RESUMO
Dolutegravir is recommended for all people living with HIV because of its efficacy, high barrier to resistance, favourable safety and tolerability profile, and affordability. Dolutegravir has the highest rates of viral suppression in pregnancy, therefore preventing perinatal HIV transmission. In view of these benefits, particularly for pregnant women, an important question is if dolutegravir is safe in pregnancy. Dolutegravir has been associated with metabolic complications, including weight gain and rare events of hyperglycaemia, that could affect maternal, fetal, and postnatal health. We review the current clinically and experimentally based literature on the implications of dolutegravir use for pregnant women and for developing embryos and fetuses. Possible effects on folate status, energy metabolism, adipogenesis, and oxidative stress are considered. In many instances, insufficient data are available, pointing to the need for additional research in this important area of HIV treatment.
Assuntos
Infecções por HIV , Gravidez , Humanos , Feminino , Infecções por HIV/tratamento farmacológico , Oxazinas , Compostos Heterocíclicos com 3 Anéis/efeitos adversos , PiperazinasRESUMO
BACKGROUND: Dolutegravir (DTG) is a recommended first-line regimen for all people with Human Immunodeficiency Virus (HIV) infection. Initial findings from Botswana, a country with no folate fortification program, showed an elevated prevalence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. Here we explore whether a low folate diet influences the risk of DTG-associated foetal anomalies in a mouse model. METHODS: C57BL/6 mice fed a folate-deficient diet for 2 weeks, were mated and then randomly allocated to control (water), or 1xDTG (2.5 mg/kg), or 5xDTG (12.5 mg/kg) both administered orally with 50 mg/kg tenofovir disoproxil fumarate 33.3 mg/kg emtricitabine. Treatment was administered once daily from gestational day (GD) 0.5 to sacrifice (GD15.5). Foetuses were assessed for gross anomalies. Maternal and foetal folate levels were quantified. FINDINGS: 313 litters (103 control, 106 1xDTG, 104 5xDTG) were assessed. Viability, placental weight, and foetal weight did not differ between groups. NTDs were only observed in the DTG groups (litter rate: 0% control; 1.0% 1xDTG; 1.3% 5xDTG). Tail, abdominal wall, limb, craniofacial, and bleeding defects all occurred at higher rates in the DTG groups versus control. Compared with our previous findings on DTG usage in folate-replete mouse pregnancies, folate deficiency was associated with higher rates of several defects, including NTDs, but in the DTG groups only. We observed a severe left-right asymmetry phenotype that was more frequent in DTG groups than controls. INTERPRETATION: Maternal folate deficiency may increase the risk for DTG-associated foetal defects. Periconceptional folic acid supplementation could be considered for women with HIV taking DTG during pregnancy, particularly in countries lacking folate fortification programs. FUNDING: This project has been funded by Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I and award #R01HD104553. LS is supported by a Tier 1 Canada Research Chair in Maternal-Child Health and HIV. HM is supported by a Junior Investigator award from the Ontario HIV Treatment Network.
Assuntos
Deficiência de Ácido Fólico , Infecções por HIV , Defeitos do Tubo Neural , Feminino , Gravidez , Humanos , Camundongos , Animais , Incidência , Placenta , Camundongos Endogâmicos C57BL , Ácido Fólico , Deficiência de Ácido Fólico/complicações , Defeitos do Tubo Neural/etiologia , Modelos Animais de Doenças , Infecções por HIV/tratamento farmacológico , Infecções por HIV/complicações , Troca Materno-Fetal , Feto , OntárioRESUMO
BACKGROUND: Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that exposure during early fetal development may be detrimental. METHODS: The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception were analyzed in pregnant mice. RESULTS: At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested. CONCLUSIONS: Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.
Assuntos
Infecções por HIV , Inibidores de Integrase de HIV , Células-Tronco Embrionárias Humanas , Exposição Materna , Animais , Feminino , Humanos , Camundongos , Gravidez , Farmacorresistência Viral/genética , Reabsorção do Feto/induzido quimicamente , Reabsorção do Feto/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/toxicidade , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/toxicidade , Células-Tronco Embrionárias Humanas/metabolismo , Piridonas/uso terapêutico , Raltegravir Potássico/toxicidade , Recém-NascidoRESUMO
Animal pregnancy models can be useful tools to study HIV antiretroviral safety and toxicity and to perform mechanistic studies that are not easily performed in humans. Utilization of clinically relevant dosing in these models improves the relevance of the findings. Cabotegravir and bictegravir are new integrase strand transfer inhibitors (INSTIs), recently approved for the treatment of people living with HIV. Studies of these drugs in pregnancy are very limited. The objective of this study was to perform a dose-optimization study of cabotegravir and bictegravir in a mouse pregnancy model with the goal of determining the dose that would yield plasma drug concentrations similar those observed in humans. Pregnant mice were administered increasing doses of cabotegravir or bictegravir in combination with emtricitabine and tenofovir by oral gavage from gestational day 11.5 to 15.5. Drug concentrations in the maternal plasma at 1 h and 24 h post drug administration and in the amniotic fluid at 1 h post drug administration were determined using high-performance liquid chromatography coupled with tandem mass spectrometry. A review of cabotegravir and bictegravir human pharmacokinetic studies are also reported. We hope these data will encourage studies of HIV antiretroviral safety/toxicity and mechanistic studies in animal pregnancy models.
RESUMO
MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor ß subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.
Assuntos
Hipotálamo/metabolismo , Insulina/farmacologia , Metformina/farmacologia , MicroRNAs/genética , Receptor de Insulina/genética , Adulto , Animais , Linhagem Celular , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Hipotálamo/citologia , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina/genética , Masculino , Camundongos , MicroRNAs/sangue , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Obesidade/sangue , Obesidade/genética , Obesidade/metabolismo , Receptor de Insulina/metabolismoAssuntos
Compostos Heterocíclicos com 3 Anéis , Oxazinas , Animais , Piperazinas , Piridonas , RatosRESUMO
BACKGROUND: Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta. METHODS: Human placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays. FINDINGS: In placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions. INTERPRETATION: These findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy. FUNDING: Funded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.
Assuntos
Placenta , Roedores , Animais , Feminino , Transportadores de Ácido Fólico/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Camundongos , Oxazinas , Piperazinas , Placenta/metabolismo , Gravidez , Piridonas , Estados UnidosRESUMO
Omega-3 fatty acid prescription drugs, Vascepa (≥96% eicosapentaenoic acid [EPA] ethyl ester) and Lovaza (46.5% EPA and 37.5% docosahexaenoic acid ethyl ester) are known therapeutic regimens to treat hypertriglyceridemia. However, their impact on glucose homeostasis, progression to type 2 diabetes, and pancreatic beta cell function are not well understood. In the present study, mice were treated with Vascepa or Lovaza for one week prior to six weeks of high-fat diet feeding. Vascepa but not Lovaza led to reduced insulin resistance, reduced fasting insulin and glucose, and improved glucose intolerance. Vascepa improved beta cell function, reduced liver triglycerides with enhanced expression of hepatic fatty acid oxidation genes, and altered microbiota composition. Vascepa has protective effects on diet-induced insulin resistance and glucose intolerance in mice.
RESUMO
BACKGROUND: Dolutegravir (DTG) is a preferred regimen for all people with HIV including pregnant women, but its effects on the fetus are not fully understood. Periconceptional exposure to DTG has been associated with increased rates of neural tube defects (NTDs), although it is unknown whether this is a causal relationship. This has led to uncertainty around the use of DTG in women of reproductive potential. METHODS: Pregnant C57BL/6J mice were randomly allocated to control (water), 1x-DTG (2.5 mg/kg-peak plasma concentration ~3000 ng/ml - therapeutic level), or 5x-DTG (12.5 mg/kg-peak plasma concentration ~12,000 ng/ml - supratherapeutic level), once daily from gestational day 0.5 until sacrifice. DTG was administered with 50 mg/kg tenofovir+33.3 mg/kg emtricitabine. Fetal phenotypes were determined, and maternal and fetal folate levels were quantified by mass-spectrometry. FINDINGS: 352 litters (91 control, 150 1x-DTG, 111 5x-DTG) yielding 2776 fetuses (747 control, 1174 1x-DTG, 855 5x-DTG) were assessed. Litter size and viability rates were similar between groups. Fetal and placenta weights were lower in the 1x-DTG vs. control. Placental weight was higher in the 5x-DTG vs. control. Five NTDs were observed, all in the 1x-DTG group. Fetal defects, including microphthalmia, severe edema, and vascular/bleeding defects were more frequent in the 1x-DTG group. In contrast, defect rates in the 5x-DTG were similar to control. Fetal folate levels were similar between control and 1x-DTG, but were significantly higher in the 5x-DTG group. INTERPRETATION: Our findings support a causal relationship of DTG at therapeutic doses with increased risk for fetal defects, including NTDs at a rate that is similar that reported in the Tsepamo study for women exposed to DTG-based ART from conception. The non-monotonic dose-response relationship between DTG and fetal anomalies could explain the previous lack of fetal toxicity findings from pre-clinical DTG studies. The fetal folate levels suggest that DTG is unlikely to be an inhibitor of folate uptake. FUNDING: This project has been funded with Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN275201800001I.
Assuntos
Anormalidades Congênitas/etiologia , Infecções por HIV/complicações , Inibidores de Integrase de HIV/efeitos adversos , Compostos Heterocíclicos com 3 Anéis/efeitos adversos , Oxazinas/efeitos adversos , Piperazinas/efeitos adversos , Piridonas/efeitos adversos , Animais , Anormalidades Congênitas/diagnóstico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores de Integrase de HIV/uso terapêutico , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Imuno-Histoquímica , Exposição Materna/efeitos adversos , Camundongos , Defeitos do Tubo Neural/diagnóstico , Defeitos do Tubo Neural/etiologia , Razão de Chances , Oxazinas/uso terapêutico , Fenótipo , Piperazinas/uso terapêutico , Gravidez , Piridonas/uso terapêutico , Medição de Risco , Fatores de RiscoRESUMO
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RESUMO
AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) affects up to 20% of pregnancies, and almost half of the women affected progress to type 2 diabetes later in life, making GDM the most significant risk factor for the development of future type 2 diabetes. An accurate prediction of future type 2 diabetes risk in the early postpartum period after GDM would allow for timely interventions to prevent or delay type 2 diabetes. In addition, new targets for interventions may be revealed by understanding the underlying pathophysiology of the transition from GDM to type 2 diabetes. The aim of this study is to identify both a predictive signature and early-stage pathophysiology of the transition from GDM to type 2 diabetes. METHODS: We used a well-characterised prospective cohort of women with a history of GDM pregnancy, all of whom were enrolled at 6-9 weeks postpartum (baseline), were confirmed not to have diabetes via 2 h 75 g OGTT and tested anually for type 2 diabetes on an ongoing basis (2 years of follow-up). A large-scale targeted lipidomic study was implemented to analyse ~1100 lipid metabolites in baseline plasma samples using a nested pair-matched case-control design, with 55 incident cases matched to 85 non-case control participants. The relationships between the concentrations of baseline plasma lipids and respective follow-up status (either type 2 diabetes or no type 2 diabetes) were employed to discover both a predictive signature and the underlying pathophysiology of the transition from GDM to type 2 diabetes. In addition, the underlying pathophysiology was examined in vivo and in vitro. RESULTS: Machine learning optimisation in a decision tree format revealed a seven-lipid metabolite type 2 diabetes predictive signature with a discriminating power (AUC) of 0.92 (87% sensitivity, 93% specificity and 91% accuracy). The signature was highly robust as it includes 45-fold cross-validation under a high confidence threshold (1.0) and binary output, which together minimise the chance of data overfitting and bias selection. Concurrent analysis of differentially expressed lipid metabolite pathways uncovered the upregulation of α-linolenic/linoleic acid metabolism (false discovery rate [FDR] 0.002) and fatty acid biosynthesis (FDR 0.005) and the downregulation of sphingolipid metabolism (FDR 0.009) as being strongly associated with the risk of developing future type 2 diabetes. Focusing specifically on sphingolipids, the downregulation of sphingolipid metabolism using the pharmacological inhibitors fumonisin B1 (FB1) and myriocin in mouse islets and Min6 K8 cells (a pancreatic beta-cell like cell line) significantly impaired glucose-stimulated insulin secretion but had no significant impact on whole-body glucose homeostasis or insulin sensitivity. CONCLUSIONS/INTERPRETATION: We reveal a novel predictive signature and associate reduced sphingolipids with the pathophysiology of transition from GDM to type 2 diabetes. Attenuating sphingolipid metabolism in islets impairs glucose-stimulated insulin secretion.
Assuntos
Biomarcadores/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Gestacional/sangue , Adulto , Animais , Área Sob a Curva , Asiático , Estudos de Casos e Controles , Árvores de Decisões , Diabetes Mellitus Tipo 2/etnologia , Diabetes Gestacional/etnologia , Progressão da Doença , Feminino , Teste de Tolerância a Glucose , Hispânico ou Latino , Humanos , Ilhotas Pancreáticas/metabolismo , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Período Pós-Parto , Gravidez , Estudos Prospectivos , Fatores de Risco , Esfingolipídeos/metabolismo , Estados UnidosRESUMO
AIM: Omega-3 fatty acid ethyl ester supplements, available by prescription, are common in the treatment of dyslipidaemia in humans. Recent studies show that 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), a metabolite formed from fish oil supplementation, was able to prevent and reverse high fat diet (HFD)-induced fatty liver in mice. In the present study, we investigated the underlying molecular mechanisms responsible for CMPF's hepatic lipid-lowering effects. MATERIALS AND METHODS: CD1 male mice were i.p. injected with CMPF (dosage, 6 mg/kg) for 7 days, followed by 5 weeks of a 60% HFD to induce a fatty liver phenotype. Metabolic parameters, liver morphology, lipid content, protein expression and microarray analysis were assessed. We also utilized primary hepatocytes, an in vitro model, to further investigate the direct effects of CMPF on hepatic lipid utilization and biosynthesis. RESULTS: CMPF-treated mice display enhanced hepatic lipid clearance while hepatic lipid storage is prevented, thereby protecting against liver lipid accumulation and development of HFD-induced hepatic insulin resistance. Mechanistically, as CMPF enters the liver, it acts as an allosteric acetyl-coA carboxylase (ACC) inhibitor, which directly induces both fatty acid oxidation and hepatic production of fibroblast growth factor 21 (FGF21). A feed-back loop is initiated by CMPF, which exists between ACC inhibition, fatty acid oxidation and production of FGF21. As a consequence, an adaptive decrease in Insig2/SREBP-1c/FAS protein expression results in priming of the liver to prevent a HFD-induced fatty liver phenotype. CONCLUSION: CMPF is a potential driver of hepatic lipid metabolism, preventing diet-induced hepatic lipid deposition and insulin resistance in the long term.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Furanos/farmacologia , Resistência à Insulina/fisiologia , Fígado , Propionatos/farmacologia , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Metabolismo dos Lipídeos , Fígado/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , CamundongosRESUMO
Specific circulating metabolites have emerged as important risk factors for the development of diabetes. The acylcarnitines (acylCs) are a family of metabolites known to be elevated in type 2 diabetes (T2D) and linked to peripheral insulin resistance. However, the effect of acylCs on pancreatic ß-cell function is not well understood. Here, we profiled circulating acylCs in two diabetes cohorts: 1) women with gestational diabetes mellitus (GDM) and 2) women with recent GDM who later developed impaired glucose tolerance (IGT), new-onset T2D, or returned to normoglycemia within a 2-year follow-up period. We observed a specific elevation in serum medium-chain (M)-acylCs, particularly hexanoyl- and octanoylcarnitine, among women with GDM and individuals with T2D without alteration in long-chain acylCs. Mice treated with M-acylCs exhibited glucose intolerance, attributed to impaired insulin secretion. Murine and human islets exposed to elevated levels of M-acylCs developed defects in glucose-stimulated insulin secretion and this was directly linked to reduced mitochondrial respiratory capacity and subsequent ability to couple glucose metabolism to insulin secretion. In conclusion, our study reveals that an elevation in circulating M-acylCs is associated with GDM and early stages of T2D onset and that this elevation directly impairs ß-cell function.
Assuntos
Carnitina/análogos & derivados , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Gestacional/metabolismo , Intolerância à Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Adulto , Animais , Carnitina/metabolismo , Carnitina/farmacologia , Estudos de Casos e Controles , Respiração Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Período Pós-Parto , GravidezRESUMO
Prescription ω-3 fatty acid ethyl ester supplements are commonly used for the treatment of hypertriglyceridemia. However, the metabolic profile and effect of the metabolites formed by these treatments remain unknown. Here we utilized unbiased metabolomics to identify 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) as a significant metabolite of the ω-3-acid ethyl ester prescription Lovaza™ in humans. Administration of CMPF to mice before or after high-fat diet feeding at exposures equivalent to those observed in humans increased whole-body lipid metabolism, improved insulin sensitivity, increased beta-oxidation, reduced lipogenic gene expression, and ameliorated steatosis. Mechanistically, we find that CMPF acutely inhibits ACC activity, and induces long-term loss of SREBP1c and ACC1/2 expression. This corresponds to an induction of FGF21, which is required for long-term steatosis protection, as FGF21KO mice are refractory to the improved metabolic effects. Thus, CMPF treatment in mice parallels the effects of human Lovaza™ supplementation, revealing that CMPF may contribute to the improved metabolic effects observed with ω-3 fatty acid prescriptions.
Assuntos
Suplementos Nutricionais , Ésteres/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/prevenção & controle , Furanos/uso terapêutico , Metaboloma , Propionatos/uso terapêutico , Adulto , Animais , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/metabolismo , Furanos/metabolismo , Humanos , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Propionatos/metabolismoRESUMO
Ghrelin and nesfatin-1 are regulators of blood glucose and energy balance. Prohormone convertases (PCs) enable processing of ghrelin and nesfatin-1 from its precursors. An acylation, enabled by ghrelin O-acyl transferase (GOAT) is critical for many of the biological actions of ghrelin. To date, there is no research on the developmental expression of GOAT, and the co-expression of both NUCB2/nesfatin-1 and prohormone convertases in the pancreas. The objective of this research was to immunolocalize ghrelin, GOAT, NUCB2/nesfatin-1, PC1/3 and PC2 in the pancreas during fetal and postnatal periods of Sprague Dawley (SD) rats using immunohistochemical analysis. GOAT mRNA in the rat pancreas during development was also determined. In the pancreas, not all islet cells immunopositive for GOAT are immunoreactive for ghrelin on postnatal (P) days 20, 27 and adult. GOAT mRNA expression in the pancreas at P27 was higher than the expression levels in rest of the developmental stages tested. Both PC1/3 and PC2 are co-expressed with NUCB2/nesfatin-1 on embryonic (E) day E21, P13, P20. While similar co-localization was also found in P27 for NUCB2/nesfatin-1 and PC1/3, NUCB2/nesfatin-1 and PC2 were found in distinct populations of cells in P27. Some ghrelin and GOAT positive cells stained for nesfatin-1 as well. Meanwhile, no co-localization of somatostatin and glucaon with nesfatin-1 was found in the pancreas of SD rats. Our findings suggest that the endocrine pancreas can produce and process precursors of ghrelin and nesfatin-1 to make bioactive forms of both peptides.
Assuntos
Aciltransferases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Grelina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organogênese , Pró-Proteína Convertases/metabolismo , Aciltransferases/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Grelina/genética , Imuno-Histoquímica , Proteínas do Tecido Nervoso/genética , Nucleobindinas , Especificidade de Órgãos/genética , Organogênese/genética , Pâncreas/metabolismo , Pró-Proteína Convertases/genética , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Nesfatin-1 (82 amino acid) is an anorexigenic and insulinotropic peptide encoded in a secreted precursor, nucleobindin-2 (NUCB2). Nucleobindin-1 (NUCB1) is a protein with very high sequence similarity to NUCB2. We hypothesized that a nesfatin-1 like peptide (NLP) is encoded in NUCB1, and this peptide is biologically active. In silico analysis found a signal peptide cleavage site at position 25 (Arginine) and 26 (Valine) preceding the NLP region in NUCB1 sequence, and potential proprotein convertase cleavage sites at Lys-Arg (KR), forming a 77 amino acid NLP. RT-PCR studies found NUCB1 mRNA in both pancreas and MIN6 cells. NUCB1-like immunoreactivity was detected in mouse insulinoma (MIN6) cells, and pancreatic islet beta cells of mice. In order to determine the biological activity of NLP, MIN6 cells were incubated with synthetic rat NLP. NLP (10nM and 100nM) upregulated preproinsulin mRNA expression and insulin secretion at 1h post-incubation. In identical experiments using MIN6 cells, a scrambled peptide based on the NLP sequence did not elicit any effects on preproinsulin mRNA expression or insulin secretion. From this result, it is clear that an intact NLP sequence is required for its biological activity. NLP appears as another endogenous insulinotropic peptide encoded in NUCB1.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Insulinoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Técnicas Imunoenzimáticas , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/tratamento farmacológico , Insulinoma/genética , Insulinoma/patologia , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Nucleobindinas , Precursores de Proteínas/genética , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Nesfatin-1 is secreted, meal-responsive anorexigenic peptide encoded in the precursor nucleobindin-2 [NUCB2]. Circulating nesfatin-1 increases post-prandially, but the dietary components that modulate NUCB2/nesfatin-1 remain unknown. We hypothesized that carbohydrate, fat and protein differentially regulate tissue specific expression of nesfatin-1. NUCB2, prohormone convertases and nesfatin-1 were detected in mouse stomach ghrelinoma [MGN3-1] cells. NUCB2 mRNA and protein were also detected in mouse liver, and small and large intestines. MGN3-1 cells were treated with glucose, fatty acids or amino acids. Male C57BL/6 mice were chronically fed high fat, high carbohydrate and high protein diets for 17 weeks. Quantitative PCR and nesfatin-1 assays were used to determine nesfatin-1 at mRNA and protein levels. Glucose stimulated NUCB2 mRNA expression in MGN3-1 cells. L-Tryptophan also increased NUCB2 mRNA expression and ghrelin mRNA expression, and nesfatin-1 secretion. Oleic acid inhibited NUCB2 mRNA expression, while ghrelin mRNA expression and secretion was enhanced. NUCB2 mRNA expression was significantly lower in the liver of mice fed a high protein diet compared to mice fed other diets. Chronic intake of high fat diet caused a significant reduction in NUCB2 mRNA in the stomach, while high protein and high fat diet caused similar suppression of NUCB2 mRNA in the large intestine. No differences in serum nesfatin-1 levels were found in mice at 7 a.m, at the commencement of the light phase. High carbohydrate diet fed mice showed significantly elevated nesfatin-1 levels at 1 p.m. Serum nesfatin-1 was significantly lower in mice fed high fat, protein or carbohydrate compared to the controls at 7 p.m, just prior to the dark phase. Mice that received a bolus of high fat had significantly elevated nesfatin-1/NUCB2 at all time points tested post-gavage, compared to control mice and mice fed other diets. Our results for the first time indicate that nesfatin-1 is modulated by nutrients.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/genética , Alimentos , Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Neoplasias Gástricas/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Ritmo Circadiano/fisiologia , Proteínas de Ligação a DNA/metabolismo , Dieta , Grelina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Nucleobindinas , Especificidade de Órgãos , Neoplasias Gástricas/metabolismoRESUMO
Ghrelin is a gut hormone with potent orexigenic and growth hormone release stimulatory effects, and is the first known endogenous ligand of the growth hormone secretagogue receptor. A notable feature of ghrelin is that it carries an acyl group, in most cases an octanoyl group, in the third serine. While it has been shown that the acylation is critical for the majority of ghrelin functions, the mechanisms of acylation of ghrelin remained poorly understood. In 2008, it was discovered that ghrelin O-acyltransferase (GOAT) is the enzyme responsible for acylating ghrelin. GOAT is highly conserved from zebrafish to humans. It is most abundant in the stomach and pancreas. GOAT mRNA expression is regulated by energy balance, being upregulated by energy restriction and downregulated by energy abundance. GOAT attenuation using synthetic inhibitors enhances insulin secretion and reduces body weight. GOAT inhibitors are currently being developed for the treatment of metabolic disorders. In addition to its ghrelin mediated effects, GOAT is also known to directly regulate bile acid secretion. The discovery of GOAT helped to redefine the ghrelin research field and enabled the development of another target molecule for potential therapies aimed to prevent/treat diabetes and obesity.
Assuntos
Aciltransferases/história , Grelina/história , Acilação , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Grelina/metabolismo , História do Século XXI , Humanos , Processamento de Proteína Pós-Traducional , Distribuição TecidualRESUMO
Nesfatin-1 is an eighty two amino acid, naturally occurring multifunctional protein encoded in the precursor nucleobindin-2 (NUCB2). A comparison of sequences indicates that NUCB2 is present in a number of animals, from hydra to humans. The 30 amino acid mid-segment of nesfatin-1 is considered to be the bioactive core of the protein, and this region displays the highest identity among nesfatin-1 sequences reported thus far. Similar to the sequence relationships observed, the tissue-specific expression and biological actions of nesfatin-1 also appear to be highly conserved across species. For example, brain is a major tissue abundantly expressing nesfatin- 1 in several species. It has been shown that various key regions of the rat, mouse and goldfish brain, which are involved in the regulation of feeding and metabolism express nesfatin-1. Exogenous administration of nesfatin-1 results in a decrease in the food intake of rats, mice and goldfish. In addition, nesfatin-1 has been shown to regulate a number of other physiological processes including hormone secretion from the pancreatic islets and pituitary gland, stress and behavior. While nesfatin-1 research still remains an emerging area in physiology, the literature available thus far clearly shows that nesfatin-1 is an important regulator of homeostasis in animals.
Assuntos
Proteínas de Ligação ao Cálcio/classificação , Proteínas de Ligação a DNA/classificação , Proteínas do Tecido Nervoso/classificação , Filogenia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , NucleobindinasRESUMO
Nesfatin-1 is a novel metabolic hormone that has glucose-responsive insulinotropic actions. Islet ß-cells and gastrointestinal tissues have been reported as abundant sources of nesfatin-1 and its precursor hormone nucleobindin-2 (NUCB2). While nesfatin-1 is emerging as a multifunctional hormone, there are no reports on the developmental expression of NUCB2/nesfatin-1. The main objective of this study was to examine the ontogenic expression of NUCB2 mRNA, and NUCB2/nesfatin-1 immunoreactivity in the pancreas, stomach and duodenum, and the circulating levels NUCB2/nesfatin-1 in Sprague Dawley rats. In addition, we also determined the co-localization of NUCB2/nesfatin-1 and insulin immunoreactivity during development. NUCB2/nesfatin-1 immunoreactivity was found in the rat stomach from postnatal days 13-27. Furthermore, NUCB2/nesfatin-1 immunoreactivity was also detected in the enteroendocrine cells of the duodenum at postnatal days 13 and 27. Duodenal NUCB2 mRNA expression at postnatal day 27 was highest. Serum NUCB2/nesfatin-1 levels on embryonic day 21 and postnatal day 1 were lower than serum NUCB2/nesfatin-1 levels of adults and neonates at postnatal days 13, 20 and 27, gradually increasing with growth, suggesting an increase in its production and secretion from tissues including the gastrointestinal tract and pancreas. Our findings indicate that NUCB2/nesfatin-1 colocalizes with insulin in the islet ß-cells at all developmental stages, but the percentage of colocalization varies in an age-dependent manner. These findings suggest that NUCB2/nesfatin-1 has potential age- and tissue-specific role in the developmental physiology of rats during growth.