Introdução: A doxorrubicina (DOX) é um quimioterápico que gera efeitos tóxicos no tecido adiposo (T.A.) e reduz a qualidade de vida dos pacientes. Objetivos: Investigar os efeitos metabólicos do tratamento com DOX no T.A. branco e propor terapia adjuvante que atenue efeitos deletérios. Métodos: Procedimento experimental 1: ratos Wistar foram tratados com dose única de DOX (15mg/kg). Cultura de células: 3T3L1 foram incubadas por 24h, 96h e 12 dias com DOX. Procedimento experimental 2: animais C57/BL6 receberam doses fracionadas de DOX associado ao uso de metformina (MET) (300mg/kg, diário) ou não. Conclusão: A DOX gera um alto impacto sobre a homeostasia do T.A. branco tanto no tratamento agudo com dose única, como no tratamento crônico com doses mais baixas. Os processos fisiológicos do tecido adiposo sofreram profundas alterações, o que resultou em menor tamanho do adipócito, maior fibrose, diminuição das vias metabólicas e redução da adiponectina e leptina circulantes, e o tratamento com MET não reverteu esses efeitos, só prevenindo o processo de fibrose do TA. / Introduction: Doxorubicin (DOX) is a chemotherapy that generates toxic effects on adipose tissue (AT) and reduces the quality of life of patients. Objectives: To investigate the metabolic effects of treatment with DOX on AT white and to propose adjuvant therapy to mitigate deleterious effects. Methods: Experimental Procedure 1: Wistar rats were treated with a single dose of doxorubicin (15mg/ kg). Cell Culture: 3T3-L1 were incubated for 24h, 96h and 12 days with doxorubicin. Experimental procedure 2: C57/BL6 mice received fractionated doses of DOX associated with the use of metformin (MET) (300 mg/kg daily) or not. Conclusion: DOX generates a high impact on the homeostasis of white AT in both acute single dose treatment, such as in chronic treatment with lower doses. The physiological processes of AT have undergone major changes, resulting in a smaller of adipocytes, increased fibrosis, reduction in metabolic pathways and decreased circulating adiponectin and leptin, and the treatment with MET did not reverse these effects, only prevent the fibrosis process on AT.

Dados recentes apontam para a participação do sistema renina-angiotensina (SRA) em processos metabólicos, devido a sua presença local em tecidos metabolicamente ativos, como o tecido adiposo, e sugerem que tais tecidos também poderiam ser alvos dos bloqueadores do SRA. Por isso, investigamos possíveis efeitos diretos de bloqueadores do SRA no metabolismo celular de adipócitos isolados. Para isso, adipócitos isolados foram tratados com doses não tóxicas de Alisquireno ou Captopril ou Losartan. Após 24 horas, as capacidades lipolíticas, lipogênicas e oxidativas foram. Como resultados, o fármaco Alisquireno, aumentou a relação entre oxidação de glicose e incorporação desse substrato em lipídeos, enquanto o Captopril diminuiu a incorporação de glicose em lipídeos, particularmente na fração glicerol do TAG mediante estímulo com insulina, bem como diminuiu a expressão gênica de receptor de (pró) renina. Como conclusão, os fármacos Captopril e Alisquireno podem modular o metabolismo lipogênico e oxidativo de adipócitos isolados, mas de maneiras diferentes. / Recent data indicate a participation of the renina-angiotensin system (RAS) in metabolic process, due its local presence in tissues, like the adipose tissue, and suggests that these tissues could be targets of RAS blockers. Therefore, we have studied the possible effects of pharmacological RAS blockers in isolated fat cells. Therefore, fat cells were isolated of epididymal fat pad and treated with non toxic doses of Aliskiren or Captopril or Losartan. After 24 hours, the lipolytic, lipogenic and oxidative capacity were tested in their respective spontaneous and stimulated states. Also, gene expression of PPARg and RAS components were verified. The results showed Aliskiren increases the relation between oxidation and lipogenesis from glucose, whereas Captopril decreased glucose lipid incorporation, especially in glicerol fraction of triglyceride when insulin stimulus exist, and the Renin receptor gene expression. As a conclusion, Captopril and Aliskiren can directly modulate lipogenic and oxidative metabolism of isolated fat cells, but in a different way.

Nós investigamos se o ácido palmitoléico modula o metabolismo de glicose e triacilglicerol (TAG) em adipócitos. Assim, células 3T3-L1 tratadas com ácido palmitoleico (16:1n7 , 200 mM) ou palmítico (16:0, 200 mM) por 24h e adipócitos epididimais de camundongos selvagem ou deficientes para PPARa tratados com 16:1n7 o ácido oleico (18:1n9, 300 mg / kg / dia), via gavagem durante 10 dias, foram avaliados. O tratamento com palmitoleico aumenta a captação de glicose e o conteúdo de GLUT4 e pThr172AMPKa. O aumento de GLUT4 foi abolido pela inibição da AMPK. Palmitoleico aumenta a conversão de glicose em lactato e CO2 e diminui a síntese de novo de ácidos graxos. O tratamento de células 3T3-L1 com ácido palmitoleico aumentou a lipólise o mRNA da ATGL e HSL, além do conteúdo proteico da ATGL e pSer660HSL. O aumento na lipólise foi abolido pela inibição de PPARa. Também, o tratamento de camundongos selvagens, mas não os deficientes para PPARa, com palmitoleico aumentou a lipólise e o mRNA da ATGL e HSL em adipócitos. Em resumo, o ácido palmitoleico aumenta a captação de glicose e sua utilização pelos adipócitos, um efeito que está associado com a expressão de GLUT4 e AMPK. Além disso, este ácido aumenta a lipólise e lipases em adipócitos viA PPARa. / We investigated whether palmitoleic acid modulates glucose and triacylglycerol (TAG) metabolism in white adipocytes. For this, 3T3-L1 cells treated with palmitoleic (16:1n7, 200 µM) or palmitic acid (16:0, 200 mM) for 24h and epididimal adipocytes from wild type or PPARa deficient mice treated with 16:1n7 or oleic acid (18:1n9, 300 mg/kg/day) by gavage for 10 days were evaluated. Thus, treatment with palmitoleic increases glucose uptake and the content of GLUT4 and pThr172AMPKa. The increase in GLUT4 was prevented by AMPK inhibition. Also, palmitoleic increases glucose conversion into lactate and CO2, and decreases de novo fatty acids synthesis. Furthermore, treatment of 3T3-L1 cells with palmitoleic increased lipolysis, mRNA levels of ATGL and HSL and protein content of ATGL and pSer660-HSL. Such increase in lipolysis can be prevented by PPARa inhibition. Treatment of wild type, but not PPARa deficient mice, with palmitoleic increased adipocytes lipolysis and ATGL and HSL mRNA levels. In conclusion, palmitoleic acid increases glucose uptake and utilization by adipocytes, associated with GLUT4 expression and AMPK activation. Furthermore, palmitoleic acid increases adipocyte lipolysis and lipases via PPARa.

Introdução. O estoque excessivo de lipídeos no organismo está associado à diversas doenças crônicas não transmissíveis. O exercício físico aumenta a utilização dos lipídeos. A mobilização dos ácidos graxos (AG) é a primeira etapa para que eles sejam utilizados como fonte energética pelo músculo esquelético. Para otimizar esse processo, têm sido estudadas substâncias que poderiam aumentar a lipólise, como a cafeína. O café e o chá-mate contêm cafeína em sua composição. Objetivo. Comparar os efeitos da ingestão de cafeína, café e chá-mate sobre o desempenho físico, parâmetros metabólicos e lipólise em resposta ao exercício físico agudo em esteira rolante, em ratos Wistar. Métodos. O estudo foi divido em etapas 1 (exercício até a exaustão, n=15) e 2 (exercício com duração de 60 minutos, n=45). A amostra foi composta por cinco grupos: controle (C), controle exercício (CE), cafeína exercício (CFNE), café exercício (CAFE) e chá-mate exercício (CME). Na etapa 1, foram comparados o desempenho, as diferenças na massa corporal e na glicemia (pós versus pré-exercício) e a atividade lipolítica. Na etapa 2, foram comparadas as diferenças na massa corporal e na glicemia (pós versus pré-exercício), a atividade lipolítica, o lactato sanguíneo e o glicogênio muscular. Os dados foram apresentados segundo a estatística descritiva (média ± erro padrão). Os dados foram analisados através de modelos gerais lineares e os deltas através da técnica de contrastes ortogonais. Para verificar associação entre as variáveis de interesse foi utilizada a correlação linear de Pearson. Resultados. Na etapa 1 não foram observadas diferenças entre os grupos com relação ao desempenho. A massa corporal pós-exercício, quando comparada à pré-exercício, diminuiu nos grupos CE (188 por cento ), CFNE (273 por cento ), CAFE (319 por cento ) e CME (204 por cento ), quando comparados ao C. Não houve diferença para a variação de glicemia entre os grupos. Observou-se aumento de 92 por cento da lipólise no grupo CAFE, quando comparado ao grupo C. Na etapa 2, houve diminuição da massa corporal (pós versus pré-exercício) nos grupos CE e CFNE (263 por cento ), CAFE (230 por cento ) e CME (183 por cento ), quando comparados ao C. A glicemia aumentou nos grupos CFNE (variação pós versus pré-exercício de 343 por cento e 220 por cento , quando comparada aos grupos C e CE, respectivamente) e CME (179 por cento , quando comparada ao C). A lipólise estava aumentada nos grupos CFNE e CAFE, quando comparados aos grupos C (150 por cento e 233 por cento , respectivamente) e CE (51 por cento e 101 por cento , respectivamente). Não foram observadas diferenças significantes entre os grupos para a concentração sanguínea de lactato e para o conteúdo de glicogênio muscular. Não houve correlação entre as variáveis, em ambas as etapas. Conclusão. Após o exercício até a exaustão, o grupo que ingeriu café apresentou aumento da atividade lipolítica. Após 60 minutos de exercício, o grupo que ingeriu cafeína, assim como o que ingeriu chá-mate, apresentou aumento da glicemia. A cafeína e o café promoveram aumento da lipólise / Introduction. The excess of body fat is associated with the development of chronic non-communicable diseases. Physical exercise enhance lipolysis. The mobilization of fatty acids (FA) is the first stage for the use of lipids as a source of energy by skeletal muscle. In order to increase the use of FA, substances have been used. Objective. This work compared the effects of caffeine, coffee and maté tea on the performance, metabolic parameters and lipolysis in response to acute physical exercise on a treadmill, in Wistar rats. Methods. The study was developed in stage 1 (exercise until exhaustion, n=15) and stage 2 (exercise lasting for 60 minutes, n=45). The design consisted of groups: control (C), control exercise (CE), caffeine exercise (CFNE), coffee exercise (CAFE) and maté tea exercise (CME). For stage 1, the performance, the differences in the total body mass and glucose (post- versus pre-exercise), and lipolytic activity were compared. For the stage 2, comparisons were made among the differences in the total body mass and glucose (post- versus pre-exercise), lipolytic activity, blood lactate level and muscular glycogen content. The data were presented as average ± standard error. The data were analyzed by means of general linear models and the deltas by the orthogonal contrasts technique. Pearsons linear correlation was used to check the association between the variables of interest. Results. In stage 1, no differences were observed between the groups in terms of performance. The post-exercise total body mass, when compared to the pre-exercise, decreased for the CE (188 per cent ), CFNE (273 per cent ), CAFE (319 per cent ) e CME (204 per cent ) groups, when compared to C. There was no difference for glucose (post- versus pre-exercise) between groups. An increase of 92 per cent in lipolysis was observed in the CAFE group, when compared to C. In stage 2, there was a decrease in the post-exercise total body mass, when compared with pre-exercise, in the CE and CFNE (263 per cent ), CAFE (230 per cent ) and CME (183 per cent ) groups. An increase in post-exercise glucose, in comparison with the pre-exercise, was observed in groups CFNE (343 per cent and 220 per cent , when compared to C and CE, respectively) and CME (179 per cent ), when compared to C. Lipolysis increased for the CFNE and CAFE groups when compared to groups C (150 per cent and 233 per cent , respectively) and CE (51 per cent and 101 per cent , respectively). No significant differences were observed between the groups for the blood lactate and the muscular glycogen levels. It wasn\'t observed correlation between the different variables, for stage 1 and 2. Conclusion. Following exercise until exhaustion, the group which ingested coffee presented an increase in the lipolytic activity. After exercise lasting 60 minutes, the group which ingested caffeine, as well as that which ingested maté tea, presented increased levels of glucose. The animals which ingested caffeine or coffee presented increased levels of lipolysis

Lorsque l'allaitement est impossible, du lait maternel pasteurisé (LMP) est préférentiellement administré, en particulier aux nouveau-nés hospitalisés. La pasteurisation de Holder (62,5°, 30 min) est appliquée pour des raisons sanitaires mais pourrait réduire l'absorption des lipides via l'inactivation des lipases endogènes du lait. L’homogénéisation du LMP pourrait contrer cet effet négatif en augmentant la surface disponible pour l’adsorption des enzymes. L’objectif de cette thèse était d’étudier l’impact de la pasteurisation de Holder et de l’homogénéisation par ultrasons sur la digestion du LM chez le nouveau-né. Un modèle de digestion in vitro a été mis en place pour évaluer la digestion gastro-intestinale de LM cru (LMC) vs. LMP aux stades « nouveau-né à terme » ou « prématuré ». Une étude clinique a été menée chez des nouveau-nés prématurés pour comparer la digestion gastrique de (A) LMC vs. LMP et (B) LMP vs. LM pasteurisé et homogénéisé (LMPH).La pasteurisation et l’homogénéisation ont modifié la structure initiale du LM, ses cinétiques digestives et sa désintégration structurale. In vitro, la pasteurisation a réduit la lipolyse gastrique au stade à terme, alors qu’aucun impact n'a été observé au stade prématuré. La lipolyse intestinale, in vitro, a été réduite. In vivo, la pasteurisation a accélérée la protéolyse gastrique de la lactoferrine et a réduit celle de l’a-lactalbumine. L'homogénéisation a accéléré la lipolyse et la protéolyse de l'albumine sérique. Concernant les conditions physiologiques, l’activité lipolytique postprandiale était augmentée après adm / When breastfeeding is not possible, pasteurized human milk (PHM) from milk banks is preferentially administered, especially for vulnerable hospitalized newborns. Holder pasteurization (62.5°, 30 min) is applied for sanitary reasons but may reduce fat absorption through inactivation of milk endogenous lipases. This could be counteracted by homogenization of PHM through an increase of the specific surface available for enzyme adsorption. The objective of this thesis was to study the impact of Holder pasteurization and ultrasonic homogenization on the digestion of HM in the newborn. An in vitro dynamic model was used to evaluate the gastrointestinal digestion of raw HM (RHM) vs. PHM at preterm and term stages. A clinical trial was conducted on hospitalized preterm newborns for comparing the gastric digestion of (A) RHM vs. PHM and (B) PHM vs. pasteurized-homogenized HM (PHHM). Pasteurization and homogenization affected the HM initial structure and its digestive kinetics and structural disWhile gastric lipolysis was reduced after pasteurization in term in vitro study, no impact was observed at the preterm stage. Intestinal lipolysis, in vitro, was reduced by pasteurization. Gastric proteolysis was selectively affected by pasteurization, being, in vivo, faster for lactoferrin and slower for a-lactalbumin. Homogenization increased lipolysis and proteolysis of serum albumin. Some physiological gastric conditions were affected by treatments: RHM had enhanced postprandial lipolytic activity and PHHM had a reduced gastric emptying time. The in vivo data described here may help to i

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

In subclinical hypothyroidism (SH), high levels of circulating thyroid stimulating hormone (TSH) maintain normal thyroid hormone levels, despite mild thyroid failure. SH is associated with cardiovascular disease and insulin resistance, although the underlying pathophysiology is not fully understood. We hypothesized that TSH may inhibit insulin action in adipocytes. To investigate this relationship, we studied primary human differentiated adipocytes. Abdominal subcutaneous adipose tissue samples were obtained (approved by OHSN-REB) from 16 weight-stable patients undergoing elective abdominal surgery. We stimulated adipocytes differentiated from stromal preadipocytes with 5 mU/ml TSH and/or 100 nM insulin, and assessed acute insulin signaling, lipogenesis and glucose uptake. Immunoblot analysis revealed that TSH suppressed insulin-stimulated Akt phosphorylation by 45% (n=5; p = 0.01). When adipocytes were pre-incubated with conventional protein kinase C (cPKC) inhibitor Gö6976, TSH inhibition was blocked. Our data indicate that TSH inhibits insulin-stimulated lipogenesis (up to 37%), but depends on BMI. Insulin-stimulated glucose uptake was enhanced by 36% and also correlated with BMI. This data suggests that TSH can modulate adipocyte insulin signaling.

Une augmentation de l’hypertrophie et l'hyperplasie des adipocytes est au coeur du développement de l'obésité. Nous avons déjà constaté que 14-3-3zeta (14-3-3ζ), une protéine d’échafaudage moléculaire, a plusieurs rôles essentiels dans l'adipogenèse. Cependant, les contributions de 14-3-3ζ dans la fonction des adipocytes matures ne sont pas connues. Les cellules 3T3-L1 et souris dépourvues de 14-3-3ζ dans les adipocytes (adi14-3-3ζKO) ont été utilisés pour examiner le rôle de 14-3-3ζ dans la lipolyse. L’élimination de 14-3-3ζ dans les cellules 3T3-L1 par l’ARNi a réduit significativement la lipolyse stimulée par l'isoprotérénol (un agoniste bêta adrénergique), la forskoline (un activateur de l’adénylate cyclase) et le dibutyryl AMPc (dbcAMP). Les analyses par qPCR ont démontré des réductions significatives d’adipose triglyceride lipase (Atgl) et lipase hormonsensible (Hsl) au niveau transcriptionnel. De plus, une réduction au niveau des substrats de la PKA phosphorylés et totaux tels que HSL et CREB, a été détectée par Western Blot dans les 3T3-L1 appauvris en 14-3-3ζ. Ces résultats in vitro ont été récapitulés in vivo, car des diminutions des taux phosphorylés et totaux de HSL ont été observés dans le tissu adipeux gonadique des souris adi14-3-3ζKO. Les souris adi14-3-3ζKO et les explants gonadiques ont également montré une lipolyse affaiblie après des injections i.p de l’agoniste bêta 3-adrénergique CL-316,243 et un traitement de l’isoprotérénol respectivement. De manière intéressante, une diminution de l’expression de 14-3-3ζ dans les cellules 3T3-L1 et les souris adi14-3-3ζKO a mené à une diminution des caractéristiques des adipocytes matures telles que les niveaux d’ARNm de Pparg, Lpl et Fabp4, les niveaux de PPARγ, le contenu en triglycérides et l'incorporation de Oil Red-O. Collectivement, ces résultats démontrent que 14-3-3ζ joue un rôle essentiel en facilitant la lipolyse et en déterminant la maturité des adipocytes. / Altered hypertrophy and hyperplasia of adipocytes lie at the core of the development of obesity. We previously demonstrated that the molecular scaffold 14-3-3zeta (14-3-3ζ) had essential roles in adipogenesis. However, the contributions of 14-3-3ζ to mature adipocyte function are not known. 3T3-L1 cells and tamoxifen-inducible adipocyte-specific 14-3-3ζ knockout mice (adi14-3-3ζKO) models were used to examine the roles of 14-3-3ζ in lipolysis. siRNA-mediated knockdown of 14-3-3ζ impaired lipolysis in 3T3-L1 cells stimulated by the beta-adrenergic agonist isoproterenol (Iso), forskolin (an activator of adenylyl cyclase) and dibutyryl cAMP (dbcAMP). qPCR analyses revealed significant reductions in lipase transcript levels (Atgl and Hsl). Furthermore, reductions in the phosphorylated and total levels of PKA substrates such as HSL and CREB were detected in 14-3-3ζ-depleted 3T3-L1 lysates by immunoblotting. These findings were recapitulated in vivo, as reductions in phosphorylated and total HSL levels were detected in the gonadal adipose tissue of adi14-3-3ζKO mice. adi14-3-3ζKO mice and gonadal explants also displayed impaired lipolysis following i.p CL-316,243 (a beta-3 adrenergic agonist) injections and Iso treatment respectively. Interestingly, decreased 14-3-3ζ expression in 3T3-L1 cells and mice revealed reductions in characteristics of a mature adipocyte, such as Pparg, Lpl, and Fabp4 transcript levels, PPARγ levels, triglyceride content, and Oil Red O (ORO) incorporation. Collectively, these results demonstrate that 14-3-3ζ has essential roles in facilitating lipolysis and determining adipocyte maturity.

Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. Given the importance of appropriately timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

Rainbow trout (Oncorhynchus mykiss) is an important freshwater fish whose glucose intolerance, white muscle lactate retention and high lipolytic inertia, have interested comparative physiologists for decades. Recent advancements in mammalian G-protein coupled receptor deorphanization research have identified many endogenous metabolites as regulators of energy metabolism, including lactate and long-chain fatty acids. In addition to being essential metabolic fuels, lactate and long-chain fatty acids regulate lipolysis and lipogenesis by binding to hydroxycarboxylic acid receptor 1 (HCAR1) and the free fatty acid receptors (FFAR1 and 4), respectively. Therefore, the goal of this thesis was to quantify the effects of exogenous lactate and lipids on glucose and fatty acid mobilization in rainbow trout and identify potential signaling mechanisms by monitoring the expression and activity of key glycolytic, gluconeogenic, lipolytic, lipogenic and β-oxidation targets in the liver, muscle and adipose tissue. In Chapter 2, in vivo measurements of metabolic fuel kinetics show that lactate (i) strongly reduced hepatic glucose production by substituting glucose for lactate and (ii) exhibited no lipolytic suppression suggesting HCAR1 signaling is weak in trout. In Chapter 3, in vivo measurements of energy mobilization show that Intralipid strongly induced lipolysis by saturating circulating lipases while transcriptional induction of gluconeogenesis compensates for the acute reduction in hepatic glucose production. Intralipid infusion increased total fatty acid concentration and altered fatty acid composition while suppressing lipid metabolism of trout liver and adipose tissue. In Chapter 4, I identify the presence (hcar1 and ffar1) and absence (ffar4) of these G-protein coupled receptor genes in the rainbow trout genome and describe their evolutionary origins, using in silico approaches of microsynteny, amino acid sequence similarity and critical residue conservation. However, their importance in fish physiology remains relatively unknown, thus future studies are warranted to further investigate such metabolic signals.