The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32P from γ-32P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32P-ribosylation of Gi catalysed by pertussis toxin (PTx) suggested that the abundance of Gi in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of Giα2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of Giα2 in the 100k pellet. No change in Gsα was observed in the l6k pellet but a 35% loss of Gsα was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32P-NAD ribosylation. Giα2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of Gsα were not seen. The distribution of 32P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of Gi on adenylyl cyclase.

There is an ongoing worldwide obesity epidemic. As a consequence, prevalence of obesity- related diseases and conditions are rapidly increasing. One of these related conditions is type 2 diabetes (T2D), which alone caused 1.5 million deaths in 2012. Thus, it is of upmost importance to develop a more complete understanding of these interrelated diseases. At the heart of all these diseases lies the adipose tissue. This tissue is a major endocrine organ, and one of the key secreted cytokines is adiponectin. Adiponectin interplays with the complex insulin signaling network, and adiponectin levels are inversely related with increased adiposity. The presence of these complex dependencies argues for the usage of mathematical modelling. In the work of Brännmark et al, a model of short-term adiponectin release has been validated. However, this model did not include adrenergic signaling, which is the canonical pathway for in situ regulation of adiponectin secretion. To fill this gap, herein, a mechanistic model describing adrenaline-induced short-term adiponectin exocytosis in white adipocytes has been constructed. The newly constructed model is capable of describing experimental data depicting adiponectin release due to adrenergic stimulation as well as data for different mediator combinations. By implementing adrenergic receptor components, the transition to a more physiological model has been initiated. By finding the smallest possible model capable of describing data, one can argue that the model depicts, to some degree, the fundamental mechanisms for short-term adiponectin secretion. Thus, this work has contributed to solidifying the framework of the mechanisms behind short-term adiponectin secretion from white adipocytes. The result of the model work upholds the role of adrenergic signaling as a central regulatory mechanism for adiponectin release. The constructed model could be used as a fundament for creating a model describing adiponectin release under diabetic conditions.

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No. of bitstreams: 1 analuizadasilvaalbertoni.pdf: 1260028 bytes, checksum: cc0af4aab8dc00d61833430e0eb2ce88 (MD5) Previous issue date: 2017-08-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A obesidade é uma doença crônica multifatorial caracterizada pelo excesso de gordura corporal e por um estado inflamatório de baixo grau, conhecido como metainflamação. Atualmente, é um dos principais problemas de saúde pública do mundo. Dados da OMS indicam que 13% da população mundial é obesa e no Brasil, o índice subiu para 17,9% da população em 2014. Fatores genéticos, dieta, desordens metabólicas, como intolerância à glicose, dislipidemias, hipertensão arterial sistêmica, desbalanço hormonal e mudanças na microbiota intestinal, são gatilhos da inflamação envolvendo adipócitos. O tecido adiposo obeso é caracterizado pelo aumento da infiltração de macrófagos, sendo estes uma fonte importante de inflamação neste tecido. Fatores transcricionais com propriedades imunorregulatórias estão envolvidos em processos inflamatórios e na adipogênese. PPARγ é um importante receptor ativado por ligantes lipídicos, regulador central da adipogênese, com funções na ativação de células do sistema imune e no metabolismo lipídico. Dados de nosso grupo demonstraram que ativação de PPARγ induz a biogênese de organelas dinâmicas denominadas Corpúsculos Lipídicos (CLs) durante infeção micobacteriana. Estas organelas possuem funções ativas no estoque de lipídios para geração de energia, síntese de membrana, síntese de mediadores inflamatórios, sinalização celular e inflamação. Além disso, os CL em macrófagos funcionam como sítios para sobrevivência de patógenos, como as micobactérias. Apesar do impacto da obesidade em doenças metabólicas e cardiovasculares ser bem compreendido, os mecanismos envolvidos na relação entre adipócitos e macrófagos infectados com patógenos intracelulares não são conhecidos, sendo este esclarecimento o objetivo de nosso estudo. Para isso, diferencianciamos células NIH3T3-L1 em adipócitos e utilizamos o sobrenadante obtido, para estimular macrófagos peritoneais infectados ou não com M. bovis BCG. Nós avaliamos a biogênese de corpúsculos lipídicos, expressão de PPARγ, síntese e secreção de citocinas, adipocinas e NO. Os resultados comprovaram que a diferenciação de células NIH3T3-L1 em adipócitos é um processo eficiente que envolve mudanças na morfologia celular e acúmulo de Corpúsculos Lipídicos. O estímulo de macrófagos com sobrenadante de adipócitos potencializou a biogênese de CLs, assim como a expressão de PPARγ, na presença de infecção micobacteriana. Nos tempos de 6 e 48 horas de estímulo com sobrenadante e infecção, a produção de TNF-α também foi potencializada, porém um decréscimo significativo foi observado no tempo de 24 horas. Quanto aos níveis de IL-10, um aumento foi observado na presença de infecção e estímulo com sobrenadante nos três tempos analisados. Além disso, apenas no tempo de 24 h observamos a secreção de nitrito de modo significativo pelos macrófagos peritoneais infectados, efeito este independente do estímulo com sobrenadante. Quanto às adipocinas, importantes no desenvolvimento da obesidade e síndrome metabólica, analisamos os níveis de leptina produzida pelos macrófagos que não foram significativos, enquanto a produção de adiponectina, apresentou-se aumentada nos macrófagos controles estimulados com sobrenadante, efeito este, que foi inibido durante a infecção por BCG. Assim, nossos resultados sugerem, um efeito modulador de fatores secretados por adipócitos na ativação de macrófagos, atribuídos a formação de corpúsculos lipídicos, expressão de PPARγ, síntese de citocinas como TNF-α e IL-10 e uma modulação negativa da produção de adiponectina durante a infecção por M. bovis BCG. / Obesity is a chronic multifactorial disease characterized by excess of body fat and a low- grade inflammatory state, known as meta-inflammation. It is currently one of the world's leading public health problems. WHO data indicate that 13% of the world population is obese, in Brazil, the obesity index was 17.9% of the population in 2014. In addition to genetic factors, diet and metabolic disorders, such as glucose intolerance, dyslipidemia, systemic arterial hypertension, hormonal imbalance and changes in the intestinal microbiota have been proposed as triggers for inflammation involving adipocytes. Recent studies have shown that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that these are an important source of inflammation in this tissue. Transcriptional factors with immunoregulatory properties are involved in inflammatory processes and adipogenesis. Such as the PPARγ, is a receptor activated by lipid ligands, central regulator of adipogenesis, with functions in the immune cells activation and lipid metabolism. Data from our group, demonstrated that PPARγ activation induces the biogenesis of dynamic organelles, the Lipid Droplets (LD) during mycobacterial infection. These organelles have functions in lipid storage for energy generation, membrane synthesis, inflammatory mediators synthesis, cell signaling and inflammation. Moreover, the LD in macrophages are niches for the pathogens survival. Although the impact of obesity on metabolic and cardiovascular diseases is understood, the mechanisms involved in the interactions between adipocytes and macrophages infected with intracellular pathogens are not known. In this study we differentiated NIH3T3-L1 cells into adipocytes and used the supernatant obtained to stimulate peritoneal macrophages infected or not with M. bovis BCG. We analyze the lipids droplets formation, PPARγ expression, cytokine, adipokines and NO synthesis. The results have shown that NIH3T3-L1 cells differentiation into adipocytes is an efficient process involving changes in cell morphology and LD storage. The macrophages stimulation with adipocyte supernatant was able to potentiated LD biogenesis, as well as, PPARγ activation, during mycobacterial infection. After 6 and 48 hours of stimulation and infection, the TNF-α levels were also potentiated, although a significant decrease was observed after 24 hours of infection. The IL-10 levels, was increase in the presence of infection and supernatant stimulation at 6, 24 and 48 hours after infection. To nitrite analysis, we observe that after 24 h of infection, there was an increase of levels secreted by macrophages, however this effect was independent of the supernatant stimulation. The adipokines, as leptin and adiponectin, are important factors in the obesity and metabolic syndrome development. Therefore, we analyzed the levels of leptin and adiponectin produced by macrophages during BCG infection. The Leptin levels were not detect, while adiponectin production, was increased in the control macrophages in presence of supernatant, an effect that was inhibited during BCG infection. Thus, our results suggest a modulating effect of secreted factors by adipocytes on the macrophages activation, attributed to the lipid droplets formation, PPARγ expression, cytokines synthesis as TNF-α and IL-10 and a dowmodulation of adiponectin during M. bovis BCG, infection.

Orientador: Dora Maria Grassi-Kassisse / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T06:42:56Z (GMT). No. of bitstreams: 1 Arouca_AlineBarbedo_M.pdf: 2816326 bytes, checksum: 7245a319bb7b00c52b0d7ae0cc6d6b02 (MD5) Previous issue date: 2012 / Resumo: O ginseng é um dos suplementos herbais mais populares do mundo. Investigações revelaram que o ginseng e seus produtos relacionados reduzem o processo inflamatório, produzem atividade adaptogênica promovendo a melhora na performance em ratos e camundongos. Das diferentes espécies de ginseng, o Siberiano (Eleutherococcus senticosus) é o que apresenta menor índice de efeitos colaterais, não produzindo excitação em pacientes, exercendo efeito protetor mais intenso sobre o sistema imune, e não desenvolvendo síndrome semelhante ao estresse, conforme já observado no Panax ginseng (ginseng Coreano). Desta forma, propomos neste trabalho, avaliar a eficácia da administração crônica de Eleutherococcus senticosus (ginseng Siberiano) sobre a performance de ratos adaptados à esteira de corrida 3 x/semana (10 m/minuto, durante 10 minutos) e submetidos a treinamento de corrida (incremento de velocidade e duração do treino até a 4ª semana, e depois mantendo 25 m/minuto, durante 60 minutos/dia até a 8ª semana), aplicando três testes de performance (teste incremental de velocidade sem inclinação) ao longo de 8 semanas de tratamento. Os resultados indicaram que não houve melhora da performance dos ratos dos grupos adaptados e corrida no segundo e no terceiro teste (meio e final do tratamento, respectivamente), e a performance do grupo corrida só foi maior que a do grupo adaptado quando os ratos foram suplementados com 100 mg/kg de peso/dia de extrato padronizado de Eleutherococcus senticosus (ES). O ganho de peso foi semelhante entre os grupos adaptado e corrida, entretanto o tratamento com o ES induziu a um menor ganho de peso dos ratos quando comparados com seus respectivos controles. A ingesta alimentar, por sua vez, foi significativamente menor no grupo Corrida e o tratamento com ES não alterou este resultado. A morfometria dos adipócitos indicou menor área dos adipócitos dos ratos suplementados em relação aos seus controles, sendo significativo no panículo epididimal para o grupo Adaptado. Em relação aos biomarcadores de estresse oxidativo, no grupo Corrida Suplementado foi observado uma tendência a menor produção de espécies reativas de oxigênio analisados pela concentração de TBARS plasmático e muscular, porém não estatisticamente diferentes. Quanto aos outros biomarcadores metabólicos (lactato e glicemia plasmáticos, e glicogênio hepático e muscular), e ao perfil lipídico (triacilglicerol e colesterol total plasmáticos), não observamos alterações significativas que demonstrassem a eficácia do tratamento com ginseng no exercício proposto. Especificamente os outros biomarcadores de estresse oxidativo (catalase e superóxido dismutase) não apontaram aumento significativo na atividade antioxidante enzimática. Concluímos, portanto, que o tratamento com Eleutherococcus senticosus na forma de tratamento proposto foi eficiente em melhorar a performance no grupo treinado em esteira (Corrida Suplementado) / Abstract: Ginseng is one of the most popular herbal supplements in the world. Investigations revealed that ginseng and its related products reduce inflammation, produce adaptogenic activity promoting improvement in performance in rats and mice. Of different species of ginseng, Siberian (Eleutherococcus senticosus) is due to a lower rate of side effects, not producing arousal in patients, exerting stronger protective effect on the immune system, and not developing like syndrome to stress, as observed in Panax ginseng (Korean ginseng). Thus, we propose in this work was to evaluate the efficacy of chronic administration of Eleutherococcus senticosus (Siberian ginseng) on the performance of rats adapted to treadmill running 3 x/week (10 m/min for 10 min) and subjected to running training (increment of speed and duration of the training until the 4th week, and then maintaining 25 m/minute for 60 minutes / day to 8 weeks) by applying three performance tests (Test incremental speed without inclination) over 8 weeks treatment. The results indicated that there was no improvement of the performance of rats in groups adapted and running in the second and third test (middle and end of treatment, respectively) and running performance only group was higher than the group adapted when the rats were treated with 100 mg/kg/day of standardized extract of Eleutherococcus senticosus (ES). Weight gain was similar between groups adapted and race, however treatment with ES induced a lower weight gain of rats when compared to their respective controls. The food intake, in turn, was significantly lower in the group racing and ES treatment did not alter this result. The adipocyte morphometry showed the smallest area of adipocytes supplemented rats compared to their controls, with significant difference in panniculus epididymal isolated of Adapted group. Regarding biomarkers of oxidative stress in Race Supplemented group was observed a trend to lower production of reactive oxygen species analyzed by plasma and muscular TBARS concentration, but not statistically different. As for other metabolic biomarkers (plasma lactate and glucose, and liver and muscle glycogen), and lipid profile (total cholesterol and plasma triacylglycerol), we observed no significant changes to demonstrate the efficacy of treatment with ginseng in the proposed exercise. Specifically other biomarkers of oxidative stress (catalase and superoxide dismutase) showed no significant increase in antioxidant enzyme activity. We therefore conclude that treatment with Eleutherococcus senticosus as proposed treatment was effective in improving performance in the group trained on a treadmill (Race Supplemented) / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Au cours de l'obésité, le Tissu Adipeux blanc (TAB) est le siège d'un important remaniement de sa Matrice Extracellulaire avec des amas fibrotiques autour des adipocytes et des vaisseaux. Cette organisation caractéristique semble avoir une incidence dans la physiopathologie de l'obésité. Les composés spécifiques à la Membrane Basale ont été mis en évidence autour des adipocytes et des cellules endothéliales et leur expression est fortement induite dans l'adipocyte obèse. L'expression de COL4A1 est positivement corrélée à l'insulino-résistance de sujets présentant une obésité modérée. De plus, dans un autre groupe de sujets massivement obèses candidats à la chirugie bariatrique, la diminution de l'expression génique de COL4A1 est à relier avec l'amélioration de l'insulino-résistance après l'intervention. Enfin, nous observons que, dans le TAB, l'expression de COL4A1 est positivement associée à l'expression génique de deux facteurs de croissance pro-fibrotiques, le TGF 1 et le TGF 3, dans le TAB. La culture tridimensionnelle d'adipocytes ou de cellules endothéliales exposés à ces deux facteurs induit un phénotype fibro-inflammatoire dans ces types cellulaires. Néanmoins, le traitement par le TGF 1 ou TGF 3 induit uniquement dans les cellules endothéliales une sur-expression de COL4A1 et non dans les adipocytes.En conclusion, nos données proposent un nouvel acteur de la fibrose du TAB au cours de l'obésité, la Membrane Basale adipocytaire et vasculaire, participant ainsi à la dysfonction tissulaire et métabolique. / During obesity, White Adipose Tissue (WAT) undergoes an important remodeling of its Extracellular Matrixwith fibrotic depots around adipocytes and vessels. This typical organization seems to have an impact in the pathophysiology of obesity. Basement Membrane components were detected around adipocytes and endothelial cells and their expression were significantly increased in obese adipocytes. COL4A1 expression in WAT is positively correlated to insulin-resistance parameters in moderate obese subjects, and its reduction is associated to insulin-resistance improvement after gastric bypass in a group of morbidly obese subjects. Finally, we demonstrated a postive correlation between COL4A1 expression and two pro-fibrotic growth factor (TGF1 and TGF3) in obese WAT. In vitro treatment of isolated adipocytes and endothelial cells with these TGF isoforms induced inflammatory and fibrotic phenotype. However, TGF1 and TGF3 exposure only provoked COL4A1 over-expression in endothelial cells, and not in adipocytes. In conclusion, our work have highlighted a new actor in WAT fibrosis during obesity, adipocytes and endothelial cells Basement Membrane, participating in the pathological alterations of obese adipose tissue and metabolism.

Persistent organic pollutants (POPs) and adipose tissue hypoxia have been shown to independently affect adipocyte functions. The goals of this study were to (1) determine the effect of PCB-77, PCB-153, and DDE on the differentiation of human preadipocytes, and (2) investigate the cross-talk between PCB-77 and hypoxia in differentiated human adipocytes. First, human preadipocytes were exposed to PCB-77, PCB-153, or DDE during the entire 14-day differentiation period. We found no effect of low POP levels on lipid accumulation. Second, differentiated human adipocytes were exposed to a combination of PCB-77 and hypoxia. We demonstrated gene-specific cross-talk between PCB-77 and hypoxia, showing an additive effect of PCB-77 on VEGF, MCP-1, and adiponectin, as well as an inhibition of PCB-77-induced expression of CYP1A1 by hypoxia. This work has expanded our understanding of the role of POPs and hypoxia in differentiated human adipocytes.

Metabolic disorders are commonly associated with obesity, a condition where excess caloric intake leads to massive adipose tissue (AT) expansion and eventual dysfunction. When adipose tissue loses its ability to store excess energy properly, lipids accumulate in non-adipose tissues such as liver, and muscle. This ectopic lipid deposition is a significant risk factor in the development of a collection of disorders described as metabolic syndrome. While metabolic syndrome is typically linked with obesity, patients who have an inability to develop adipose tissue depots (lipodystrophy) develop similar clinical outcomes. There is evidence that aberrant mTORC1 signaling may occur in both settings, and may be a factor that contributes to adipose dysfunction. I find that adipocyte specific loss of Raptor, a key mTORC1 subunit, leads to progressive lipoatrophy, and associated metabolic dysfunction including AT inflammation, hepatosteatosis, and insulin resistance. Interestingly, inhibition of autophagy, a pathway upregulated during Raptordeletion, prevents lipoatrophy but does not protect from ectopic lipid deposition and AT inflammation. These results suggest that outputs of mTORC1 in adipocytes individually regulate adipocyte storage capacity, and AT health. Furthermore, ablation of the amino acid sensing RagGTPases, thought to be necessary for mTORC1 activity, does not phenocopy Raptor KO, suggesting RagGTPase independent functions of mTORC1 in adipocytes. RagA/B deletion, however, did consistently increase Ucp1 expression in WAT, indicating a possible noncanonical role of the Rags in regulating Ucp1. Overall, these studies advance our understanding of regulation of adipose tissue metabolism, and shed light on previously unstudied nutrient specific signaling pathways in adipocytes.

The prevalence of type 2 diabetes mellitus is increasing worldwide and is considered one of the top health concerns globally. The occurrence of type 2 diabetes is linked to the rapidly increasing trend of obesity in both adults and children, which is proposed to be a contributing factor in the development of insulin resistance and type 2 diabetes. White adipose tissue, an insulin target tissue, is an important endocrine organ involved in the control of energy homeostasis through its direct influence on metabolism, insulin sensitivity and food intake. To better understand these functions, we studied adipocyte differentiation in 3T3-Ll cells, a white adipose tissue cell line. Many mitochondrial proteins exhibit an increase in expression levels during adipogenesis as identified by mass spectrometry. Moreover, increased mitochondrial mass and altered morphology was observed by light microscopy. Qualitative changes in mitochondrial gene expression were also observed during adipogenesis as revealed by Affymetrix GeneChip analysis. Additionally, striking changes in mitochondrial protein expression and morphology were identified following treatment with the insulin sensitizing agent, rosiglitazone. These results suggest that mitochondrial biogenesis and remodeling is inherent to white adipocyte differentiation. To investigate the physiological relevance of these findings, mRNA and protein expression profiles and mitochondrial morphology were studied during the development of insulin resistance and obesity and following treatment with rosiglitazone in ob/ob mice. These studies reveal a marked decrease in transcript levels for over 50% of mitochondrial genes with the onset of obesity in ob/ob mice. Rosiglitazone treatment stimulates enhanced expression in approximately half of these genes, as well as changes in mitochondrial mass and remodeling. Furthermore, these studies reveal that depressed oxygen consumption and fatty acid oxidation occur with obesity development and these alterations can be reversed with rosiglitazone treatment. This work identifies the previously underscored plasticity of mitochondria in white fat and suggests that mitochondrial biogenesis and remodeling in white adipose tissue may lead to systemic changes in insulin sensitivity and energy homeostasis. Lastly, these studies suggest that mitochondria may be an important therapeutic target for antidiabetic drugs.

White adipose plays a major role in the regulation of whole body metabolism through the storage and hydrolysis of triglycerides and by secretion of adipokines. The function of endocrine cells is highly dependent on the unfolded protein response (UPR), a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the cell's secretory protein load. Here we demonstrate that the adipocyte UPR pathway is necessary for its secretory functions, and can thus play a crucial role in the control of whole body energy homeostasis. ER protein folding capacity is dependent both on the number of available chaperones as well as on their activity, which requires a sufficient ATP supply. In 3T3-L1 adipocytes, mitochondrial biogenesis occurred in parallel with induction of the UPR; therefore, we tested whether it was necessary for efficient ER function. Inhibition of mitochondrial ATP synthesis through depletion of Tfam, a mitochondrial transcription factor, or treatment with inhibitors of oxidative phosphorylation, demonstrate that ER function is sensitive to acute changes in adenine nucleotide levels. In addition, adenylate kinase 2 (AK2), which regulates mitochondrial adenine nucleotide interconversion, is markedly induced during adipocyte and B cell differentiation. AK2 depletion impairs induction of the UPR and secretion in both cell types. Interestingly, cytosolic adenylate kinase 1 (AK1) does not have the same effect upon UPR induction. We show that adenine nucleotides promote proper ER function and alterations in specific aspects of ATP synthesis can impair UPR signaling. Understanding the complex energetic regulation of the UPR may provide insight into the relationship between UPR and disease.