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1.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
2.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33690219

RESUMO

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.


Assuntos
Carcinoma Hepatocelular , Quinase do Ponto de Checagem 2/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neoplasias Hepáticas , Fosfoenolpiruvato Carboxiquinase (GTP)/deficiência , Acilação/efeitos dos fármacos , Acilação/genética , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Quinase do Ponto de Checagem 2/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo
3.
Gene ; 768: 145263, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33122078

RESUMO

Translationally controlled tumor protein (TCTP) has various cellular functions and molecular interactions, many related to its growth-promoting and antiapoptotic properties. Recently, TCTP expression was reported to increases in insulin-resistant mice fed with high-fat diet. TCTP is a multifunctional protein, but its role in liver metabolism is unclear. Here, we investigated the function and mechanism of TCTP in HepG2 cells. Knock-down of TCTP led to 287 differentially expressed genes (DEGs) that were highly associated with cellular apoptosis and signal response, TNF and NF-κB signaling pathways, glycolysis/gluconeogenesis, insulin resistance, FoxO and insulin signaling pathways, adipocytokine and AMPK signaling pathways. shTCTP downregulated the expression of the key gluconeogenesis enzyme phosphoenolpyruvate carboxykinase (PCK1). Furthermore, TCTP regulated the alternative splicing of genes enriched in the phospholipid biosynthetic process and glycerophospholipid metabolism. We further showed that shTCTP down-regulated the intracellular levels of triglyceride and total cholesterol. Our results showed that TCTP regulates the liver cell transcriptome at both the transcriptional and alternative splicing levels. The TCTP regulatory network predicts the biological functions of TCTP in glucose and lipid metabolism, and also insulin resistance, which may be associated with liver metabolism and diseases such as nonalcoholic fatty liver disease.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação da Expressão Gênica/genética , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Processamento Alternativo/genética , Apoptose/genética , Glicemia/genética , Linhagem Celular Tumoral , Colesterol/sangue , Dieta Hiperlipídica , Gluconeogênese/genética , Glucose/metabolismo , Glicerofosfolipídeos/metabolismo , Glicólise/genética , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Ativação Transcricional/genética , Transcriptoma/genética , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/genética
4.
J Biol Chem ; 296: 100205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33334880

RESUMO

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Domínio Catalítico , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Simulação de Acoplamento Molecular , Fosfoenolpiruvato Carboxiquinase (GTP)/química
5.
Molecules ; 25(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182581

RESUMO

Diabetes mellitus is a disease characterized by persistent high blood glucose levels and accompanied by impaired metabolic pathways. In this study, we used zebrafish to investigate the effect of crocins isolated from Crocus sativus L., on the control of glucose levels and pancreatic ß-cells. Embryos were exposed to an aqueous solution of crocins and whole embryo glucose levels were measured at 48 h post-treatment. We showed that the application of crocins reduces zebrafish embryo glucose levels and enhances insulin expression. We also examined whether crocins are implicated in the metabolic pathway of gluconeogenesis. We showed that following a single application of crocins and glucose level reduction, the expression of phosphoenolpyruvate carboxykinase1 (pck1), a key gene involved in glucose metabolism, is increased. We propose a putative role for the crocins in glucose metabolism and insulin management.


Assuntos
Carotenoides/farmacologia , Crocus/química , Hiperglicemia/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Glicemia/metabolismo , Carotenoides/análise , Gluconeogênese , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Íons , Pâncreas/embriologia , Pâncreas/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Extratos Vegetais/farmacologia , Peixe-Zebra
6.
Nutrients ; 12(10)2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33036430

RESUMO

Fructose consumption by rodents modulates both hepatic and intestinal lipid metabolism and gluconeogenesis. We have previously demonstrated that in utero exposure to dexamethasone (DEX) interacts with fructose consumption during adult life to exacerbate hepatic steatosis in rats. The aim of this study was to clarify if adult rats born to DEX-treated mothers would display differences in intestinal gluconeogenesis after excessive fructose intake. To address this issue, female Wistar rats were treated with DEX during pregnancy and control (CTL) mothers were kept untreated. Adult offspring born to CTL and DEX-treated mothers were assigned to receive either tap water (Control-Standard Chow (CTL-SC) and Dexamethasone-Standard Chow (DEX-SC)) or 10% fructose in the drinking water (CTL-fructose and DEX-fructose). Fructose consumption lasted for 80 days. All rats were subjected to a 40 h fasting before sample collection. We found that DEX-fructose rats have increased glucose and reduced lactate in the portal blood. Jejunum samples of DEX-fructose rats have enhanced phosphoenolpyruvate carboxykinase (PEPCK) expression and activity, higher facilitated glucose transporter member 2 (GLUT2) and facilitated glucose transporter member 5 (GLUT5) content, and increased villous height, crypt depth, and proliferating cell nuclear antigen (PCNA) staining. The current data reveal that rats born to DEX-treated mothers that consume fructose during adult life have increased intestinal gluconeogenesis while recapitulating metabolic and morphological features of the neonatal jejunum phenotype.


Assuntos
Dexametasona/efeitos adversos , Carboidratos da Dieta/efeitos adversos , Células Epiteliais/patologia , Frutose/efeitos adversos , Gluconeogênese , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Exposição Materna/efeitos adversos , Troca Materno-Fetal/fisiologia , Efeitos Tardios da Exposição Pré-Natal , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Animais , Feminino , Transportador de Glucose Tipo 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Gravidez , Ratos Wistar
7.
Food Funct ; 11(9): 7696-7706, 2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32914810

RESUMO

Monk fruit extract (MFE) is a natural sweetener that has been used as an ingredient of food and pharmaceutical products. The effects of feeding synbiotic yogurt fortified with MFE to rats with type 2 diabetes induced by high-fat diet and streptozotocin on serum lipid levels and hepatic AMPK signaling pathway were evaluated. Results showed that oral administration of the synbiotic yogurt fortified with MFE could improve serum lipid levels, respiratory exchange rate, and heat level in type 2 diabetic rats. Transcriptome analysis showed that synbiotic yogurt fortified with MFE may affect the expression of genes involved in binding, catalytic activity, and transporter activity. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that these differentially expressed genes were related to AMPK signaling pathway, linoleic acid metabolism, and α-linolenic acid metabolism. Western blotting confirmed that synbiotic yogurt fortified with MFE could activate AMPK signaling and improve the protein level of the hepatic gluconeogenic enzyme G6Pase in diabetic rats. The results indicated that MFE could be a novel sweetener for functional yogurt and related products.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cucurbitaceae , Diabetes Mellitus Tipo 2/metabolismo , Lipídeos/sangue , Fígado/enzimologia , Simbióticos , Iogurte , Animais , Peso Corporal , Diabetes Mellitus Tipo 2/dietoterapia , Diabetes Mellitus Tipo 2/prevenção & controle , Perfilação da Expressão Gênica , Glucose-6-Fosfatase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácido Linoleico/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Extratos Vegetais , Ratos , Respiração , Transdução de Sinais , Edulcorantes , Ácido alfa-Linoleico/metabolismo
8.
Genes (Basel) ; 11(7)2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32610475

RESUMO

Obesity is associated with an increased risk of developing cardiovascular disease (CVD), with limited alterations in cardiac genomic characteristics known. Cardiac transcriptome analysis was conducted to profile gene signatures in high-fat diet (HFD)-induced obese mice. A total of 184 differentially expressed genes (DEGs) were identified between groups. Based on the gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs, the critical role of closely interlocked glucose metabolism was determined in HFD-induced cardiac remodeling DEGs, including Nr4a1, Fgf21, Slc2a3, Pck1, Gck, Hmgcs2, and Bpgm. Subsequently, the expression levels of these DEGs were evaluated in both the myocardium and palmitic acid (PA)-stimulated H9c2 cardiomyocytes using qPCR. Nr4a1 was highlighted according to its overexpression resulting from the HFD. Additionally, inhibition of Nr4a1 by siRNA reversed the PA-induced altered expression of glucose metabolism-related DEGs and hexokinase 2 (HK2), the rate-limiting enzyme in glycolysis, thus indicating that Nr4a1 could modulate glucose metabolism homeostasis by regulating the expression of key enzymes in glycolysis, which may subsequently influence cardiac function in obesity. Overall, we provide a comprehensive understanding of the myocardium transcript molecular framework influenced by HFD and propose Nr4a1 as a key glucose metabolism target in obesity-induced CVD.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Miocárdio/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Obesidade/metabolismo , Transcriptoma , Animais , Linhagem Celular , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Obesidade/etiologia , Obesidade/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos
9.
Int J Biochem Cell Biol ; 125: 105789, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32522621

RESUMO

Hepatocellular carcinoma (HCC) is a fatal disease characterized by poor liver function with increasing morbidity and poor prognosis. Extracellular vesicles, released by different cells, have been associated with HCC development. Nevertheless, the mechanisms beyond extracellular vesicles in HCC remain uncharacterized. Therefore, the current study aimed to clarify the mechanism of pro-angiogenic microRNA-584-5p in hepatocellular carcinoma. Our results showed that miR-584-5p was highly-expressed in both cancer cells (Hep3B) and their extracellular vesicles. Hep3B and extracellular vesicles were then respectively co-cultured with human vascular endothelial cell line (Ea.hy926), and they both accelerated Ea.hy926 proliferation and migration. Ea.hy926 cells could internalize extracellular vesicles carrying microRNA-584-5p. Of note, microRNA-584-5p could bind to phosphoenolpyruvate carboxykinase 1 to promote nuclear factor E2-related factor 2. Moreover, silencing microRNA-584-5p was found to decline microvessel density, vascular endothelial growth factor A, and tumor growth in vivo and in vitro. Taken altogether, our findings demonstrated that extracellular vesicles-derived microRNA-584-5p promotes angiogenesis by inhibiting PCK1 -mediating NRF2 activation, which highlights the theoretical basis for potential treatments for HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Vesículas Extracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neovascularização Patológica/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , Fator 2 Relacionado a NF-E2/genética , Neovascularização Patológica/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Transdução de Sinais/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nature ; 580(7804): 530-535, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322062

RESUMO

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/patologia , Proliferação de Células , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Nus , Oxisteróis/metabolismo , Fosforilação , Prognóstico , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
11.
Can J Diabetes ; 44(5): 401-406.e1, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32279935

RESUMO

OBJECTIVES: Metabolic surgery has been proven to be widely effective for the control of glucose and weight in patients with type 2 diabetes and obesity. However, the effects of bariatric surgery on nonobesity type 2 diabetes and its metabolism are still unclear. This study aimed to measure the effects of duodenal-jejunal exclusion on glycometabolism in nonobese rats with type 2 diabetes and to investigate its mechanisms. METHODS: Goto-Kakizaki rats and Sprague-Dawley rats were divided into duodenal-jejunal exclusion operation groups and sham operation groups, respectively. The glucose-relative parameters were measured before and after operation. Eight weeks postoperation, the levels of the key regulators of intestinal gluconeogenesis and the crucial proteins of hepatic insulin signalling were evaluated. RESULTS: Postoperatively, the concentrations of blood glucose declined, and the insulin sensitivity increased significantly in rats with diabetes. However, there was no obvious reduction in weight. Eight weeks postoperatively, the mRNA levels of glucose-6-phosphatase and phosphoenolpyruvate pyruvate kinase in the jejunum and the levels of insulin receptor substrate-2 and glucose transporter-2 in the liver were significantly increased compared with the rats that had undergone the sham operation. CONCLUSIONS: Duodenal-jejunal exclusion surgery is an effective procedure for improving glucose metabolism independent of weight loss in nonobese rats with diabetes. The molecular mechanisms might be associated with a series of processes, including intestinal gluconeogenesis and the hepatic insulin signaling pathway.


Assuntos
Cirurgia Bariátrica , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/cirurgia , Duodeno/cirurgia , Gluconeogênese/genética , Jejuno/cirurgia , Fígado/metabolismo , Estômago/cirurgia , Anastomose Cirúrgica , Animais , Peso Corporal , Diabetes Mellitus Tipo 2/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Jejuno/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , RNA Mensageiro/metabolismo , Ratos
12.
Biochimie ; 171-172: 31-37, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045650

RESUMO

Although up to 25% of glucose released into circulation in the postabsorptive state comes from renal gluconeogenesis, the regulatory mechanisms of this process are still poorly recognized, comparing to hepatic ones. The aim of the present study was to examine if hypoxia-inducible factor-1 (HIF-1) might be involved in the regulation of glucose de novo synthesis in kidneys. It was found that HK-2 cells (immortalized human kidney proximal tubules, capable of gluconeogenesis/glycogen synthesis) cultured with gluconeogenic substrates either in hypoxia (1% O2) or in the presence of DMOG (an inhibitor of HIF-1α degradation) exhibited increased glycogen content. This phenomenon was not correlated with augmented glucose intake and the effects were reversed by echinomycin (an inhibitor of HIF-1 binding to HRE sequence). As concluded from the measurement of the intracellular content of gluconeogenic intermediates followed by Western blot analysis, under conditions of hypoxia/increased HIF-1 level the activity of phosphoenolpyruvate carboxykinase (PEPCK) was elevated, as a result of increased expression of the cytosolic isoform of PEPCK (PEPCK-C). Chromatin immunoprecipitation (ChIP) analysis proved HIF-1 ability to bind to the promoter region of PEPCK-C gene. The final conclusion that hypoxia/HIF-1 accelerates the rate of renal glucogenesis via the mechanism engaging activation of PEPCK-C expression might be useful in terms of e.g. diabetes treatment, as it is commonly accepted that under diabetic conditions kidneys and liver seem to be equally important sources of glucose synthesized de novo.


Assuntos
Gluconeogênese , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Hipóxia Celular , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos
13.
J Cell Physiol ; 235(1): 166-175, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31180589

RESUMO

The pancreatic islets of Langerhans, mainly formed by glucagon-producing α-cells and insulin-producing ß-cells, are critical for glucose homeostasis. Insulin and glucagon oppositely modulate blood glucose levels in health, but a combined decline in insulin secretion together with increased glucagon secretion contribute to hyperglycemia in diabetes. Despite this bi-hormonal dysregulation, most studies have focused on insulin secretion and much less is known about glucagon secretion. Therefore, a deeper understanding of α-cell metabolism and glucagon secretion is of great interest. Here, we show that phosphoenolpyruvate carboxykinase (PCK1), an essential cataplerotic enzyme involved in metabolism and long considered to be absent from the pancreatic islet, is expressed in pancreatic α-cells of both murine and human. Furthermore, PCK1 transcription is induced by fasting and diabetes in rat pancreas, which indicates that the PCK1 activity is required for α-cell adaptation to different metabolic states. To our knowledge, this is the first evidence implicating PCK1 expression in α-cell metabolism.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Células Secretoras de Glucagon/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Pâncreas/enzimologia , Pâncreas/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Ratos
14.
Am J Physiol Cell Physiol ; 318(1): C137-C149, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31721616

RESUMO

Reactive oxygen species (ROS) are important signaling molecules mediating the exercise-induced adaptations in skeletal muscle. Acute exercise also drives the expression of genes involved in reesterification and glyceroneogenesis in white adipose tissue (WAT), but whether ROS play any role in this effect has not been explored. We speculated that exercise-induced ROS would regulate acute exercise-induced responses in WAT. To address this question, we utilized various models to alter redox signaling in WAT. We examined basal and exercise-induced gene expression in a genetically modified mouse model of reduced mitochondrial ROS emission [mitochondrial catalase overexpression (MCAT)]. Additionally, H2O2, various antioxidants, and the ß3-adrenergic receptor agonist CL316243 were used to assess gene expression in white adipose tissue culture. MCAT mice have reduced ROS emission from WAT, enlarged WAT depots and adipocytes, and greater pyruvate dehydrogenase kinase-4 (Pdk4) gene expression. In WAT culture, H2O2 reduced glyceroneogenic gene expression. In wild-type mice, acute exercise induced dramatic but transient increases in Pdk4 and phosphoenolpyruvate carboxykinase (Pck1) mRNA in both subcutaneous inguinal WAT and epididymal WAT depots, which was almost completely absent in MCAT mice. Furthermore, the induction of Pdk4 and Pck1 in WAT culture by CL316243 was markedly reduced in the presence of antioxidants N-acetyl-cysteine or vitamin E. Genetic and nutritional approaches that attenuate redox signaling prevent exercise- and ß-agonist-induced gene expression within WAT. Combined, these data suggest that ROS represent important mediators of gene expression within WAT.


Assuntos
Adipócitos/enzimologia , Tecido Adiposo Branco/enzimologia , Metabolismo Energético , Mitocôndrias/enzimologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia , Tecido Adiposo Branco/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Antioxidantes , Catalase/genética , Catalase/metabolismo , Metabolismo Energético/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Oxidantes/farmacologia , Oxirredução , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Esforço Físico , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Transdução de Sinais , Fatores de Tempo , Técnicas de Cultura de Tecidos
15.
J Cell Physiol ; 235(1): 349-363, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31237701

RESUMO

Metabolism homeostasis plays an important role in progenitor-cell differentiation to adipocytes, but less is known about the whole transcriptional profiling of cellular metabolism during adipogenesis. We got the first insight into the whole transcriptional profiling of cellular metabolism during adipogenesis from human mesenchymal stem cells (hMSCs) by the RNA-Seq technique. There were 1,998, 2,629, 3,112, and 3,054 differentially expressed genes (DEGs) at Days 7, 14, 21, and 28, respectively, during adipogenesis. The most enriched phosphatidylinositol 3' kinase-serine/threonine kinase (PI3K-Akt) signaling pathway stimulated and directly regulated cellular metabolism by priming glucose aerobic glycolysis, arginine and proline metabolism, glutathione metabolism, and arachidonic acid metabolism during adipogenesis, targeting the potential key genes, such as fatty acid synthase (FABP4), phosphoenolpyruvate carboxykinase 1 (PKC1), stearoyl-CoA desaturase (SCD), and solute carrier family 2 member 1 of Gluts (SLC2A1). And it confirmed PCK1 as the key player for cellular metabolism by small interfering RNA. A comprehensive understanding of cellular metabolism and its regulatory axis of the signaling pathway during adipogenesis would reveal new study and therapy targets for fat metabolism disorders.


Assuntos
Adipócitos/citologia , Adipogenia/genética , Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Células-Tronco Mesenquimais/citologia , Ácido Araquidônico/metabolismo , Arginina/metabolismo , Células Cultivadas , Proteínas de Ligação a Ácido Graxo/metabolismo , Perfilação da Expressão Gênica , Transportador de Glucose Tipo 1/metabolismo , Glutationa/metabolismo , Glicólise/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Prolina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Estearoil-CoA Dessaturase/metabolismo , Adulto Jovem
16.
Elife ; 82019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31841108

RESUMO

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with the liver being the main organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic colonization capacity upregulated the gluconeogenic enzyme PCK1, which enhanced liver metastatic growth by driving pyrimidine nucleotide biosynthesis under hypoxia. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with leflunomide substantially impaired CRC liver metastatic colonization and hypoxic growth. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis under hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in CRC metastatic progression.


Assuntos
Neoplasias Colorretais/fisiopatologia , Hipóxia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/fisiopatologia , Neoplasias Hepáticas/secundário , Nucleotídeos/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Modelos Teóricos
17.
Stem Cells ; 37(12): 1542-1555, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31574189

RESUMO

Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a rate-limiting enzyme that plays critical roles in multiple physiological processes. The decompensation of PCK2 leads to various energy metabolic disorders. However, little is known regarding the effects of PCK2 on osteogenesis by human mesenchymal stem cells (hMSCs). Here, we report a novel function of PCK2 as a positive regulator of MSCs osteogenic differentiation. In addition to its well-known role in anabolism, we demonstrate that PCK2 regulates autophagy. PCK2 deficiency significantly suppressed autophagy, leading to the impairment of osteogenic capacity of MSCs. On the other hand, autophagy was promoted by PCK2 overexpression; this was accompanied by increased osteogenic differentiation of MSCs. Moreover, PCK2 regulated osteogenic differentiation of MSCs via AMP-activated protein kinase (AMPK)/unc-51 like autophagy activating kinase 1(ULK1)-dependent autophagy. Collectively, our present study unveiled a novel role for PCK2 in integrating autophagy and bone formation, providing a potential target for stem cell-based bone tissue engineering that may lead to improved therapies for metabolic bone diseases. Stem Cells 2019;37:1542-1555.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteogênese/fisiologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Desenvolvimento Ósseo/fisiologia , Osso e Ossos/citologia , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Interferência de RNA , RNA Interferente Pequeno/genética
18.
Med Sci Monit ; 25: 6023-6033, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31406102

RESUMO

BACKGROUND The PCK1 gene encodes phosphoenolpyruvate carboxykinase (PEPCK), which has been shown have a role in metabolic events in hepatocellular carcinoma (HCC). This study aimed to investigate the role of the SHH gene and its encoded protein, sonic hedgehog (SHH), in two human hepatocellular carcinoma (HCC) cell lines. MATERIAL AND METHODS The human HCC cell lines Hep3B and SMMC-7721 were cultured. Cells were transfected with plasmids carrying specific SHH gene short-hairpin RNA (shRNA) and negative control (NC) shRNA. The effects of knockdown of expression levels of the SHH gene were studied on cell survival, cell apoptosis, the cell cycle, gluconeogenesis, and the expression of PCK1. Anchorage-independent growth, a characteristic of transformed cells, was detected by the colony formation assay. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot were performed 24 h after transfection. RESULTS Knockdown of expression levels of the SHH gene reduced cell proliferation and growth of HCC cells and induced cell apoptosis and G1 cell cycle arrest in both HCC cell lines. Knockdown of the SHH gene decreased the levels of glycolysis products and increased the production of glucose and reduced the phosphorylation of PI3K and Akt but induced the expression of PCK1. CONCLUSIONS Knockdown of the SHH gene reduced cell survival of HCC cells by increasing apoptosis, reducing cell proliferation, inducing G1 cell cycle arrest, and restoring gluconeogenesis, and was associated with the inhibition of the PI3K/Akt axis and induced the expression of PCK1.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise , Proteínas Hedgehog/metabolismo , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia
19.
Adv Exp Med Biol ; 1155: 113-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468390

RESUMO

We previously showed that taurine administration contributed to the extension of time to exhaustion through exercise-induced hypoglycemia restraint, and we suggested that the activation of hepatic gluconeogenesis was initiated before the exercise with the taurine administration. We hypothesize that the extension effect of exercise duration with the taurine administration is restrained in the rats which inhibited hepatic gluconeogenesis. In this study, we aimed to produce a rat model that inhibited hepatic gluconeogenesis as a first step in testing our hypothesis.F344 male rats of 8 weeks after birth were purchased. The blood samples were collected via jugular vein catheter to perform the pyruvate tolerance test (PTT) with the intraperitoneal administration, and to determine the optimal time point of blood glucose measurement. 3-mercaptopicolinic acids (3MPA) was used as an inhibitor of PEPCK. The rats were divided into three groups, Non-dosage control (CON) group, 30 mg/kg・BW 3MPA (3MPA 30) group, and 300 mg/kg・BW 3MPA (3MPA 300) group.The blood glucose level showed a significant peak 15 min after pyruvate administration. The change of the blood glucose level after the PTT in 3MPA 300 group was significantly smaller than that of the CON group at this time point. These results show we could prepare the rat model that inhibited hepatic gluconeogenesis.


Assuntos
Modelos Animais de Doenças , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/fisiopatologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Glicemia , Masculino , Condicionamento Físico Animal , Ratos , Ratos Endogâmicos F344 , Taurina
20.
Sci Rep ; 9(1): 10181, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308441

RESUMO

Exposure to maternal diabetes during pregnancy results in diabetes in offspring, but its underlying mechanisms are unclear. Here, we investigated the phenotype and molecular defects of the offspring of poorly controlled diabetic female mice generated by streptozotocin (STZ) administration. Offspring was exposed to maternal diabetes during pregnancy and lactation. The body weight of STZ offspring was lower than that of control offspring at birth and in adulthood, and glucose tolerance was impaired in adult STZ offspring. Interestingly, the phenotype was more pronounced in male offspring. We next investigated the morphology of islets and expression of ß cell-related genes, but no significant changes were observed. However, transcriptome analysis of the liver revealed activation of the fork head box protein O1 (Foxo1) pathway in STZ male offspring. Notably, two key gluconeogenesis enzyme genes, glucose 6 phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), were upregulated. Consistent with this finding, phosphorylation of Foxo1 was decreased in the liver of STZ male offspring. These changes were not obvious in female offspring. The activation of Foxo1 and gluconeogenesis in the liver may have contributed to the impaired glucose tolerance of STZ male offspring.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Proteína Forkhead Box O1/metabolismo , Intolerância à Glucose/etiologia , Animais , Glicemia/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Proteína Forkhead Box O1/fisiologia , Gluconeogênese/genética , Intolerância à Glucose/metabolismo , Glucose-6-Fosfatase/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Lactação/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Gravidez , Estreptozocina/farmacologia
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