MODY 2 causado por mutaciones en la glucoquinasa: una aproximación a nivel molecular / MODY 2 caused by glucokinase mutations: a molecular approach

La diabetes MODY 2 es un tipo de diabetes monogénica producida por una mutación en la enzima glucoquinasa, generando un fenotipo hiperglicémico. Para posibles fines terapéuticos o de diagnóstico, se debe conocer esta proteína, una enzima monomérica de la familia de las hexoquinasas, encargadas de convertir glucosa en glucosa-6-fosfato, el primer paso de la glicolisis. La glucoquinasa se caracteriza por sus propiedades cinéticas únicas: tiene una afinidad mucho menor por el sustrato que las demás hexoquinasas y no es inhibida por su producto. Se encuentra principalmente en páncreas e hígado (ßGK y LGK, respectivamente), donde como sensor regula los distintos estados metabólicos de estos tejidos, y controla la glicemia a nivel sistémico. Las formas ßGK y LGK se diferencian a nivel transcripcional, pues el gen posee dos promotores distintos, específicos para cada tejido. A nivel hormonal, la actividad de esta enzima es regulada selectivamente de manera tejido-específica por glucosa, insulina y otras proteínas reguladoras. La isoforma hepática puede ser secuestrada hacia el núcleo por la proteína reguladora de glucoquinasa (GKRP, por su sigla en inglés). La principal característica de la enzima glucoquinasa es su inusual regulación alostérica, propiedad que le permite adoptar dos conformaciones principales, una cerrada (activa) y otra súper-abierta (inactiva). Se han desarrollado distintas drogas activadoras de glucoquinasa, las cuales se unen al sitio alostérico de la enzima y estabilizan a la proteína en su estado cerrado. En esta revisión se describen las características estructurales y propiedades regulatorias que posee la enzima glucoquinasa, relacionándolas con su rol en el desarrollo de la diabetes MODY 2. También se profundiza en las implicancias moleculares de algunas mutaciones descritas que originan MODY 2, y se abordan los efectos de moléculas activadoras de glucoquinasa.

Diabetes MODY 2 or GCK-MODY is a type of monogenic diabetes produced by a mutation in the glucokinase enzyme, generating a hyperglycemic phenotype. This protein, a monomeric enzyme of the hexokinase family, is responsible for converting glucose into glucose-6-phosphate, the first step of glycolysis. Glucokinase is characterized by its unique kinetic properties: it has a much lower affinity for its substrate than other hexokinases and is not inhibited by its product. It is found mainly in pancreas (ßGK) and liver (LGK), where it acts as a sensor regulating the different metabolic states of these tissues, and ultimately, controlling systemic glycemia. The two forms ßGK and LGK differ at a transcriptional level, because the gene presents two different tissue-specific promoters. The activity of glucokinase in liver and pancreas is regulated by glucose, insulin and other regulatory proteins. The liver isoform can be sequestered to the nucleus by the glucokinase regulatory protein (GKRP). The main characteristic of the enzyme is its unusual allosteric regulation, a property that allows the protein to adopt a closed (active) conformation, and a super-open (inactive) conformation. Different glucokinase activating drugs have been developed, which bind to the allosteric site of the enzyme and stabilize glucokinase in its closed state. This review describes the structural and regulatory properties of the glucokinase enzyme, and its role in the development of MODY 2 diabetes. The molecular implications of some mutations that originate MODY 2 are also described, and the effects of glucokinase activating molecules are addressed.

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia

ABSTRACT Objective To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. Subjects and methods We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. Results We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. Conclusion The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

Expression profile of mitrogen-activated protein kinase (MAPK) signaling genes in the skeletal muscle & liver of rat with type 2 diabetes: Role in disease pathology.

Background & objectives: Type 2 diabetes (T2D) is characterized as hyperglycaemia caused by defects in insulin secretion, and it affects target tissues, such as skeletal muscle, liver and adipose tissue. Therefore, analyzing the changes of gene expression profiles in these tissues is important to elucidate the pathogenesis of T2D. We, therefore, measured the gene transcript alterations in liver and skeletal muscle of rat with induced T2D, to detect differentially expressed genes in liver and skeletal muscle and perform gene-annotation enrichment analysis. Methods: In the present study, skeletal muscle and liver tissue from 10 streptozotocin-induced diabetic rats and 10 control rats were analyzed using gene expression microarrays. KEGG pathways enriched by differentially expressed genes (DEGs) were identified by WebGestalt Expander and GATHER software. DEGs were validated by the method of real-time PCR and western blot. Results: From the 9,929 expressed genes across the genome, 1,305 and 997 differentially expressed genes (DEGs, P<0.01) were identified in comparisons of skeletal muscle and liver, respectively. large numbers of DEGs (200) were common in both comparisons, which was clearly more than the predicted number (131 genes, P<0.001). For further interpretation of the gene expression data, three over-representation analysis softwares (WebGestalt, Expander and GATHER) were used. All the tools detected one KEGG pathway (MAPK signaling) and two GO (gene ontology) biological processes (response to stress and cell death), with enrichment of DEGs in both tissues. In addition, PPI (protein-protein interaction) networks constructed using human homologues not only revealed the tendency of DEGs to form a highly connected module, but also suggested a “hub” role of p38-MAPK-related genes (such as MAPK14) in the pathogenesis of T2D. Interpretation & conclusions: Our results indicated the considerably aberrant MAPK signaling in both insulin-sensitive tissues of T2D rat, and that the p38 may play a role as a common “hub” in the gene module response to hyperglycaemia. Furthermore, our research pinpoints the role of several new T2D-associated genes (such as Srebf1 and Ppargc1) in the human population.

Incidental mild hyperglycemia in children: two MODY 2 families identified in Brazilian subjects / Hiperglicemia incidental em crianças: duas famílias com MODY 2 identificadas em brasileiros

Maturity-onset diabetes of the young (MODY) is characterized by an autosomal dominant mode of inheritance, early onset of hyperglycemia, and defects of insulin secretion. MODY subtypes described present genetic, metabolic, and clinical differences. MODY 2 is characterized by mild asymptomatic fasting hyperglycemia, and rarely requires pharmacological treatment. Hence, precise diagnosis of MODY is important for determining management and prognosis. We report two heterozygous GCK mutations identified during the investigation of short stature. Case 1: a prepubertal 14-year-old boy was evaluated for constitutional delay of growth and puberty. During follow-up, he showed abnormal fasting glucose (113 mg/dL), increased level of HbA1c (6.6%), and negative β-cell antibodies. His father and two siblings also had slightly elevated blood glucose levels. The mother had normal glycemia. A GCK heterozygous missense mutation, p.Arg191Trp, was identified in the proband. Eighteen family members were screened for this mutation, and 11 had the mutation in heterozygous state. Case 2: a 4-year-old boy investigated for short stature revealed no other laboratorial alterations than elevated glycemia (118 mg/dL); β-cell antibodies were negative. His father, a paternal aunt, and the paternal grandmother also had slightly elevated glycemia, whereas his mother had normal glycemia. A GCK heterozygous missense mutation, p.Glu221Lys, was identified in the index patient and in four family members. All affected patients had mild elevated glycemia. Individuals with normal glycemia did not harbor mutations. GCK mutation screening should be considered in patients with chronic mild early-onset hyperglycemia, family history of impaired glycemia, and negative β-cell antibodies. Arq Bras Endocrinol Metab. 2012;56(8):519-24.

O diabetes do tipo MODY (maturity-onset diabetes of the young) caracteriza-se por herança autossômica dominante, início precoce da hiperglicemia e defeitos na secreção de insulina. Os subtipos de MODY apresentam diferenças genéticas, metabólicas e clínicas. O MODY 2 é caracterizado por hiperglicemia leve assintomática e raramente requer tratamento farmacológico. O diagnóstico preciso de MODY é importante para se determinar o tratamento e o prognóstico. Relatamos duas mutações no gene GCK em heterozigose identificadas durante investigação de baixa estatura. Caso 1: paciente do sexo masculino, com 14 anos, pré-púbere, avaliado por atraso constitucional do crescimento e da puberdade. Durante o acompanhamento, apresentou glicemia de jejum alterada (113 mg/dL), aumento de HbA1c (6,6%) e anticorpos anticélulas β negativos. Seu pai e dois irmãos também apresentavam glicemia levemente elevada. A mãe tinha glicemia normal. Foi identificada no gene GCK uma mutação missense em heterozigose, p.Arg191Trp. Dezoito membros da família foram rastreados e 11 apresentavam essa mutação. Caso 2: paciente do sexo masculino, com 4 anos, em avaliação por baixa estatura. Não apresentou alterações laboratoriais, exceto por glicemia elevada (118 mg/dL). Anticorpos anticélulas β foram negativos. Seu pai, uma tia paterna e a avó paterna também apresentavam glicemia discretamente elevada, e a mãe, glicemia normal. A mutação missense em heterozigose, p.Glu221Lys, foi identificada no paciente-índice e em 4 membros da família. Todos os pacientes afetados apresentavam hiperglicemia leve. Essas mutações não foram identificadas nos indivíduos com glicemia normal. O rastreamento de mutações no gene GCK deve ser considerado em pacientes com hiperglicemia crônica leve e de início precoce, história familiar de glicemia elevada e anticorpos anticélulas β negativos. Arq Bras Endocrinol Metab. 2012;56(8):519-24.

Repression of Ppargcla gene in liver of hyperglycemic rats induced with high fat diet combined with streptozotocin / Represión del gen Ppargc1a en hígado de ratas con hiperglicemia inducida por dieta alta en grasas combinada con estreptozotocina

Type 2 diabetes mellitus implies deregulation of multiple metabolic processes, being the maintenance of glycemia one of the most important. Many genes are involved in the deregulation of this particular process. Therefore, the aim of this study was to evaluate gene expression of genes related to type 2 diabetes mellitus, in the liver and pancreas of rats with hyperglycemia induced by high fat diet along with a low single dose of streptozotocin. Ahsg and Ppargc1a genes were studied in liver, whereas Kcnj11 and Slc2a2 genes were analyzed in pancreas. For this purpose, 210-240 g female rats were fed a high fat diet or a control diet for three weeks. At day 14, animals fed with high fat diet were injected with a single low dose of streptozotocin (35 mg/kg) and the control group rats were injected only with the vehicle. Plasmatic glucose, triglycerides and total cholesterol levels were measured at the beginning, day 14 and end of treatment. Body weight was also measured. Once the treatment was complete, rats were appropriately euthanized and then, pancreas and liver were surgically removed and frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent, treated with DNase I and reversely transcribed to cDNA. Gene expression analysis was performed using SYBR Green ­ Real time PCR and comparative Cq method, using three reference genes. Rats fed with high fat diet and treated with streptozotocin showed higher values of plasmatic glucose (17.09 +/- 0.43 vs. 5.91 +/- 0.29 mmol/L, p < 0.01) and a minor expression of Ppargc1a versus the control group (2-fold less expressed, p < 0.05) in liver. We conclude that repression of Ppargc1a gene may be an important process in the establishment of chronic hyperglycemia, probably through deregulation of hepatic gluconeogenesis. However, further studies need to be performed in order to clarify the role of Ppargc1a deregulation in liver glucose homeostasis.

La diabetes mellitus tipo 2 implica desregulación de varios procesos metabólicos, siendo la mantención de la glicemia uno de los más importantes. Varios genes están involucrados en este proceso. Así, el objetivo del presente estudio fue evaluar la expresión génica de genes relacionados a diabetes mellitus tipo 2 en hígado y páncreas de ratas con hiperglicemia inducida con una dieta alta en grasas junto con una baja dosis de estreptozotocina. Ratas hembra de 210 - 240 g fueron alimentadas con una dieta alta en grasas o con una dieta control por tres semanas. Al día 14, los animales alimentados con dieta alta en grasas fueron inyectados con una dosis baja de estreptozotocina (35 mg/kg), mientras las del grupo control fueron inyectadas con el vehículo. El peso corporal y las concentraciones plasmáticas de glucosa, triglicéridos y colesterol total, fueron medidos al inicio, al día 14 y al final del tratamiento. Al finalizar el tratamiento, los animales fueron sacrificados, el hígado y páncreas fueron quirúrgicamente removidos e inmediatamente congelados en nitrógeno liquido. El RNA fue extraído usando el reactivo TRIzol, tratado con DNasa I y posteriormente convertido a cDNA. Los análisis de expresión génica fueron realizados usando SYBR Green - real time PCR y el método del Cq comparativo. En hígado se estudiaron los genes Ahsg y Ppargc1a, mientras que en páncreas se analizaron los genes Slc2a2 y Kcnj11. Las ratas alimentadas con dieta alta en grasas y tratadas con streptozotocina mostraron niveles mayores de glucosa plasmática (17,09 +/- 0,43 vs. 5,91 +/- 0,29 mmol/L, p < 0,01) y una menor expresión hepática de Ppargc1a versus el grupo control (2 veces menor expresión, p < 0,05). En conclusión, la represión del gen Ppargc1a puede ser un importante proceso en el establecimiento de la hiperglicemia crónica, probablemente debido a una desregulación de la gluconeogénesis hepática. Sin embargo, estudios adicionales son necesarios para esclarecer el rol de esta...

Hiperglicemia, tendencia genética y las complicaciones de la diabetes mellitus / Hiperglicemia genetic trend and complications in diabetes mellitus

Las complicaciones crónicas de la diabetes mellitus constituyen la principal causa de muerte y de limitación social en la población diabética; en este trabajo realizamos un análisis teórico acerca de las hipótesis más recientes que explican el desarrollo de estas complicaciones. Se hace especial mención a la importancia de la hiperglicemia y su control