes, but they may affect other signaling pathways in adipogenesis. 4-OH-PCB 11 decreased adiponectin expression compared to the increase that was seen in unexposed differentiating human and mouse adipocytes. Adiponectin is secreted from adipose tissue, and this decrease could indicate a form of dysfunction. This finding is consistent with the results of the purified SULT1E1 study, where 4-OH-PCB 11 potently inhibited SULT1E1, but 4’-OH-PCB 3 did not (Figure 3-3 and Table 3-1). Thus, there is a potential for OH-PCBs to disrupt the expression of adiponectin and perhaps other vital adipokines and this could negatively affect adipose tissue function. Future studies will be needed to determine if these effects are indeed mediated by intracellular estradiol and SULT1E1. Moreover, the potential for in vivo disruption of circulating adiponectin by OH-PCBs and other toxicants that inhibit SULTs remains to be studied.

Prostate Apoptosis Response-4 (Par-4) is a conserved and ubiquitous tumor-suppressor factor which can selectively induce apoptosis in tumor cells, while leaving normal cells unaffected. While Par-4 is well established as a tumor-suppressor, there have yet been no formal investigations as to whether it has a physiologic role in normal tissues. Early observations of Par-4 knockout mouse lines yielded that the adult mice displayed significant weight gain and fat accumulation compared to their wild-type counterparts while on a conventional chow diet. Interestingly, obese mouse and human subjects were found to exhibit reduced expression of Par-4 in adipose tissue as well as lower levels of secreted Par-4 in their plasma, compared to samples collected from lean human subjects. Subsequent in vitro experiments would show that loss of Par-4 has significant impact upon adipogenesis. Mechanistically, Par-4 loss during adipogenesis in cell culture correlated inversely with expression of the adipogenic transcription factor PPARγ. Subsequent experiments would demonstrate that Par-4 transcriptionally represses PPARγ at the promoter level. Thereby, we conclude that Par-4 regulates adipogenesis and lipid accumulation through transcriptional repression of the PPARγ promoter. This research utilizes novel models and may be used as the basis for Par-4-mediated therapies for obesity and metabolic disease.

The formation of adipocytes is a complicated process in which insulin and IGF-1 signaling pathways and numerous transcription factors control the conversion of precursor cells to mature fat cells. The Rab5 protein acts as a rate-limiting protein during receptor-mediated endocytosis by switching between a GDP-bound inactive form and a GTP-bound active form. The inactivation and activation of Rab5 are regulated by several Rab5 GTPase activating proteins (GAPs) and Rab5 guanine nucleotide exchange factors (GEFs), respectively. This dissertation demonstrated that the activity of the small GTPase Rab5 and its regulators are essential for the differentiation of 3T3-L1 pre-adipocytes. Specifically, it showed that Rab5 activation is detrimental to the differentiation process. The overexpression of a dominant-negative Rab5:S34N mutant, but not an active counterpart (Rab5:Q79L), stimulated the differentiation of 3T3-L1 pre-adipocytes. Consequently, the expression of Rab5:S34N increased the expression of two adipogenic-specific transcriptional factors, PPARγ and C/EBPα. siRNA-mediated depletion of Rab5 inhibited the differentiation of 3T3-L1 pre-adipocytes, providing further evidence for the requirement of Rab5 in the process of adipogenesis. A dramatic decrease of the Rab5-GTP level is also observed during the differentiation of 3T3-L1 pre-adipocytes. Consistent with these observations, I found that the expression of Rab5 GEFs (i.e., RIN1, Rabex-5, and RAP6), which increased the GTP-bound form of Rab5, blocked the differentiation process. In contrast, the expression of Rab5 GAPs (i.e., RN-tre and RabGAP-5), which decreased the GTP-bound form of Rab5, stimulated differentiation of 3T3-L1 pre-adipocytes. I also found a novel interaction between the VPS9 domain of the Rab5 GEFs and the activated insulin receptor. This interaction is specific since the VPS9 domain did not interact with the catalytic inactive mutant of the insulin receptor and the Rab5 GAPs (no VPS9 domain) did not bind to the activated insulin receptor. The data point out that a reduction on the GTP-bound form of Rab5 is required for the rapid differentiation of 3T3-L1 pre-adipocytes, identifying Rab5 inactivation as an important contributor of adipogenesis. Also, these observations suggest a novel cellular mechanism of Rab5 activity in the adipogenesis process in connection with the insulin receptor, the Rab5 GAPs, and the Rab5 GEFs.

<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