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Purpose Observational studies show that longer breastfeeding periods reduce maternal risk of type 2 diabetes mellitus. However, it is currently unknown if the long-term benefits of breastfeeding for maternal glucose homeostasis are linked to changes in the endocrine pancreas. Methods We presently evaluated functional, morphological and molecular aspects of the endocrine pancreas of mice subjected to two sequential cycles of pregnancy and lactation (L21). Age-matched mice not allowed to breastfeed (L0) and virgin mice were used as controls. Results L21 mice exhibited increased tolerance and increased glucose-stimulated insulin secretion (GSIS) by isolated islets. Pancreatic islets of L21 mice did not present evident morphological changes to justify the increased GSIS. On the other hand, islets of L21 mice exhibited a reduction in Cavb3 and Kir6.2 expression with concordant increased intracellular Ca2+ levels after challenge with glucose. Conclusion Altogether, the present findings show the breastfeeding exerts long-term benefits for maternal endocrine pancreas by increasing intracellular Ca2+ levels and GSIS.

Antenatal corticosteroid therapy is used to reduce neonatal mortality in preterm infants but it is currently unknown whether this intervention affects lipid metabolism at the peripartum. This study aimed to evaluate if antenatal corticosteroid therapy in pregnant rats and women affects lipid metabolism during early lactation. We evaluated women at risk of preterm delivery that received corticosteroid therapy (CASE) and women that were not exposed to corticosteroid and were not at risk of preterm delivery (CONTROL). Samples were collected to measure serum and milk triacylglycerol (TAG) three days after delivery. Rats were treated with dexamethasone (DEX) between the 15th and the 20th days of pregnancy. Samples were collected at different days after delivery (L3, L8 and L14). TAG was measured in serum, liver and mammary gland (MG). TAG appearance rates were measured after tyloxapol injection and gavage with olive oil. We also evaluated the expression of key genes related to lipid metabolism in the liver and in the MG and hepatic phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). CASE volunteers delivered earlier than CONTROL but presented unaltered milk and serum TAG concentrations. Early lactating DEX rats exhibited increased TAG in serum, MG and milk. No changes in CD36 and LPL were detected in the MG and liver. Early lactating DEX rats displayed increased TAG appearance rate and reduced hepatic AMPK/ACC phosphorylation. Our data revealed that antenatal corticosteroid therapy reduces hepatic AMPK/ACC phosphorylation during early lactation that reflects in increased TAG concentration in serum, MG and milk.

Pancreatic islets from pregnant rats develop a transitory increase in the pancreatic β-cell proliferation rate and mass. Increased apoptosis during early lactation contributes to the rapid reversal of those morphological changes. Exposure to synthetic glucocorticoids during pregnancy has been previously reported to impair insulin secretion, but its impacts on pancreatic islet morphological changes during pregnancy and lactation have not been described. To address this issue, we assessed the morphological and molecular characteristics of pancreatic islets from rats that underwent undisturbed pregnancy (CTL) or were treated with dexamethasone between the 14th and 19th days of pregnancy (DEX). Pancreatic islets were analyzed on the 20th day of pregnancy (P20) and on the 3rd, 8th, 14th and 21st days of lactation (L3, L8, L14 and L21, respectively). Pancreatic islets from CTL rats exhibited transitory increases in cellular proliferation and pancreatic β-cell mass at P20, which were reversed at L3, when a transitory increase in apoptosis was observed. This was followed by the appearance of morphological features of pancreatic islet neogenesis at L8. Islets from DEX rats did not demonstrate an increase in apoptosis at L3, which coincided with an increase in the expression of M2 macrophage markers relative to M1 macrophage and T lymphocyte markers. Islets from DEX rats also did not exhibit the morphological characteristics of pancreatic islet neogenesis at L8. Our data demonstrate that maternal pancreatic islets undergo a renewal process during lactation that is impaired by exposure to DEX during pregnancy.

Aims The present investigation evaluated whether pregnancy followed by lactation exerts long-term impacts on maternal hepatic lipid metabolism. Main methods Female mice were subjected to two pregnancies, after which they were either allowed to breastfeed their pups for 21 days (L21) or had their litter removed (L0). Age-matched virgin mice were used as controls (CTL). Three months after the second delivery, serum was collected for lipid profiling, and fragments of liver were used to assess lipid content and to evaluate the key steps of de novo non-esterified fatty acid (NEFA) synthesis, esterification and β-oxidation, very low density lipoprotein (VLDL) assembly and secretion and autophagy. Key findings L0 exhibited a significant increase in hepatic TG and reduced apolipoprotein B-100 (ApoB-100) expression. L21 mice had increased ATP citrate lyase (ACLY) activity and reduced acetyl-CoA carboxylase (ACC) phosphorylation but no increased hepatic TG. On the other hand, L21 mice had reduced hepatic sequestosome 1 (SQSTM1/p62) levels. Increased high density lipoprotein (HDL) cholesterol and hepatic apolipoprotein A-1 (ApoA-1) expression were found exclusively in L21. Significance The present study reveals that long-term hepatic lipid accumulation is induced by the history of pregnancy without lactation. On the other hand, reduced SQSTM1/p62 levels indicate that increased autophagic flux during life may prevent hepatic fat in dams subjected to lactation. Lactation after pregnancy is also obligatory for a long-term increase in maternal HDL. The present data may contribute to the understanding of the mechanisms leading to elevated cardiometabolic risk in women limited to short periods of lactation.

The progeny of rats born and breastfed by mothers receiving dexamethasone (DEX) during pregnancy exhibits permanent reduction in body weight and adiposity but the precise mechanisms related to this programming are not fully understood. In order to clarify this issue, the present study investigated key aspects of lipoprotein production and lipid metabolism by the liver and the intestine that would explain the reduced adiposity seen in the adult offspring exposed to DEX in utero. Female Wistar rats were treated with DEX (0.1 mg/kg/day) between the 15th and the 21st days of pregnancy, while control mothers were treated with vehicle. Male offspring born to control mothers were nursed by either adoptive control mothers (CTL/CTL) or DEX-treated mothers (CTL/DEX). Male offspring born to DEX-treated mothers were nursed by either control mothers (DEX/CTL) or adoptive DEX-treated mothers (DEX/DEX). We found that only the male DEX/DEX offspring had reduced adiposity. Additionally, male DEX/DEX progeny had lower circulating triacylglycerol (TAG) levels only in fed-state. The four groups of offspring presented similar energy expenditure, respiratory quotient and very low-density lipoprotein (VLDL) production. On the other hand, DEX/DEX rats displayed reduced TAG levels after gavage with olive oil and reduced expression of fatty acid translocase Cd36 (Fat/Cd36) and peroxisome proliferator-activated receptor γ (Pparg) in the jejunum. Altogether, our study supports the notion that reduced fat absorption by the jejunum may contribute to the lower adiposity of the adult offspring born and breastfed by mothers treated with DEX during pregnancy.

Orientador: Gabriel Forato Anhê Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Adaptações fisiológicas durante a gravidez são fundamentais para garantir o desenvolvimento fetal adequado. Entretanto, o ambiente adverso intrauterino, em ratos e humanos, ocasionado por uma restrição calórica ou o excesso de glicocorticoides, está relacionado ao baixo peso ao nascer. Embora os glicocorticoides sejam amplamente utilizados em partos prematuros, alguns estudos demonstram a relação entre o excesso de glicocorticoide (GC) e menor absorção de ácidos graxos, além de menor expressão da lipase lipoproteica no tecido adiposo e menor comprimento do intestino delgado nos primeiros três meses de vida. Assim, foi levantada a hipótese de que o repetidas doses de GC, no final da gestação, poderia provocar alterações no metabolismo lipídico na vida adulta de proles nascidas de mães que foram tratadas com dexametasona. Este trabalho utilizou ratas Wistar com 12 semanas de vida e o tratamento com dexametasona, 100µg/kg/dia subcutânea do 15º ao 21º dia de prenhez, ocorreu em metade das ratas prenhes e na outra metade foi administrado injeções subcutâneas de salina. Após o parto foi realizada a metodologia de cross-fostering (adoção cruzada) entre as proles para que, assim, fosse possível observar a relação entre o nascimento e a amamentação, frente ao tratamento proposto, e os possíveis impactos metabólicos das proles. Para alcançar o objetivo deste trabalho as expressões gênicas foram analisadas pela Reação de Cadeia de Polimerase (PCR), além de analisar a expressão de micro RNA (miR). Juntamente foram realizados testes para a verificação da absorção de gordura, motilidade gastrointestinal, acompanhamento da evolução de peso desde o primeiro dia de lactação até a 12ª semanas de vida da prole de machos. Em paralelo foram realizados testes para acompanhar a produção de VLDL e também dosagens de triglicérides, além da quantificação da atividade enzimática relacionada com a oxidação mitocondrial e o teste de respirometria (para avaliar o gasto calórico indireto). Nossos resultados demonstraram que proles de machos nascidas e a amamentadas por uma rata tratada com repedidas doses de dexametasona (grupo DD) no período perinatal foi capaz de provocar redução da expressão de CD 36, gene relacionado com o transporte de ácidos graxos, em jejuno, e também foi constatado menor comprimento intestinal, maior motilidade entérica e redução da trigliceridemia durante o estado alimentado. Não sendo observadas alterações de atividade enzimática mitocondrial ou maior gasto calórico, assim como ausência de modificações na expressão dos genes relacionados aos miR quando efetuada a comparação entre o grupo DD e o controle (prole nascida e amamentada por rata sem tratamento com CG) Abstract: Physiological adaptations during pregnancy are essential to ensure adequate fetal development. However, the adverse intrauterine environment, in rats and humans, caused by caloric restriction or excess of glucocorticoids, is related to low birth weight. Although glucocorticoids are widely used in premature births, some studies demonstrate a relationship between excess glucocorticoid (GC) and reduced-fat absorption, in addition to lower expression of lipoprotein lipase in adipose tissue and shortest length at small intestine in rats with tree months of life. Thus, the hypothesis was raised that repeated doses of GC, at the end of pregnancy, could cause changes in lipid metabolism in the adult life of children born from mothers who were treated with dexamethasone. This work used Wistar rats with 12 weeks of age and treatment with dexamethasone, 100µg / kg / day subcutaneously from 15 to 21 days of pregnancy, occurred in half of the rats that we considered pregnant, the other half was administered injections of saline (subcutaneously). We used the cross-fostering methodology, among the offspring so that, therefore, it is possible to observe the relationship between birth and breastfeeding and the possible metabolic effects of the offspring. To achieve the objective of this work, we analyzed the gene expressions by the Polymerase Chain Reaction (PCR), in addition we analyzed the micro RNA (miR) expression, the fat absorption and the gastrointestinal motility were verified. We also checked the weight gain from the first day of lactation until the 12th week of the life of male rat. In parallel, tests were carried out to monitor the production of VLDL and also triglyceride measurements, in addition to quantifying enzyme activity, which is useful with mitochondrial oxidation and respirometry test (to assess indirect caloric expenditure). Our results showed that birth and breastfeeding through repeated doses of dexamethasone (group DD) in the perinatal period were able to reduce the expression of CD 36, a gene related to the transport of free fat acid, jejunum, and it was also found a shorter intestinal length, greater enteric mobility and reduced triglyceridemia during food status. It was not observed change in mitochondrial enzyme activity neither increased caloric expenditure among the group DD and the control (birth and breastfeeding form a rat without treatment with GC). The genes related to miR remained unchanged between the groups analyzed Doutorado Farmacologia Doutor em Farmacologia CNPQ 142047/2016-3

Aims The aim of the present study was to clarify if in utero exposure to DEX would affect the development of different types of pancreatic endocrine cells during postnatal life. Main methods We investigated morphological and transcriptional features of both pancreatic β- and α-cell populations within the pancreatic islets during the early postnatal life of rats born to mothers treated with DEX (0.1 mg/kg) from day 14 to 19 of pregnancy. Untreated pregnant Wistar rats of the same age (12-week-old) were used as control (CTL). Pups were euthanized on the 1st, 3rd and 21st (PND1, PND3 and PND21, respectively) days of life, regardless of sex. Serum insulin and glucagon levels were also evaluated. Key findings Rats born to DEX-treated mothers exhibited increased pancreatic α-cell mass, circulating glucagon levels and Gcg, Pax6, MafB and Nkx2.2 expression. Rats born to DEX-treated mothers also presented a rise in serum insulin levels on the PND3 that was paralleled by reduced β-cell mass. Such increase in serum insulin levels, instead, was associated with increased expression of genes associated to insulin secretion such as Gck and Slc2a2. Significance Altogether, the present data reveals yet unknown changes in endocrine pancreas during early postnatal life of rats exposed to DEX in utero. Such data may contribute to the understanding of the metabolic features of rats born to DEX-treated mothers.

Agomelatine (AGO) is an antidepressant drug with agonistic activity at melatonin receptor 1 (MT1) and MT2 and with neutral antagonistic activity at serotonin receptor 5-HT2C. Although experimental studies show that melatonin reduces hypertriglyceridemia and hepatic steatosis induced by excessive fructose intake, no studies have tested if AGO exerts similar actions. To address this issue we have treated male Wistar rats with fructose (15% in the drinking water) and/or AGO (40 mg/kg/day) for two weeks. AGO reduced body weight gain, feeding efficiency and hepatic lipid levels without affecting caloric intake in fructose-treated rats. AGO has also decreased very low-density lipoprotein (VLDL) production and circulating TAG levels after an oral load with olive oil. Accordingly, treatment with AGO reduced the hepatic expression of fatty acid synthase (Fasn), a limiting step for hepatic de novo lipogenesis (DNLG). The expression of apolipoprotein B (Apob) and microsomal triglyceride transfer protein (Mttp) in the ileum, two crucial proteins for intestinal lipoprotein production, were also downregulated by treatment with AGO. Altogether, the present data show that AGO mimics the metabolic benefits of melatonin when used in fructose-treated rats. This study also suggests that it is relevant to evaluate the potential of AGO to treat metabolic disorders in future clinical trials.

Aims: Rats born to mothers subjected to caloric restriction (CR) during pregnancy and lactation exhibit reduced birth weight and adiposity in adult life. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that play important roles in adipocytes differentiation. The aim of our study is to evaluate if antenatal exposition PPAR-gamma (Pioglitazone) or PPAR-alpha (Gemfibrozil) activators are able to affect energy metabolism in the offspring born from rats subjected to RC during pregnancy and lactation. Methods: Wistar rats were kept under CR from the 11th day of pregnancy until the 21st day after delivery. Pioglitazone (10mg/kg) or Gemfibrozil (90mg/kg) were offered during the period of CR (offspring were CR+Pio and CR+Gem, respectively). Adult female offspring were subjected to indirect calorimetry, thermal infra-red imaging of BAT, VLDL production assay and DEXA for body composition. Results: The body weight in adult CR+Gem, but not in CR+Pio, was higher than that of offspring born to CR mother. CR+Pio had reduced energy expenditure (EE) and higher relative weight of the perigonadal and retroperitoneal fat pads. On the other hand, adult CR+Gem had no changes in EE and visceral adiposity when compared to RC but presented increased whole-body adiposity as revealed by DEXA analysis. Although hepatic triglycerides (TG) or VLDL production were not altered in offspring born to CR mothers and in CR+Pio, these parameters were downregulated in RC+Gem. Both RC+Pio and RC+Gem had reduced temperature of the interscapular brown adipose tissue when compared to CR. Conclusion: Maternal activation of PPAR-gamma increases visceral adiposity and reduces EE in the offspring born to mothers subjected to CR during pregnancy. Maternal activation of PPAR-alpha instead, increased body weight and reduced hepatic steatosis of the offspring born to mother exposed to CR. Disclosure V.B. Veronesi: None. J.C. Santos-Silva: None. D.N. Souza: None. C.J. Teixeira: None. F.B. Hecht: None. N. Tobar: None. M. Rodrigues Pioli: None. G.F. Anhe: None. Funding Funda??o de Amparo ? Pesquisa do Estado de S?o Paulo

Uma série de estudos mostra que a duração do período de amamentação tem uma relação inversa com o risco das mães desenvolverem Diabetes Mellitus tipo 2. O presente estudo dividiu fêmeas de camundongo em três grupos: Controle (Virgem), sem lactação após o parto (L0) e 21 dias de lactação após o parto (L21). Esses animais foram submetidos a dois ciclos de gestação e desmame. Dois meses depois do último desmame, as fêmeas foram submetidas ao teste de tolerância à glicose (GTT) e tolerância à insulina (ITT). Após o sacrifício dos animais, foram coletados fígado e pâncreas para rastreamento de lipídeos no tecido hepático, secreção de insulina e investigação de microRNAs (miRNAs) nas ilhotas pancreáticas. As fêmeas do grupo L21 apresentaram maior tolerância à glicose e não houve diferença na tolerância à insulina entre os grupos. No grupo L0, ocorreu maior acúmulo de gordura no fígado. As fêmeas do grupo L21 adquiriram maior capacidade de secretar insulina e o miRNA 7b-3p e 7a-3p estava menos expresso nesses mesmos animais.

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

Recent studies show that the metabolic effects of fructose may vary depending on the phase of its consumption along with the light/dark cycle. Here, we investigated the metabolic outcomes of fructose consumption by rats during either the light (LPF) or the dark (DPF) phases of the light/dark cycle. This experimental approach was combined with other interventions, including restriction of chow availability to the dark phase, melatonin administration or intracerebroventricular inhibition of adenosine monophosphate-activated protein kinase (AMPK) with Compound C. LPF, but not DPF rats, exhibited increased hypothalamic AMPK phosphorylation, glucose intolerance, reduced urinary 6-sulfatoxymelatonin (6-S-Mel) (a metabolite of melatonin) and increased corticosterone levels. LPF, but not DPF rats, also exhibited increased chow ingestion during the light phase. The mentioned changes were blunted by Compound C. LPF rats subjected to dark phase-restricted feeding still exhibited increased hypothalamic AMPK phosphorylation but failed to develop the endocrine and metabolic changes. Moreover, melatonin administration to LPF rats reduced corticosterone and prevented glucose intolerance. Altogether, the present data suggests that consumption of fructose during the light phase results in out-of-phase feeding due to increased hypothalamic AMPK phosphorylation. This shift in spontaneous chow ingestion is responsible for the reduction of 6-S-Mel and glucose intolerance.

Orientadores: Silvana Auxiliadora Bordin da Silva, Gabriel Forato Anhê Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A gravidez é caracterizada por uma série de modificações fisiológicas maternas para se adaptar tanto à presença do feto quanto às alterações características da gravidez. Uma das alterações ocorridas nesse período é a resistência periférica à insulina, que leva ao aumento do volume e proliferação de células ?. O período pós-parto é caracterizado por um aumento da apoptose deste tipo celular e, considerando que o remodelamento funcional do pâncreas ocorre numa estreita janela para a manutenção da homeostasia energética materna, aventamos que o excesso de glicocorticoides pode alterar esse remodelamento e, a longo prazo, impactar na resposta secretora apropriada. Neste trabalho, verificamos a participação da UPR (Unfolded Protein Response) e apoptose frente ao excesso de glicocorticoide, no remodelamento pancreático pós-natal. Para isso, foram realizadas dosagens bioquímicas (glicemia randômica, colesterol, triglicérides, insulina), hormonal no periparto (estradiol) e, além disso, ilhotas pancreáticas foram utilizadas para avaliação da apoptose (citometria, PCR, Western Blot). Nossos resultados mostram que o excesso de glicocorticoides no final da prenhez reduz a expressão da via da UPR (ATF4, CHOP), associado com a diminuição da expressão de TRB3 e aumento da pAKT, com consequente redução da fragmentação de DNA. O tratamento com glicocorticoides leva também à redução da expressão de genes pró-apoptóticos (CD40L e Cidea) tanto no período gestacional (20 dias de prenhez) quanto no terceiro dia de lactação Abstract: Pregnancy is characterized by a series of physiologicalmaternal changes to adapt to the fetus presence and the characteristic changes of pregnancy. One of the changes in this period is peripheral insulin resistance, which leads to increased volume and proliferation of ? cells. The postpartum period is characterized by increased apoptosis of this cell type and, considering that the functional remodeling of the pancreashappens ina close time window to maintain maternal energy homeostasis, we hypothesized that excess of glucocorticoids can change this remodeling and, in the long-term, impact the appropriate secretory response. In this work we see the participation of the UPR (Unfolded Protein Response) and apoptosis compared to excess glucocorticoids, in postpartum pancreatic remodeling. Forthis was carried out biochemical tests (random glucose, cholesterol, triglycerides, insulin), peripartum hormone (estradiol) and, moreover, pancreatic islets were used for assessment of apoptosis (flow cytometry, PCR, Western blot). Our results show that excess glucocorticoid reduces the expression of important UPR pathway (ATF4, CHOP), associated with decreasedTRB3 expression and increased pAKT, with consequent reduction in DNA fragmentation. Treatment with glucocorticoids also leads to reduced expression of pro-apoptotic genes (CD40L and Cidea) both during pregnancy (20 days of pregnancy) and atthe third day of lactation Mestrado Farmacologia Mestre em Farmacologia CNPQ 159658/2015-2 CAPES 01-P-3488/2014, 01-P-4348/2015 FAPESP