OBJECTIVE: T2D is a metabolic disease that is a significant health problem throughout many populations. Increased incidence of T2D across the age spectrum makes preventive measures for this disease a top healthcare priority. Physiological changes such as expression of pro-inflammatory T cell cytokines, insulin resistance, and pancreatic beta cell dysfunction play major roles in the onset of T2D. Current treatments include lifestyle changes with oral medications and/or synthetic insulin therapy. While treatments aim to normalize blood glucose and increase insulin sensitivity in patients diagnosed with T2D, efforts are growing to find preventative therapies for prediabetes, a condition where blood glucose levels are higher than normal but are under the threshold determined for a diabetes diagnosis. Metformin, a well-known first-line recommendation for treating T2D, in conjunction with lifestyle modification may be a viable preventative measure to delay the onset of T2D. Previous study results have created momentum to generate data promoting metformin use as an off-label preventative drug for T2D. To identify a therapeutic intervention that may help to shift T cell cytokine profiles from being pro-inflammatory and diabetogenic to anti-inflammatory, we investigated the effects of metformin on immune cell function in prediabetic patients. It is known that one effect of metformin is activating AMPK, which secondarily decreases inflammation. We therefore hypothesized metformin affects immune cell function by modulating genes in the AMPK pathway. METHODS: We recruited 49 subjects using EPIC database to screen patients with appointments at the Nutrition and Weight Management Center at Boston Medical Center. Forty-nine pre-metformin and 13 post- metformin blood samples were collected from subjects at baseline and after three months of taking metformin, respectively. Ficoll was utilized to separate and extract PBMCs. I activated PBMCs with LPS or CpG for 24 hours, and anti-CD3/CD28 for 24 or 40 hours. Then I isolated and reverse transcribed RNA, producing cDNA. We ran a human AMPK signaling qRT-PCR array on the 40-hour anti-CD3/CD28 activated PBMCs from 4 randomly chosen subjects and analyzed data to investigate candidate targets in the AMPK pathway possibly modulated by metformin. I designed primers for six chosen targets and ran qRT-PCR comparing the pre- and post-metformin dataset of 13 subjects, using the generated human gene-specific primers to see if these genes were affected across the dataset. RESULTS: Total sample population was n=13. The majority of subjects were African American females. The study participants were considered prediabetic when A1C measured between 5.7-6.4%. Median A1C and BMI averaged at 5.8% and 38.6 kg/m2  2.48 (mean  SEM), respectively. There was an expected decrease in BMI as metformin is associated with weight loss. To understand how metformin may affect genes in the AMPK pathway, qRT-PCR array analysis of the 40-hour anti-CD3/CD28 activated PBMCs in a subset of 4 subjects was used to create a volcano plot. The plot demonstrated that out of the possible gene candidates, SLC2A4, LIPE, INSR, CRY1, GAPDH, and STK11 had the greatest log2 fold change and –log (p-value). Further analysis on the 4 subjects compared delta Ct values and relative gene expression showing CRY1, a circadian function gene, had a significant decrease in expression (p=0.03, n=4, paired t-test). Primers were designed for the six candidate genes and used to run qRT-PCR on the entire dataset of 13 subjects. There was a significant decrease in expression of STK11 in 24-hour non-stimulated PBMCs (p=0.008, n=12, paired t-test) and CRY1 in 24-hour anti-CD3/CD28 activated PBMCs (p=0.04, n=12, paired t-test). There was a significant increase in expression of SLC2A4 in 24-hour CpG activated PBMCs (p=0.02, n=12, paired t-test). Furthermore, GLUT4 was detected in CpG activated immune cells and gene expression was increased in cells from subjects post-metformin treatment. CONCLUSIONS: Further investigation is required to examine how metformin decreases the expression of CRY1 and how this decrease associates with pro-inflammatory cytokine expression. STK11 expression was decreased in non-stimulated cells but did not show any trend in the activated conditions. Additional research is warranted to see if these results can be repeated, and if so, more work will be needed to define the link between CRY1/STK11 and metformin-driven AMPK activation in immune cells. Protein expression analysis will be required to support our gene expression data. Overall, these findings initiate our understanding of how AMPK activation and changes in cellular metabolism activate pathways leading to cytokine secretion by immune cells. Further study of the downstream effects of metformin and how it may change inflammatory cytokine profiles will strengthen the evidence identifying metformin as a viable preventative therapy for prediabetic patients.

In seasonal mammals the duration of nocturnal melatonin secretion accurately reflects the environmental photoperiod. The endocrine rhythm is decoded by a specialised portion of the pituitary gland (the pars tuberalis, PT) which then relays this information to the pars distalis and hypothalamus, precipitating huge annual changes in physiology and behaviour. However how the PT decodes the melatonin signal is currently unknown. Melatonin influences gene transcription in the ovine PT at its onset and offset, and the phase relationship of these two groups is believed to form the underlying mechanism by which the PT integrates seasonal time. The transcripts induced at melatonin offset are understood to be under the control of a seasonally gated cAMP mechanism. Conversely processes involved in melatonin-mediated gene induction are currently not understood.The work in this thesis ultimately aims to reveal how the seasonal melatonin signal is decoded by the PT. To this end melatonin-mediated gene induction has been characterised through RNAseq, the highly displaced cohorts submitted to bioinformatic promoter analysis and the paradigm tested though in vitro modelling techniques.In this study a 1.5 h infusion with melatonin acutely regulated 219 transcripts in the ovine PT (115 induced, 104 repressed, >1.5 fold change), confirming previous association of several genes (including Cry1, MT1, Gadd45g, Nampt and Npas4) to rapid melatonin control. Gross promoter analysis of these groups indicated that the induced gene cohort was significantly enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transcriptional control. Further bioinformatic analysis specifically implicated the importance of transcription factors ZFP161 and PAX5 in melatonin-mediated gene induction in the PT. Several immortalised cell lines were screened for the presence of a functional melatonin receptor. Two strains (MCF7 oMT1 and NES2Y) exhibited significant attenuation of forskolin-mediated cAMP accumulation when co-treated with melatonin, a hallmark of melatonin Gαi-coupled protein receptor signalling. These lines were subsequently evaluated as models of melatonin-mediated gene induction of the sheep PT through ovine promoter reporter assays of Cry1, Nampt, NeuroD1 and Npas4. However, treatment with melatonin failed to evoke a reporter response suggesting that the cell line models were inadequately equipped to reflect PT biology. Subsequently a protocol was established to culture ovine PT explants culture which faithfully recapitulated melatonin mediated transcriptional dynamics in vitro, providing a possible tool for the future investigation of the PT. Lastly, previous work has shown the transcriptional profile of Npas4 to peak highly and transiently, pre-empting the expression of other melatonin-induced genes. Using a COS7 cell line model, heterologously-expressed NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. Collectively these data indicated NPAS4 to act as an immediate activator of melatonin regulated circuits

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A saudável interação entre o indivíduo e o meio depende do alinhamento entre a dinâmica fisiológica do primeiro e os periódicos movimentos da natureza. A interação entre tais ritmos por sua vez constitui-se em base e derivação do processo de evolução. O comprometimento de tal alinhamento representa um risco para a sobrevivência das espécies. Neste contexto, os organismos alinham seus ritmos fisiológicos a diferentes ciclos externos. Desta forma, ciclos endógenos são coordenados por relógios biológicos que determinam em nosso organismo, específicos ritmos em fase com a natureza, tais como ritmos circadianos (RC), cujo período aproxima-se de 24 horas. O peso corporal, a ingestão de alimentos e o consumo de energia são processos caracterizados pelo RC e a obesidade está associada a uma dessincronização deste processo. A modulação do RC é resultado da expressão dos clock gens CLOCK e BMAL1 que formam um heterodímero responsável pela transcrição gênica de Per1, Per2, Per3, Cry1 e Cry2. As proteínas codificadas por estes genes, uma vez sintetizadas, formam dímeros (PER-CRY) no citoplasma que, a partir de determinada concentração, retornam ao núcleo, bloqueando a ação do heterodímero CLOCK/BMAL1 na transcrição dos próprios genes, formando assim uma alça de retroalimentação negativa de transcrição e tradução. Estes genes asseguram a periodicidade e são significativamente expressos no núcleo supraquiasmático (SCN) do hipotálamo. Para estudar esse processo em camundongos normais e hiperalimentados, saciados e em estado de fome, foi utilizado um método de registro do comportamento alimentar baseado no som produzido pela alimentação dos animais, e a correlação destes estados metabólicos com a expressão de CLOCK, BMAL1, Per1, Per2, Per3, bem como das proteínas Cry1 e Cry2 no SCN, por análise de imagens obtidas em microscopia confocal. Camundongos suíços controle em estado de fome (CF) e saciados (CS) foram comparados com animais hiperalimentados com fome (HF) e saciados (HS). Nenhum grupo demonstrou diferença nos conteúdos CLOCK e BMAL1, indicando capacidade potencial para modular os ritmos biológicos. No entanto, as proteínas Per1, Per2, Per3 e Cry1 apresentaram menor expressão no grupo CS, mostrando uma diferença significativa quando comparados com o grupo CF (P<0,05), diferença esta não encontrada na comparação entre os grupos HF e HS. A quantidade de proteína Cry2 não foi diferente na mesma comparação. Os resultados do estudo indicaram que as alterações dos ritmos endógenos e exógenos, refletido pelo comportamento hiperfágico observado em camundongos hiperalimentados, pode ser devido a um defeito no mecanismo de feedback negativo associado ao dímero Cry-Per, que não bloqueia a transcrição de Per1 Per2, Per3 e Cry1 pelo heterodímero CLOCK-BMAL1. / The healthy interaction between the subject and the environment depends on the alignment between the physiological dynamics of the first one and the periodical movements of nature. The interaction between these rhythms in turn is based on the derivation and evolution process. The involvement of such an alignment is a risk to the survival of species. In this context the bodies line up their physiological rhythms to different external cycles. Thus, endogenous cycles are coordinated by biological clocks which determine in our organism specific rhythms in phase with the nature, such as Circadian Rhythms (CR) whose period is close to 24 hours. The body weight, the food intake and the energy consumption are processes characterized by the CR and the obesity is associated with a different timing of this process. The CR modulation is a result of the formulation of clock-gens CLOCK and BMAL1 who form an heterodimer responsible for the gene transcription of Per1, Per2, Per3, Cry1 e Cry2. The proteins encoded by these genes, once synthesized, form dimers (PER-CRY) in the cytoplasm that, depending on a given concentration, return to the core blocking the action of the CLOCK/BMAL1 heterodimer in the transcription of its own genes, thus forming a negative feedback loop of transcription and translation. These genes secure the periodicity and are significantly expressed in the hypothalamus suprachiasmatic nucleus. In order to study this process in regular, hyper-fed, hungry and satiated mice, we used a registration method of feeding behavior based on the sound produced by animal feeding and the relation between the metabolic states with the expression CLOCK, BMAL1, Per1, Per2, Per3, as well as the Cry1 and Cry2 proteins in the SCN, by analysis of images obtained in confocal microscopy. Control Swiss mice in state of hunger/ satiated were compared to hyper-fed animals in the same conditions. None of them showed difference in the CLOCK and BMAL1 contents, showing a potential capacity to modulate the biological rhythms. However, the Per1, Per2, Per3 and Cry1 proteins showed a minor expression in the CS group and a significant difference when compared to the CF group (P<0,05). This difference cant be found in the HF and HS groups. The results of the studies indicated that the endogenous and exogenous changes, reflected by the hyperphagic behavior observed in hyper-fed mice, may be due to a defect in the mechanism of negative feedback associated to the Cry-Per dimer, which has abolished the blocking mechanism of Per1 Per2, Per3 and Cry1 by the CLOCK-BMAL1 heterodimer.