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1.
Hum Gene Ther ; 30(10): 1263-1273, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31319709

RESUMO

Glycogen storage diseases (GSDs) type I (GSDI) and type III (GSDIII), the most frequent hepatic GSDs, are due to defects in glycogen metabolism, mainly in the liver. In addition to hypoglycemia and liver pathology, renal, myeloid, or muscle complications affect GSDI and GSDIII patients. Currently, patient management is based on dietary treatment preventing severe hypoglycemia and increasing the lifespan of patients. However, most of the patients develop long-term pathologies. In the past years, gene therapy for GSDI has generated proof of concept for hepatic GSDs. This resulted in a recent clinical trial of adeno-associated virus (AAV)-based gene replacement for GSDIa. However, the current limitations of AAV-mediated gene transfer still represent a challenge for successful gene therapy in GSDI and GSDIII. Indeed, transgene loss over time was observed in GSDI liver, possibly due to the degeneration of hepatocytes underlying the physiopathology of both GSDI and GSDIII and leading to hepatic tumor development. Moreover, multitissue targeting requires high vector doses to target nonpermissive tissues such as muscle and kidney. Interestingly, recent pharmacological interventions or dietary regimen aiming at the amelioration of the hepatocyte abnormalities before the administration of gene therapy demonstrated improved efficacy in GSDs. In this review, we describe the advances in gene therapy and the limitations to be overcome to achieve efficient and safe gene transfer in GSDs.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Glucose-6-Fosfatase/genética , Doença de Depósito de Glicogênio Tipo III/terapia , Doença de Depósito de Glicogênio Tipo I/terapia , Hipoglicemia/terapia , Animais , Ensaios Clínicos como Assunto , Dependovirus/metabolismo , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose-6-Fosfatase/metabolismo , Glicogênio/biossíntese , Doença de Depósito de Glicogênio Tipo I/enzimologia , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/patologia , Doença de Depósito de Glicogênio Tipo III/enzimologia , Doença de Depósito de Glicogênio Tipo III/genética , Doença de Depósito de Glicogênio Tipo III/patologia , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Hipoglicemia/enzimologia , Hipoglicemia/genética , Hipoglicemia/patologia , Fígado/enzimologia , Fígado/patologia , Transgenes
2.
Proteins ; 87(1): 41-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367518

RESUMO

Mammalian glutamate dehydrogenase (GDH) has complex allosteric regulation and the loss of GTP inhibition causes the hyperinsulinism/hyperammonemia syndrome (HHS) where insulin is hypersecreted upon consumption of protein. The archetypical HHS lesion is H454Y and lies in the GTP binding pocket. To better understand the mechanism of HHS, we determined the crystal structure of H454Y. When the bovine GDH crystal structures were minimized to prepare for further computational analysis, unusually large deviations were found at the allosteric NADH binding site due to chemical sequence errors. Notably, 387 lies in an allosteric where several activators and inhibitors bind and should be lysine rather than asparagine. All structures were re-refined and the consequence of this sequence error on NADH binding was calculated using free energy perturbation. The binding free energy penalty going from the correct to incorrect sequence found is +5 kcal/mol per site and therefore has a significant impact on drug development. BROADER AUDIENCE ABSTRACT: Glutamate dehydrogenase is a key enzyme involved in amino acid catabolism. As such, it is heavily regulated in animals by a wide array of metabolites. The importance of this regulation is most apparent in a genetic disorder called hyperinsulinism/hyperammonemia (HHS) where patients hypersecrete insulin upon the consumption of protein. We determined the atomic structure of one of these HHS mutants to better understand the disease and also analyzed an allosteric regulatory site.


Assuntos
Glutamato Desidrogenase/química , Guanosina Trifosfato/metabolismo , Hiperinsulinismo/genética , Hipoglicemia/genética , Proteínas Mutantes/química , Mutação , Regulação Alostérica , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Humanos , Hiperinsulinismo/enzimologia , Hipoglicemia/enzimologia , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica
3.
J Pediatr Endocrinol Metab ; 31(7): 781-788, 2018 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-29858906

RESUMO

Background The phosphoglucomutase 1 (PGM1) enzyme plays a central role in glucose homeostasis by catalyzing the inter-conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, PGM1 deficiency has been recognized as a cause of the congenital disorders of glycosylation (CDGs). Methods Two Chinese Han pediatric patients with recurrent hypoglycemia, hepatopathy and growth retardation are described in this study. Targeted gene sequencing (TGS) was performed to screen for causal genetic variants in the genome of the patients and their parents to determine the genetic basis of the phenotype. Results DNA sequencing identified three variations of the PGM1 gene (NM_002633.2). Patient 1 had a novel homozygous mutation (c.119delT, p.Ile40Thrfs*28). In patient 2, we found a compound heterozygous mutation of c.1172G>T(p.Gly391Val) (novel) and c.1507C>T(p.Arg503*) (known pathogenic). Conclusions This report deepens our understanding of the clinical features of PGM1 mutation. The early molecular genetic analysis and multisystem assessment were here found to be essential to the diagnosis of PGM1-CDG and the provision of timely and proper treatment.


Assuntos
Biomarcadores/metabolismo , Doença de Depósito de Glicogênio/genética , Mutação , Fosfoglucomutase/genética , Criança , Pré-Escolar , Feminino , Doença de Depósito de Glicogênio/enzimologia , Doença de Depósito de Glicogênio/patologia , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Humanos , Hipoglicemia/enzimologia , Hipoglicemia/genética , Hipoglicemia/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Fenótipo , Prognóstico
4.
Cardiovasc Diabetol ; 16(1): 154, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29195509

RESUMO

BACKGROUND: The potential of anti-aging effect of DPP-4 inhibitors is unknown. This study was performed to determine whether linagliptin, a DPP-4 inhibitor, could protect against premature aging in klotho-/- mice. METHODS: Klotho-/- mice exhibit multiple phenotypes resembling human premature aging, including extremely shortened life span, cognitive impairment, hippocampal neurodegeneration, hair loss, muscle atrophy, hypoglycemia, etc. To investigate the effect of linagliptin on these aging-related phenotypes, male klotho-/- mice were divided into two groups: (1) control group fed the standard diet, and (2) linagliptin group fed the standard diet containing linagliptin. Treatment with linagliptin was performed for 4 weeks. The effect of linagliptin on the above mentioned aging-related phenotypes was examined. RESULTS: Body weight of klotho-/- mice was greater in linagliptin group than in control group (11.1 ± 0.3 vs 9.9 ± 0.3 g; P < 0.01), which was associated with greater gastrocnemius muscle weight (P < 0.01) and greater kidney weight (P < 0.05) in linagliptin group. Thus, linagliptin significantly prevented body weight loss in klotho-/- mice. Survival rate of klotho-/- mice was greater in linagliptin group (93%) compared to control group (67%), although the difference did not reach statistical significance (P = 0.08). None of linagliptin-treated klotho-/- mice had alopecia during the treatment (P < 0.05 vs control klotho-/- mice). Latency of klotho-/- mice in passive avoidance test was larger in linagliptin group than in control group (P < 0.05), indicating the amelioration of cognitive impairment by linagliptin. Cerebral blood flow of klotho-/- mice was larger in linagliptin group than in control group (P < 0.01), being associated with greater cerebral phospho-eNOS levels (P < 0.05) in linagliptin group. Neuronal cell number in hippocampal CA1 region was greater in linagliptin group than in control group (P < 0.05). Linagliptin group had greater cerebral phospho-Akt (P < 0.05) and phospho-CREB (P < 0.05) than control group. Thus, linagliptin ameliorated brain aging in klotho-/- mice. The degree of hypoglycemia in klotho-/- mice was less in linagliptin group than in control group, as estimated by the findings of OGTT. CONCLUSIONS: Out work provided the evidence that DPP-4 inhibition with linagliptin slowed the progression of premature aging in klotho-/- mice, and provided a novel insight into the potential role of DPP-4 in the mechanism of premature aging.


Assuntos
Senilidade Prematura , Envelhecimento/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Glucuronidase/deficiência , Linagliptina/farmacologia , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/psicologia , Alopecia/enzimologia , Alopecia/genética , Alopecia/fisiopatologia , Alopecia/prevenção & controle , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Cognição/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genótipo , Glucuronidase/genética , Hipoglicemia/sangue , Hipoglicemia/enzimologia , Hipoglicemia/genética , Hipoglicemia/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Óxido Nítrico Sintase Tipo III/metabolismo , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Perda de Peso/efeitos dos fármacos
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 34(2): 228-231, 2017 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-28397225

RESUMO

OBJECTIVE: To analyze the clinical and molecular features of a child with carnitine palmitoyltransferase 1A (CPT1A) deficiency. METHODS: Clinical data of the child was collected. Blood acylcarnitine was determined with tandem mass spectrometry. DNA was extracted from the child and his parents. All exons and flanking regions of the CPT1A gene were analyzed by PCR and Sanger sequencing. RESULTS: Analysis showed that the patient carried compound heterozygous mutations c.1787T>C and c.2201T>C of the CPT1A gene, which derived his father and mother, respectively. Both mutations were verified as novel through the retrieval of dbSNP, HGMD and 1000 genome databases. Bioinformatic analysis suggested that the mutations can affect protein function. CONCLUSION: Acyl carnitine analysis has been the main method for the diagnosis of CPT1A deficiency. The c.1787T>C and c.2201T>C mutations of the CPT1A gene probably underlie the disease in this patient. Gene testing can provide important clues for genetic counseling and prenatal diagnosis.


Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Hipoglicemia/genética , Erros Inatos do Metabolismo Lipídico/genética , Sequência de Bases , Carnitina O-Palmitoiltransferase/genética , Éxons , Feminino , Humanos , Hipoglicemia/enzimologia , Lactente , Erros Inatos do Metabolismo Lipídico/enzimologia , Masculino , Dados de Sequência Molecular , Mutação Puntual , Gravidez
6.
Neurochem Res ; 41(7): 1612-24, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26935743

RESUMO

In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.


Assuntos
Glândulas Suprarrenais/enzimologia , Encéfalo/enzimologia , Hipoglicemia/induzido quimicamente , Hipoglicemia/enzimologia , Insulina/toxicidade , Tirosina 3-Mono-Oxigenase/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Encéfalo/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley
7.
Diabetes ; 65(6): 1672-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26953163

RESUMO

Congenital hyperinsulinism of infancy (CHI) can be caused by inactivating mutations in the gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), a ubiquitously expressed enzyme involved in fatty acid oxidation. The hypersecretion of insulin may be explained by a loss of interaction between SCHAD and glutamate dehydrogenase in the pancreatic ß-cells. However, there is also a general accumulation of metabolites specific for the enzymatic defect in affected individuals. It remains to be explored whether hypoglycemia in SCHAD CHI can be uncoupled from the systemic effect on fatty acid oxidation. We therefore transplanted islets from global SCHAD knockout (SCHADKO) mice into mice with streptozotocin-induced diabetes. After transplantation, SCHADKO islet recipients exhibited significantly lower random and fasting blood glucose compared with mice transplanted with normal islets or nondiabetic, nontransplanted controls. Furthermore, intraperitoneal glucose tolerance was improved in animals receiving SCHADKO islets compared with those receiving normal islets. Graft ß-cell proliferation and apoptosis rates were similar in the two transplantation groups. We conclude that hypoglycemia in SCHAD-CHI is islet cell-autonomous.


Assuntos
3-Hidroxiacil-CoA Desidrogenase/deficiência , Hiperinsulinismo Congênito/enzimologia , Hipoglicemia/enzimologia , Células Secretoras de Insulina/metabolismo , Fenótipo , Animais , Hiperinsulinismo Congênito/genética , Glutamato Desidrogenase/metabolismo , Hipoglicemia/genética , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout
8.
Cell Metab ; 21(6): 883-90, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26039451

RESUMO

The AMP-activated protein kinase (AMPK) activates autophagy, but its role in aging and fasting-induced muscle function has not been defined. Here we report that fasting mice lacking skeletal muscle AMPK (AMPK-MKO) results in hypoglycemia and hyperketosis. This is not due to defective fatty acid oxidation, but instead is related to a block in muscle proteolysis that leads to reduced circulating levels of alanine, an essential amino acid required for gluconeogenesis. Markers of muscle autophagy including phosphorylation of Ulk1 Ser555 and Ser757 and aggregation of RFP-LC3 puncta are impaired. Consistent with impaired autophagy, aged AMPK-MKO mice possess a significant myopathy characterized by reduced muscle function, mitochondrial disease, and accumulation of the autophagy/mitophagy proteins p62 and Parkin. These findings establish an essential requirement for skeletal muscle AMPK-mediated autophagy in preserving blood glucose levels during prolonged fasting as well as maintaining muscle integrity and mitochondrial function during aging.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Envelhecimento/metabolismo , Autofagia , Jejum/efeitos adversos , Hipoglicemia/enzimologia , Músculo Esquelético/enzimologia , Doenças Musculares/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Envelhecimento/genética , Envelhecimento/patologia , Animais , Ativação Enzimática/genética , Hipoglicemia/etiologia , Hipoglicemia/genética , Hipoglicemia/patologia , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Doenças Musculares/genética , Doenças Musculares/patologia
9.
Cell Rep ; 11(6): 884-892, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25937276

RESUMO

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.


Assuntos
Endossomos/enzimologia , Fígado/enzimologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Simulação por Computador , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Técnicas de Silenciamento de Genes , Gluconeogênese/genética , Glucose/metabolismo , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio Tipo I/enzimologia , Doença de Depósito de Glicogênio Tipo I/patologia , Hepatomegalia/enzimologia , Hepatomegalia/patologia , Hiperglicemia/enzimologia , Hiperglicemia/patologia , Hipoglicemia/enzimologia , Hipoglicemia/patologia , Insulina/metabolismo , Metabolismo dos Lipídeos , Camundongos Knockout , Modelos Biológicos , Proteômica , Transdução de Sinais/genética
10.
Neuroscience ; 292: 34-45, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25701713

RESUMO

The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates glycogen content and turnover in these structures during glucose sufficiency and shortage.


Assuntos
Astrócitos/enzimologia , Catecolaminas/metabolismo , Estradiol/metabolismo , Glicogênio/metabolismo , Hipotálamo/enzimologia , Rombencéfalo/metabolismo , Animais , Estradiol/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Hipoglicemia/enzimologia , Hipoglicemiantes/administração & dosagem , Hipotálamo/efeitos dos fármacos , Insulina/administração & dosagem , Insulina/metabolismo , Neurotoxinas/toxicidade , Ovariectomia , Oxidopamina/toxicidade , Ratos Sprague-Dawley , Rombencéfalo/efeitos dos fármacos
11.
FASEB J ; 29(4): 1426-34, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25550458

RESUMO

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible "exon-switch" approach to knock in the constitutively active Pik3ca(H1047R) mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca(H1047R) mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild-type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3ca(H1047R) mutant mice died earlier (median survival 46.5 d post-mutation induction) than wild-type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.


Assuntos
Hipoglicemia/enzimologia , Hipoglicemia/genética , Insulina/sangue , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Substituição de Aminoácidos , Animais , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Glucose/metabolismo , Humanos , Hipoglicemia/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Tamanho do Órgão/genética , Tamanho do Órgão/fisiologia , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ganho de Peso
13.
Am J Physiol Regul Integr Comp Physiol ; 306(7): R457-69, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24381179

RESUMO

Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolosensory loci operate autonomously or interact to coordinate central nervous system (CNS) reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite l-lactate during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal AMPK activity, coincident with exacerbation of hypoglycemia. Hindbrain lactate repletion also blunted hypoglycemic upregulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 phosphoAMPK (pAMPK), elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Hipoglicemia/enzimologia , Hipotálamo/enzimologia , Ácido Láctico/metabolismo , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Rombencéfalo/metabolismo , Neurônios Adrenérgicos/metabolismo , Animais , Modelos Animais de Doenças , Ativação Enzimática , Comportamento Alimentar , Regulação da Expressão Gênica , Hipoglicemia/induzido quimicamente , Hipoglicemia/genética , Hipoglicemia/fisiopatologia , Hipoglicemia/psicologia , Hipotálamo/fisiopatologia , Infusões Intraventriculares , Insulina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácido Láctico/administração & dosagem , Masculino , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Neuropeptídeos/genética , Orexinas , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-Dawley , Rombencéfalo/fisiopatologia , Transdução de Sinais
14.
Cell Mol Neurobiol ; 33(8): 1065-74, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23975094

RESUMO

Neonatal hypoglycaemia initiates a series of events leading to neuronal death, even if glucose and glycogen stores return to normal. Disturbances in the cortical dopaminergic function affect memory and cognition. We recommend Bacopa monnieri extract or Bacoside A to treat neonatal hypoglycaemia. We investigated the alterations in dopaminergic functions by studying the Dopamine D1 and D2 receptor subtypes. Receptor-binding studies revealed a significant decrease (p < 0.001) in dopamine D1 receptor number in the hypoglycaemic condition, suggesting cognitive dysfunction. cAMP content was significantly (p < 0.001) downregulated in hypoglycaemic neonatal rats indicating the reduction in cell signalling of the dopamine D1 receptors. It is attributed to the deficits in spatial learning and memory. Hypoglycaemic neonatal rats treated with Bacopa extract alone and Bacoside A ameliorated the dopaminergic and cAMP imbalance as effectively as the glucose therapy. The upregulated Bax expression in the present study indicates the high cell death in hypoglycaemic neonatal rats. Enzyme assay of SOD confirmed cortical cell death due to free radical accumulation. The gene expression of SOD in the cortex was significantly downregulated (p < 0.001). Bacopa treatment showed a significant reversal in the altered gene expression parameters (p < 0.001) of Bax and SOD. Our results suggest that in the rat experimental model of neonatal hypoglycaemia, Bacopa extract improved alterations in D1, D2 receptor expression, cAMP signalling and cell death resulting from oxidative stress. This is an important area of study given the significant motor and cognitive impairment that may arise from neonatal hypoglycaemia if proper treatment is not implemented.


Assuntos
Bacopa/química , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Hipoglicemia/tratamento farmacológico , Hipoglicemia/metabolismo , Fármacos Neuroprotetores/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Animais Recém-Nascidos , Benzazepinas/farmacologia , Caspase 8/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , AMP Cíclico/metabolismo , Hipoglicemia/enzimologia , Imuno-Histoquímica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
15.
Eur J Med Genet ; 56(8): 411-5, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23751782

RESUMO

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).


Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Hipoglicemia/enzimologia , Hipoglicemia/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Mutação , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Ativação Enzimática , Éxons , Ordem dos Genes , Humanos , Hidroximetilglutaril-CoA Sintase/química , Lactente , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação Proteica
16.
Biomed Res Int ; 2013: 727143, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23586057

RESUMO

Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1-7) and 5 anthocyanins (compound 8-12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC(50), 4.78 µ M). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications.


Assuntos
Aldeído Redutase/metabolismo , Complicações do Diabetes/enzimologia , Hipoglicemia/enzimologia , Hipoglicemiantes/administração & dosagem , Extratos Vegetais/administração & dosagem , Aldeído Redutase/antagonistas & inibidores , Animais , Antocianinas/administração & dosagem , Antocianinas/química , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/patologia , Galactitol/metabolismo , Humanos , Hipoglicemia/tratamento farmacológico , Hipoglicemia/patologia , Cinética , Cristalino/efeitos dos fármacos , Extratos Vegetais/química , Ratos , Zea mays/química
17.
Anal Bioanal Chem ; 405(4): 1345-51, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23143007

RESUMO

Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency.


Assuntos
Cardiomiopatias/diagnóstico , Carnitina/análogos & derivados , Hiperamonemia/diagnóstico , Hipoglicemia/diagnóstico , Erros Inatos do Metabolismo Lipídico/diagnóstico , Doenças Musculares/diagnóstico , Espectrometria de Massas em Tandem/métodos , Transporte Biológico , Cardiomiopatias/enzimologia , Cardiomiopatias/metabolismo , Carnitina/análise , Carnitina/deficiência , Carnitina/metabolismo , Carnitina Aciltransferases/deficiência , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Ácidos Graxos/metabolismo , Fibroblastos/química , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Hiperamonemia/enzimologia , Hiperamonemia/metabolismo , Hipoglicemia/enzimologia , Hipoglicemia/metabolismo , Erros Inatos do Metabolismo Lipídico/enzimologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias/metabolismo , Doenças Musculares/enzimologia , Doenças Musculares/metabolismo , Oxirredução , Espectrometria de Massas por Ionização por Electrospray/métodos
18.
Diabetes Metab ; 38(6): 550-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22996038

RESUMO

AIM: Despite half of all type 2 diabetes mellitus (T2DM) patients being over 65 and treatment being complicated by an elevated risk of iatrogenic hypoglycaemia, information about antidiabetic treatment is scarce in this age group. This prospective observational study compares DPP4-inhibitors (DPP4-i) with conventional oral antidiabetic drugs (COAD) in the real-life treatment of elderly patients with T2DM uncontrolled on metformin alone. METHODS: Two treatment cohorts (DPP4-i and COAD, constituted on the basis of the GP decision of add-on therapy at the 1st visit) were compared after 6months. The primary objective was to assess the incidence of hypoglycaemic episodes in relationship with glycaemic control assessed by HbA(1c) level. RESULTS: Demographics and disease history were comparable between the two cohorts (DPP4-i, n=931 and COAD, n=257) at baseline. The incidence of hypoglycaemia/severe hypoglycaemia was significantly higher over 6months in the COAD cohort (20.1%/2.4% vs. 6.4%/0.1%; P<0.001) whereas similar improvements were observed in glycaemic control with HbA(1c) down from 7.9% to 7.0% (COAD) and 6.9% (DPP4-i). The 7% target was reached without hypoglycaemia in more patients in the DPP4-i than in COAD cohort (59.7% vs. 45.5%; P<0.001). Patients in both cohorts who experienced hypoglycaemia more frequently had a pre-existing diabetic complication. The COAD was more likely to be discontinued (6.6% vs. 1.6%; P<0.001). CONCLUSION: This large cohort study of elderly T2DM patients in France shows that the incidence of hypoglycaemia was three times higher in patients prescribed a COAD versus a DPP4-i after 6months while both treatments induced satisfactory glycaemic control.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Metformina/administração & dosagem , Idoso , Estudos de Coortes , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/enzimologia , Quimioterapia Combinada , Inibidores Enzimáticos/efeitos adversos , Feminino , Humanos , Hipoglicemia/sangue , Hipoglicemia/tratamento farmacológico , Hipoglicemia/enzimologia , Hipoglicemiantes/efeitos adversos , Masculino , Metformina/efeitos adversos , Estudos Prospectivos , Estatísticas não Paramétricas
19.
Neurosci Lett ; 525(2): 140-5, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22884618

RESUMO

Cerebral pyruvate depletion and lactate acidosis are common metabolic characteristics of patients with traumatic brain injury (TBI) and are associated with poor prognosis. Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme coupling glycolysis to mitochondrial tricarboxylic acid (TCA) cycle. Brain PDH activity is regulated by its phosphorylation status and other effectors. Phosphorylation of PDH E1α1 subunit by PDH kinase inhibits PDH activity while dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP1) restores PDH activity. In situ hybridization showed that PDP1 mRNA is highly expressed in the cerebral cortex, hippocampus and thalamus of rat. Controlled cortical impact (CCI) induced a significant increase in PDP1 mRNA expression in ipsilateral cerebral cortex at 4 h (P<0.05) and 24 h post CCI (P<0.01) that returned to basal level 72 h post CCI. PDP1 mRNA level increased transiently in ipsilateral hippocampal dentate gyrus and CA1-3 subfields 4 h post CCI (P<0.01) but decreased significantly 24 h and 72 h (P<0.01) post CCI, coinciding with a marked increase in neuronal apoptosis in ipsilateral hippocampus 24 h post CCI. PDP1 mRNA expression in thalamus and other subcortical regions decreased persistently post CCI. Contralateral CCI and craniotomy showed similar effects on PDP1 mRNA expression as ipsilateral CCI. Because GFAP mRNA expression was induced in brain regions where PDP1 expression was altered, further study should determine the potential relationship between astrocyte activation, PDP1 alteration, and pyruvate metabolism following TBI.


Assuntos
Lesões Encefálicas/enzimologia , Córtex Cerebral/enzimologia , Hipocampo/enzimologia , Hiperglicemia/enzimologia , Hipoglicemia/enzimologia , Neurônios/enzimologia , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismo , RNA Mensageiro/metabolismo , Tálamo/enzimologia , Animais , Apoptose , Biomarcadores/metabolismo , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Hipocampo/patologia , Hiperglicemia/etiologia , Hipoglicemia/etiologia , Hibridização In Situ , Masculino , Neurônios/patologia , Piruvato Desidrogenase (Lipoamida)-Fosfatase/genética , Ratos , Ratos Sprague-Dawley
20.
PLoS One ; 7(5): e36335, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22590531

RESUMO

Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK) activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+)/Calmodulin-dependent protein kinase kinase (CaMKK), which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO), a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG)-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV) in rats 30 min before 2DG ICV injection (40 µmol) to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol) did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Hipoglicemia/enzimologia , Hipotálamo/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Antimetabólitos/efeitos adversos , Antimetabólitos/farmacologia , Benzimidazóis/farmacologia , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Hipoglicemia/induzido quimicamente , Masculino , Naftalimidas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
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