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1.
Int J Mol Sci ; 24(9)2023 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-37175762

RESUMO

Insulin facilitates renal sodium reabsorption and attenuates gluconeogenesis. Sex differences in this regulation have not been well characterized. Using tetracycline-inducible Cre-lox recombination, we knocked out (KO) the insulin receptor (InsR) from the renal tubule in adult male (M) and female (F) mice (C57Bl6 background) with a paired box 8 (PAX8) promoter. Body weights were not affected by the KO, but mean kidney weights were reduced in the KO mice (13 and 3%, in M and F, respectively, relative to wild-type (WT) mice). A microscopic analysis revealed 25 and 19% reductions in the proximal tubule (PT) and cortical collecting duct cell heights, respectively, in KOMs relative to WTMs. The reductions were 5 and 11% for KOFs. Western blotting of renal cortex homogenates showed decreased protein levels for the ß and γ subunits of the epithelial sodium channel (ENaC) and the sodium-potassium-2-chloride cotransporter type 2 (NKCC2) in both sexes of KO mice; however, α-ENaC was upregulated in KOMs and downregulated in KOFs. Both sexes of KO mice cleared exogenously administered glucose faster than the WT mice and had lower semi-fasted, anesthetized blood glucose levels. However, KOMs (but not KOFs) demonstrated evidence of enhanced renal gluconeogenesis, including higher levels of renal glucose-6-phosphatase, the PT's production of glucose, post-prandial blood glucose, and plasma insulin, whereas KOFs exhibited downregulation of renal high-capacity sodium glucose cotransporter (SGLT2) and upregulation of SGLT1; these changes appeared to be absent in the KOM. Overall, these findings suggest a sex-differential reliance on intact renal tubular InsR signaling which may be translationally important in type 2 diabetes, obesity, or insulin resistance when renal insulin signaling is reduced.


Assuntos
Diabetes Mellitus Tipo 2 , Receptor de Insulina , Feminino , Masculino , Animais , Camundongos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Glicemia , Insulina/metabolismo , Glucose/metabolismo , Camundongos Knockout , Sódio/metabolismo , Canais Epiteliais de Sódio/metabolismo
2.
J Diabetes Res ; 2022: 5126968, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35237694

RESUMO

The prevalence of diabetes mellitus (DM) is increasing at a staggering rate around the world. In the United States, more than 30.3 million Americans have DM. Type 2 diabetes mellitus (T2DM) accounts for 91.2% of diabetic cases and disproportionately affects African Americans and Hispanics. T2DM is a major risk factor for cardiovascular disease (CVD) and is the leading cause of morbidity and mortality among diabetic patients. While significant advances in T2DM treatment have been made, intensive glucose control has failed to reduce the development of macro and microvascular related deaths in this group. This highlights the need to further elucidate the underlying molecular mechanisms contributing to CVD in the setting of T2DM. Endothelial dysfunction (ED) plays an important role in the development of diabetes-induced vascular complications, including CVD and diabetic nephropathy (DN). Thus, the endothelium provides a lucrative means to investigate the molecular events involved in the development of vascular complications associated with T2DM. microRNAs (miRNA) participate in numerous cellular responses, including mediating messages in vascular homeostasis. Exosomes are small extracellular vesicles (40-160 nanometers) that are abundant in circulation and can deliver various molecules, including miRNAs, from donor to recipient cells to facilitate cell-to-cell communication. Endothelial cells are in constant contact with exosomes (and exosomal content) that can induce a functional response. This review discusses the modulatory role of exosomal miRNAs and proteins in diabetes-induced endothelial dysfunction, highlighting the significance of miRNAs as markers, mediators, and potential therapeutic interventions to ameliorate ED in this patient group.


Assuntos
Diabetes Mellitus Tipo 2/genética , Células Endoteliais/efeitos dos fármacos , MicroRNAs/análise , MicroRNAs/farmacologia , Biomarcadores/análise , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Células Endoteliais/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/transplante , Humanos , MicroRNAs/uso terapêutico
3.
Front Physiol ; 12: 787521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35058797

RESUMO

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5-10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20-40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

4.
Am J Physiol Renal Physiol ; 319(3): F476-F486, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32715758

RESUMO

miRNAs provide fine tuning of gene expression via inhibition of translation. miR-451 has a modulatory role in cell cycling via downregulation of mechanistic target of rapamycin. We aimed to test whether chronic systemic inhibition of miR-451 would enhance renal fibrosis (associated with deranged autophagy). Adult TallyHo/Jng mice (obese insulin resistant) were randomized to two treatment groups to receive either miR-451 inhibition [via a locked nucleic acid construct] or a similar scrambled locked nucleic acid control for 8 wk. All mice were fed a high-fat diet (60% kcal from fat) ad libitum and humanely euthanized after 12 wk. Kidneys and blood were collected for analysis. Renal expression of miR-451 was sixfold lower in inhibitor-treated mice compared with control mice. miR-451 inhibition increased kidney weight and collagen and glycogen deposition. Blood chemistry revealed significantly higher Na+ and anion gap (relative metabolic acidosis) in inhibitor-treated mice. Western blot analysis and immunohistochemistry of the kidney revealed that the inhibitor increased markers of renal injury and fibrosis, e.g., kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, transforming growth factor-ß, 14-3-3 protein-ζ, mechanistic target of rapamycin, AMP-activated protein kinase-α, calcium-binding protein 39, matrix metallopeptidase-9, and the autophagy receptor sequestosome 1. In contrast, the inhibitor reduced the epithelial cell integrity marker collagen type IV and the autophagy markers microtubule-associated protein 1A/1B light chain 3B and beclin-1. Taken together, these results support a protective role for miR-451 in reducing renal fibrosis by enhancing autophagy in obese mice.


Assuntos
Autofagia/fisiologia , Rim/patologia , MicroRNAs/antagonistas & inibidores , Animais , Autofagia/efeitos dos fármacos , Dieta Hiperlipídica , Fibrose , Regulação da Expressão Gênica , Resistência à Insulina , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/induzido quimicamente , Peptídeos , Distribuição Aleatória , Transdução de Sinais
5.
Cell Physiol Biochem ; 54(4): 682-695, 2020 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-32678535

RESUMO

BACKGROUND/AIMS: Metabolic syndrome and type 2 diabetes are associated with some degree of acidosis. Acidosis has also been shown to upregulate renal gluconeogenesis. Whether impaired insulin or insulin-like-growth factor 1 receptor (IGF1) signaling alter this relationship is not known. Our aim was to determine the effects of deletion of insulin and IGF1 receptors (Insr and Igf1r) from renal proximal tubule (PT) on the gluconeogenic response to acidosis. METHODS: We developed a mouse model with PT-targeted dual knockout (KO) of the Insr/Igf1r by driving Cre-recombinase with the gamma-glutamyl transferase (gGT) promoter. Male and female mice were maintained as control or acidotic by treatment with NH4Cl in the drinking water for 1-week. RESULTS: Acidosis in both genotypes increased renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1-bisphosphatase (FBP1), but not glucose-6-phosphatase catalytic subunit (G6PC), which showed significantly lower expression in the KO regardless of treatment. Several differences between KO and WT suggested a protective role for insulin/IGF1 receptor signaling in maintaining relative euglycemia in the face of acidosis. First, the increase in FBP1 with acid was greater in the KO (significant interactive term). Secondly, proximal-tubule-associated FOXO1 and AKT overall protein levels were suppressed by acid loading in the KO, but not in the WT. Robust intact insulin signaling would be needed to reduce gluconeogenesis in PT. Third, phosphorylated FOXO1 (pS256) levels were markedly reduced by acid loading in the KO PT, but not in the WT. This reduction would support greater gluconeogenesis. Fourth, the sodium-glucose cotransporter (SGLT1) was increased by acid loading in the KO kidney, but not the WT. While this would not necessarily affect gluconeogenesis, it could result in increased circulatory glucose via renal reabsorption. Reduced susceptibility to glucose-homeostatic dysregulation in the WT could potentially relate to the sharp (over 50%) reduction in renal levels of sirtuin-1 (SIRT1), which deacetylates and regulates transcription of a number of genes. This reduction was absent in the KO. CONCLUSION: Insulin resistance of the kidney may increase whole-body glucose instability a major risk factor for morbidity in diabetes. High dietary acid loads provide a dilemma for the kidney, as ammoniagenesis liberates α-ketoglutarate, which is a substrate for gluconeogenesis. We demonstrate an important role for insulin and/or IGF1 receptor signaling in the PT to facilitate this process and reduce excursions in blood glucose. Thus, medications and lifestyle changes that improve renal insulin sensitivity may also provide added benefit in type 2 diabetes especially when coupled with metabolic acidosis.


Assuntos
Acidose Tubular Renal/metabolismo , Glucose/metabolismo , Insulina/sangue , Túbulos Renais Proximais/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Acidose Tubular Renal/enzimologia , Acidose Tubular Renal/genética , Cloreto de Amônio/administração & dosagem , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteína Forkhead Box O1/metabolismo , Frutose-Bifosfatase/metabolismo , Gluconeogênese/genética , Glucose-6-Fosfatase/metabolismo , Resistência à Insulina/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo
6.
Int J Mol Sci ; 19(10)2018 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-30249002

RESUMO

Insulin therapy is often needed to overcome insulin receptor resistance in type 2 diabetes; however, the impact of providing additional insulin to already hyperinsulinemic subjects is not clear. We infused male TALLYHO/Jng (TH) mice (insulin resistant) with insulin (50 U/kg·bw/d) or vehicle (control) by osmotic minipump for 14 days. One group of insulin-infused mice was switched to 4% NaCl diet (high-sodium diet, HSD) in the second week. Blood chemistry revealed a significantly higher anion gap and blood sodium concentrations with insulin infusion, i.e., relative metabolic acidosis. Systolic BP and heart rate were slightly (~5 mm Hg) higher in insulin-infused versus control mice. HSD resulted in a modest and transient rise in mean arterial blood pressure (BP), relative to control or insulin-infused, normal-NaCl-fed mice. In kidney, insulin infusion: (1) increased total and phosphorylated (serine-1177) endothelial nitric oxide synthase (eNOS) band densities; (2) reduced band density of the uncoupled form of eNOS; and (3) increased renal homogenate nitric oxide synthase (NOS) activity. Despite this, plasma and urine levels of nitrates plus nitrites (NOx) fell with insulin infusion, by day 14 (40⁻50%) suggesting worsening of resistance. Overall, insulin infusion ramps up the cellular means in kidney to increase vasodilatory and natriuretic NO, but in the long term may be associated with worsening of insulin receptor resistance.


Assuntos
Regulação Enzimológica da Expressão Gênica , Resistência à Insulina , Insulina/administração & dosagem , Rim/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico/urina , Animais , Frequência Cardíaca , Hipoglicemiantes/administração & dosagem , Infusões Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL
7.
Am J Physiol Renal Physiol ; 315(3): F413-F416, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29846113

RESUMO

The renal collecting duct and other postmacula densa sites are the primary tubular regions for fine-tuning of electrolyte homeostasis in the body. A role for the mechanistic target of rapamycin (mTOR), a serine-threonine kinase, has recently been appreciated in this regulation. mTOR exists in two distinct multiprotein functional complexes, i.e., mTORC1 and mTORC2. Upregulation of mTORC1, by growth factors and amino acids, is associated with cell cycle regulation and hypertrophic changes. In contrast, mTORC2 has been demonstrated to have a role in regulating Na+ and K+ reabsorptive processes, including those downstream of insulin and serum- and glucocorticoid-regulated kinase (SGK). In addition, mTORC2 can upregulate mTORC1. A number of elegant in vitro and in vivo studies using cell systems and genetically modified mice have revealed mechanisms underlying activation of the epithelial Na+ channel (ENaC) and the renal outer medullary K+ channel (ROMK) by mTORC2. Overall, mTOR in its systematic integration of phosphorylative signaling facilitates the delicate balance of whole body electrolyte homeostasis in the face of changes in metabolic status. Thus, inappropriate regulation of renal mTOR has the potential to result in electrolyte disturbances, such as acidosis/alkalosis, hyponatremia, and hypertension. The goal of this minireview is to highlight the physiological role of mTOR in its complexes in regulating electrolyte homeostasis in the aldosterone-sensitive distal nephron.


Assuntos
Aminoácidos/metabolismo , Proteínas Alimentares/metabolismo , Eletrólitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Túbulos Renais Coletores/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Cloretos/metabolismo , Eletrólitos/urina , Humanos , Capacidade de Concentração Renal , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Potássio/metabolismo , Eliminação Renal , Reabsorção Renal , Sódio/metabolismo , Serina-Treonina Quinases TOR/genética
8.
Artigo em Inglês | MEDLINE | ID: mdl-29399662

RESUMO

AIMS: The use of circulatory miRNAs as biomarkers and therapeutic targets for T2DM is an explosive area of study. However, no study has investigated circulatory miRNA expression exclusively in African-American adults. The aim of this study was to identify the expression of nine selected miRNAs in erythrocytes of pre-diabetic and type 2 diabetic African-American adults. MAIN METHODS: Patients were recruited from the Howard University Hospital Diabetes Treatment Center following an 8 to 10 hour overnight fast. Expression of the nine selected miRNAs (miRNA-499, miRNA-146, miRNA-126, miRNA-223, miRNA-15a, miRNA-15b, miRNA-224, miRNA-326, and miRNA-375) was evaluated using quantitative real time PCR. KEY FINDINGS: miRNA-15a, miRNA-15b, and miRNA-499 were significantly reduced in erythrocytes of pre-diabetic African-American adults. In the T2DM group, we found significant correlations between miRNA-15a and BMI (r=0.59, p=0.04), miRNA-15a and weight (r=0.52, p=0.01), and miRNA-15b and diastolic blood pressure (r=-0.52, p=0.02). In the pre-diabetic group, we found significant correlations between miRNA-15b and weight (r=0.90, p=0.02) and miRNA-499 and HbA1c (r=-0.89, p=0.01). SIGNIFICANCE: To our knowledge, this is the first study investigating miRNA expression in erythrocytes of non-diabetic high-risk obese--pre-diabetic and type 2 diabetic African-American adults. The findings of this study are consistent with previous reports of reduced expression of miRNA-15a, miRNA-15b, and miRNA-499 in human plasma or serum and in animal models. The current findings support the use of circulating miRNA-15a, miRNA-15b, and miRNA-499 as potential biomarkers for T2DM in African-American adults.

9.
Ethn Dis ; 23(4): 436-40, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24392605

RESUMO

OBJECTIVE: Our study aimed to evaluate total plasma ghrelin (TGh) concentration and its correlation with leptin and insulin in obese African American (AA) adolescents with a family history of type 2 diabetes. PARTICIPANTS AND METHODS: Insulin, leptin, and TGh were measured for 15 non-obese controls in fasted state and 19 obese AA adolescents on samples collected during oral glucose tolerance test (OGTT) using radioimmunoassay kits. The hormonal concentrations were compared at fasting levels between obese and non-obese AA adolescents. Insulin, leptin, and TGh concentrations were also compared during OGTT in the obese group. RESULTS: Fasting TGh was significantly lower in obese AA adolescents compared to non-obese controls, while fasting leptin and insulin were significantly higher in obese AA adolescents compared to non-obese controls. During OGTT, for the obese group, TGh increased significantly and plasma leptin decreased significantly. A significant negative correlation was found between TGh and leptin at 30 and 120 min, but at no other time points (0, 60, and 90 min). A significant positive correlation was found between TGh and insulin at 30 min during OGTT, but no other time points. CONCLUSIONS: TGh was lower in obese AA adolescents with a family history of type 2 diabetes and a significant correlation occurred between TGh and leptin and TGh and insulin during OGGT at specific time points in our obese group. These findings indicate that insulin resistant obese AA adolescents have impaired ghrelin suppression.


Assuntos
Negro ou Afro-Americano , Grelina/sangue , Teste de Tolerância a Glucose , Obesidade/sangue , Obesidade/etnologia , Adolescente , Estudos de Casos e Controles , Criança , Jejum/sangue , Feminino , Humanos , Insulina/sangue , Leptina/sangue , Masculino , Radioimunoensaio , Adulto Jovem
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