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BACKGROUND AND OBJECTIVES: Dual amylin and calcitonin receptor agonists (DACRAs) are therapeutic candidates in the treatment of obesity with beneficial effects on weight loss superior to suppression of food intake. Hence, suggesting effects on energy expenditure by possibly targeting mitochondria in metabolically active tissue. METHODS: Male rats with HFD-induced obesity received a DACRA, KBP-336, every third day for 8 weeks. Upon study end, mitochondrial respiratory capacity (MRC), - enzyme activity, - transcriptional factors, and -content were measured in perirenal (pAT) and inguinal adipose tissue. A pair-fed group was included to examine food intake-independent effects of KBP-336. RESULTS: A vehicle-corrected weight loss (23.4 ± 2.8%) was achieved with KBP-336, which was not observed to the same extent with the food-restricted weight loss (12.4 ± 2.8%) (P < 0.001). Maximal coupled respiration supported by carbohydrate and lipid-linked substrates was increased after KBP-336 treatment independent of food intake in pAT (P < 0.01). Moreover, oligomycin-induced leak respiration and the activity of citrate synthase and ß-hydroxyacetyl-CoA-dehydrogenase were increased with KBP-336 treatment (P < 0.05). These effects occurred without changes in mitochondrial content in pAT. CONCLUSIONS: These findings demonstrate favorable effects of KBP-336 on MRC in adipose tissue, indicating an increased energy expenditure and capacity to utilize fatty acids. Thus, providing more mechanistic insight into the DACRA-induced weight loss.
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The microRNA-29 family members miR-29a-3p, miR-29b-3p, and miR-29c-3p are ubiquitously expressed and consistently increased in various tissues and cell types in conditions of metabolic disease, obesity, insulin resistance, and type 2 diabetes. In pancreatic ß cells, miR-29a is required for normal exocytosis, but increased levels are associated with impaired ß-cell function. Similarly, in liver, miR-29 species are higher in models of insulin resistance and type 2 diabetes, and either knock-out or depletion using a microRNA inhibitor improves hepatic insulin resistance. In skeletal muscle, miR-29 family upregulation is associated with insulin resistance and altered substrate oxidation, and similarly, in adipocytes, overexpression of miR-29a leads to insulin resistance. Blocking miR-29a using nucleic acid antisense therapeutics show promising results in preclinical animal models of obesity and type 2 diabetes, although the widespread expression pattern of miR-29 family members complicates the exploration of single target tissues. However, in fibrotic diseases, such as in late complications of diabetes and metabolic disease (diabetic kidney disease, nonalcoholic steatohepatitis), miR-29 species expression is suppressed by TGF-ß allowing increased extracellular matrix collagen to form. In the clinical setting, circulating levels of miR-29a and miR-29b are consistently increased in type 2 diabetes and in gestational diabetes and are also possible prognostic markers for deterioration of glucose tolerance. In conclusion, miR-29 family miRNAs play an essential role in various organs relevant to intermediary metabolism and its upregulation contributes to impaired glucose metabolism, whereas it suppresses fibrosis development. Thus, a correct balance of levels of miR-29 family miRNA seems important for cellular and organ homeostasis in metabolism.
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Diabetes Mellitus Tipo 2 , Resistência à Insulina , Células Secretoras de Insulina , MicroRNAs , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Fibrose , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , MicroRNAs/genética , Obesidade/metabolismoRESUMO
In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy.
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Dieta com Restrição de Proteínas , Ilhotas Pancreáticas , Angiopoietinas/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Feminino , Desenvolvimento Fetal , Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Gravidez , RNA Mensageiro/genética , Ratos , Receptores da Prolactina/genética , Receptores da Somatotropina/metabolismo , Esteróis/metabolismo , Fatores de Transcrição/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
Inappropriate insulin secretion from ß-cells is considered as an early sign of impaired glucose tolerance and type 2 diabetes (T2D). Glucokinase (GCK) is an important enzyme that regulates glucose metabolism and ensures that the normal circulating glucose concentrations are maintained. GCK expression is induced by glucose and regulated via transcription factors and regulatory proteins. Recently, microRNA-206 (miR-206) was reported to regulate GCK and alter glucose tolerance in normal and high-fat diet-fed mice. Although the study findings have implications for human diabetes, studies in human islets are lacking. Here, we analyze human islets from individuals without or with T2D, using TaqMan-based real-time qPCR at the tissue (isolated islet) level as well as at single cell resolution, to assess the relationship between miR-206 and GCK expression in normal and T2D human islets. Our data suggest that, unlike mouse islets, human islets do not exhibit any correlation between miR-206 and GCK transcripts. These data implicate the need for further studies aimed toward exploring its potential role(s) in human islets.
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Diabetes Mellitus Tipo 2/genética , Glucoquinase/genética , Ilhotas Pancreáticas/metabolismo , MicroRNAs/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula ÚnicaRESUMO
Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides.
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Movimento Celular/efeitos dos fármacos , Insulina/farmacologia , Neurotensina/farmacologia , Substância P/farmacologia , Linhagem Celular , Citocinas/biossíntese , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Staphylococcus lugdunensis/isolamento & purificaçãoRESUMO
Mitochondria are important organelles in cellular metabolism. Several crucial metabolic pathways such as the energy producing electron transport chain or the tricarboxylic acid cycle are hosted inside the mitochondria. The proper function of mitochondria depends on the import of proteins, which are encoded in the nucleus and synthesized in the cytosol. Micro-ribonucleic acids (miRNAs) are short non-coding ribonucleic acid (RNA) molecules with the ability to prevent messenger RNA (mRNA)-translation or to induce the degradation of mRNA-transcripts. Although miRNAs are mainly located in the cytosol or the nucleus, a subset of ~150 different miRNAs, called mitomiRs, has also been found localized to mitochondrial fractions of cells and tissues together with the subunits of the RNA-induced silencing complex (RISC); the protein complex through which miRNAs normally act to prevent translation of their mRNA-targets. The focus of this review is on miRNAs and mitomiRs with influence on mitochondrial metabolism and their possible pathophysiological impact.
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MicroRNAs/metabolismo , Mitocôndrias/metabolismo , RNA Mensageiro/metabolismo , Aminoácidos/metabolismo , Animais , Ciclo do Ácido Cítrico , Humanos , Metabolismo dos Lipídeos , MicroRNAs/análise , MicroRNAs/genética , Mitocôndrias/química , Mitocôndrias/genética , Dinâmica Mitocondrial , Degradação do RNAm Mediada por Códon sem Sentido , Biossíntese de Proteínas , Transporte de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Complexo de Inativação Induzido por RNA/análise , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
Resveratrol (Resv) is a polyphenol reported to modulate mitochondrial activity. The aim was to use HeLa and 143B cells to characterize the action of Resv on mitochondrial activity, cell size and proliferation using wild type (WT) and Rho 0 cells deficient in mitochondrial DNA. In both HeLa WT and Rho 0 cells, the oxygen consumption rate (OCR) was increased at 20 µM Resv after 24 h, whereas only a non-significant increase of OCR was observed in 143B WT cells. Resv decreased cell number concentration-dependently in both WT and Rho 0 cell types. An increased cell diameter was observed in HeLa WT, but not in Rho 0 when treated with Resv. Overall, the findings presented indicate that functional mitochondria are a prerequisite for cell enlargement by Resv.
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Antineoplásicos Fitogênicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Estilbenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , DNA Mitocondrial/química , Células HeLa , Humanos , Mitocôndrias/fisiologia , Especificidade de Órgãos , Osteoblastos/citologia , Osteoblastos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , ResveratrolRESUMO
The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.
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Diabetes Gestacional/fisiopatologia , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Células Secretoras de Insulina/metabolismo , Obesidade/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Epigênese Genética/genética , Epigênese Genética/fisiologia , Feminino , Humanos , Fenômenos Fisiológicos da Nutrição Materna , Obesidade/genética , Fenótipo , Gravidez , Fenômenos Fisiológicos da Nutrição Pré-Natal , Fatores de RiscoRESUMO
BACKGROUND: TH2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G4 blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4). METHODS: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. RESULTS: During SIT, the main changes in the peripheral blood were an increase in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Tregs and a decrease in CCR4(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4(+)CD25(+)CD127(low/-) Tregs and ILT2(+)CD4(+)CD25(+)CD127(low/-) Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. CONCLUSIONS: The changes in CXCR3(+)CD4(+)CD25(+)CD127(low/-) Treg, IgG4, and sHLA-G levels in the peripheral blood and in ILT2(+) Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards TH1 during SIT.
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Alérgenos/imunologia , Antígenos HLA-G/imunologia , Imunoterapia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Linfócitos T Reguladores/imunologia , Adulto , Citocinas/biossíntese , Epitopos de Linfócito B/imunologia , Feminino , Antígenos HLA-G/sangue , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CCR4/metabolismo , Receptores CXCR3/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
Different types of small non-coding RNAs, especially miRNAs, may be found in the circulation, either protein-bound or enclosed in extracellular vesicles. During gestation, and particularly during gestational diabetes mellitus (GDM), the levels of several miRNAs are altered. Worldwide the incidence of GDM is increasing, in part driven by the current obesity epidemic. This is a point of public health concern because offspring of women with GDM frequently suffer from short- and long-term complications of maternal GDM. This has prompted the investigation of whether levels of specific miRNA species, detected early in gestation, may be used as diagnostic or prognostic markers for the development of GDM. Here, we summarize the mechanisms of RNA secretion and review circulating miRNAs associated with GDM. Several miRNAs are associated with GDM: miR-29a-3p and miR-29b-3p are generally upregulated in GDM pregnancies, also when measured prior to the development of GDM, while miR-16-5p is consistently upregulated in GDM pregnancies, especially in late gestation. miR-330-3p in circulation is increased in late gestation GDM women, especially in those with poor insulin secretion. miR-17-5p, miR-19a/b-3p, miR-223-3p, miR-155-5p, miR-125-a/b-5p, miR-210-3p and miR-132 are also associated with GDM, but less so and with more contradictory results reported. There could be a publication bias as miRNAs identified early are investigated the most, suggesting that it is likely that additional, more recently detected miRNAs could also be associated with GDM. Thus, circulating miRNAs show potential as biomarkers of GDM diagnosis or prognosis, especially multiple miRNAs containing prediction algorithms show promise, but further studies are needed.
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MicroRNA Circulante , Diabetes Gestacional , MicroRNAs , Gravidez , Feminino , Humanos , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/genética , MicroRNA Circulante/genética , Biomarcadores , MicroRNAs/genética , ObesidadeRESUMO
Obesity is an important risk factor for cardiovascular disease and type 2 diabetes mellitus. Even a modest weight loss of 5-15% improves metabolic health, but circulating markers to indicate weight loss efficiency are lacking. MicroRNAs, small non-coding post-transcriptional regulators of gene expression, are secreted from tissues into the circulation and may be potential biomarkers for metabolic health. However, it is not known which specific microRNA species are reproducibly changed in levels by weight loss. In this study, we performed a systematic review and meta-analysis to investigate the microRNAs associated with weight loss by comparing baseline to follow-up levels following intervention-driven weight loss. This systematic review was performed according to the PRISMA guidelines with searches in PubMed and SCOPUS. The primary search resulted in a total of 697 articles, which were screened according to the prior established inclusion and exclusion criteria. Following the screening of articles, the review was based on the inclusion of 27 full-text articles, which were evaluated for quality and the risk of bias. We performed systematic data extraction, whereafter the relative values for miRNAs were calculated. A meta-analysis was performed for the miRNA species investigated in three or more studies: miR-26a, miR-126, and miR-223 were overall significantly increased following weight loss, while miR-142 was significantly decreased after weight loss. miR-221, miR-140, miR-122, and miR-146 were not significantly changed by intervention-driven weight loss. These results indicate that few miRNAs are significantly changed during weight loss.
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BACKGROUND: Women with polycystic ovary syndrome (PCOS) often change their metabolic profile over time to decrease levels of androgens while often gaining a propensity for the development of the metabolic syndrome. Recent discoveries indicate that microRNAs (miRNAs) play a role in the development of PCOS and constitute potential biomarkers for PCOS. We aimed to identify miRNAs associated with the development of an impaired metabolic profile in women with PCOS, in a follow-up study, compared with women without PCOS. METHODS AND MATERIALS: Clinical measurements of PCOS status and metabolic disease were obtained twice 6 years apart in a cohort of 46 women with PCOS and nine controls. All participants were evaluated for degree of metabolic disease (hypertension, dyslipidemia, central obesity, and impaired glucose tolerance). MiRNA levels were measured using Taqman® Array cards of 96 pre-selected miRNAs associated with PCOS and/or metabolic disease. RESULTS: Women with PCOS decreased their levels of androgens during follow-up. Twenty-six of the miRNAs were significantly changed in circulation in women with PCOS during the follow-up, and twenty-four of them had decreased, while levels did not change in the control group. Four miRNAs were significantly different at baseline between healthy controls and women with PCOS; miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p, which were decreased in PCOS. After follow-up, miR-28-3p, miR-139-5p, and miR-376a-3p increased in PCOS women to the levels observed in healthy controls. Of these, miR-139-5p correlated with total testosterone levels (rho = 0.50, padj = 0.013), while miR-376-3p correlated significantly with the waist-hip ratio at follow-up (rho = 0.43, padj = 0.01). Predicted targets of miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p were enriched in pathways associated with Insulin/IGF signaling, interleukin signaling, the GNRH receptor pathways, and other signaling pathways. MiRNAs altered during follow-up in PCOS patients were enriched in pathways related to immune regulation, gonadotropin-releasing hormone signaling, tyrosine kinase signaling, and WNT signaling. CONCLUSIONS: These studies indicate that miRNAs associated with PCOS and androgen metabolism overall decrease during a 6-year follow-up, reflecting the phenotypic change in PCOS individuals towards a less hyperandrogenic profile.
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MicroRNA Circulante , MicroRNAs , Síndrome do Ovário Policístico , Humanos , Feminino , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo , MicroRNA Circulante/genética , Estudos Longitudinais , Seguimentos , MicroRNAs/genética , MicroRNAs/metabolismo , Estudos de Coortes , AndrogêniosRESUMO
Uncoupling Protein 2 (UCP2) is expressed in the pancreatic ß-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.
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Glucoquinase/fisiologia , Glucose/metabolismo , Canais Iônicos/biossíntese , Ilhotas Pancreáticas/metabolismo , Proteínas Mitocondriais/biossíntese , RNA Mensageiro/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/genética , Glucose/farmacologia , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Superóxidos/metabolismo , Proteína Desacopladora 2RESUMO
Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.
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Proliferação de Células , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , MicroRNAs/biossíntese , Animais , Linhagem Celular , Tamanho Celular , Glucose/farmacologia , Intolerância à Glucose , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ratos , Regulação para CimaRESUMO
Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1αß-/- mice that demonstrate dedifferentiation of pancreatic ß-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses.
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Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Algoritmos , Animais , Diabetes Mellitus Tipo 2/genética , Humanos , Insulina/genética , Aprendizado de Máquina , CamundongosRESUMO
BACKGROUND & AIMS: Pancreatic islet ß-cells are factories for insulin production; however, ectopic expression of insulin also is well recognized. The gallbladder is a next-door neighbor to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. METHODS: We compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assays, RNA sequencing, real-time polymerase chain reaction, chromatin immunoprecipitation, and functional studies. RESULTS: We show that the epithelial lining of developing, as well as adult, mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a non-obese diabetic (NOD) mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Interestingly, in human gallbladders, insulin splice variants are absent, although insulin splice forms are observed in human islets. CONCLUSIONS: In summary, our biochemical, transcriptomic, and functional data in mouse and human gallbladder epithelial cells collectively show the evolutionary and developmental similarities between gallbladder and the pancreas that allow gallbladder epithelial cells to continue insulin production in adult life. Understanding the mechanisms regulating insulin transcription and translation in gallbladder epithelial cells would help guide future studies in type 1 diabetes therapy.
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Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Células Epiteliais/metabolismo , Vesícula Biliar/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos NODRESUMO
Fatty acid-induced damage in pancreatic ß-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve ß-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP2, FATP1 and FATP4 were unchanged. RNAi against FAT/CD36 decreased fatty acid-induced apoptosis. Over-expression of constitutively active STAT5 was able to mimic hGH's suppression of FAT/CD36 expression, whereas dominant negative STAT5 was unable to block the effect of hGH indicating that STAT5 did not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect ß-cells against fatty acid-induced damages.
Assuntos
Antígenos CD36/metabolismo , Citoproteção , Hormônio do Crescimento Humano/fisiologia , Células Secretoras de Insulina/fisiologia , Palmitatos/metabolismo , Animais , Apoptose/genética , Transporte Biológico , Antígenos CD36/genética , Linhagem Celular , Glucose/farmacologia , Hormônio do Crescimento Humano/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Palmitatos/toxicidade , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Fator de Transcrição STAT5/metabolismoRESUMO
Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.
Assuntos
Isotiocianatos/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Envelhecimento da Pele/efeitos dos fármacos , Sulfóxidos/farmacologia , Animais , Antioxidantes/farmacologia , Suplementos Nutricionais , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
MicroRNAs (miRNAs) are elements of the gene regulatory network and manipulating their abundance is essential toward elucidating their role in patho-physiological conditions. We present a detailed workflow that identifies important miRNAs using a machine learning algorithm. We then provide optimized techniques to validate the identified miRNAs through over-expression/loss-of-function studies. Overall, these protocols apply to any field in biology where high-dimensional data are produced. For complete details on the use and execution of this protocol, please refer to Wong et al. (2021a).
Assuntos
Perfilação da Expressão Gênica/métodos , Aprendizado de Máquina , MicroRNAs/genética , Transcriptoma/genética , Algoritmos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes/genética , Humanos , Ilhotas Pancreáticas/citologia , Fluxo de TrabalhoRESUMO
Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing.