Functional Characterization of MODY2 Mutations Highlights the Importance of the Fine-Tuning of Glucokinase and Its Role in Glucose Sensing

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing

Guía clínica para el diagnóstico y seguimiento de la distrofia miotónica tipo 1, DM1 o enfermedad de Steinert

La enfermedad de Steinert o distrofia miotónica tipo 1 (DM1), (OMIM 160900) es la miopatía más prevalente en el adulto. Es una enfermedad multisistémica con alteración de prácticamente todos los órganos y tejidos y una variabilidad fenotípica muy amplia, lo que implica que deba ser atendida por diferentes especialistas que dominen las alteraciones más importantes. En los últimos anos ˜ se ha avanzado de manera exponencial en el conocimiento de la enfermedad y en su manejo. El objetivo de la guía es establecer recomendaciones para el diagnóstico, el pronóstico, el seguimiento y el tratamiento de las diferentes alteraciones de la DM1. Esta guía de consenso se ha realizado de manera multidisciplinar. Se ha contado con neurólogos, neumólogos, cardiólogos, endocrinólogos, neuropediatras y genetistas que han realizado una revisión sistemática de la literatura. Se recomienda realizar un diagnóstico genético con cuantificación precisa de tripletes CTG. Los pacientes con DM1 deben seguir control cardiológico y neumológico de por vida. Antes de cualquier cirugía con anestesia general debe realizarse una evaluación respiratoria. Debe monitorizarse la presencia de síntomas de disfagia periódicamente. Debe ofrecerse consejo genético a los pacientes con DM1 y a sus familiares. La DM1 es una enfermedad multisistémica que requiere un seguimiento en unidades especializadas multidisciplinares

Nuevo diagnóstico de diabetes en pacientes con Covid-19 ¿Cuál es la evidencia?

Sin financiaciónNo data 2021UE

Cómo valorar el riesgo cardiovascular antes del ejercicio en el paciente con diabetes

Sin financiaciónNo data 2021UE

Pituitary adenoma and meningioma simulating a single selar and paraseal injury

To analyze the importance of including axial cuts in studies of any brain region, including the selar.  Remember the possibility of the existence of two different tumors simultaneously, in the same anatomical region

Identification of a novel large CASR deletion in a patient with familial hypocalciuric hypercalcemia

Familial hypocalciuric hypercalcemia type I is an autosomal dominant disorder caused by heterozygous loss-of-function mutations in the CASR gene and is characterized by moderately elevated serum calcium concentrations, low urinary calcium excretion and inappropriately normal or mildly elevated parathyroid hormone (PTH) concentrations. We performed a clinical and genetic characterization of one patient suspected of familial hypocalciuric hypercalcemia type I. Patient presented persistent hypercalcemia with normal PTH and 25-hydroxyvitamin D levels. The CASR was screened for mutations by PCR followed by direct Sanger sequencing and, in order to detect large deletions or duplications, multiplex ligation-dependent probe amplification (MLPA) was used. One large deletion of 973 nucleotides in heterozygous state (c.1733-255_2450del) was detected. This is the first large deletion detected by the MLPA technique in the CASR gene

Effect of mutation p.Ala449Thr on the GK interaction with GKRP.

<p>A) Inhibition of glucokinase activity by human GKRP. Enzyme activity was measured at 5 mM glucose as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>, in the presence of 10 µM S6P (left panel) or 0.2 mM F1P (right panel). Results are means ± SEM for three independent enzyme purifications assayed in triplicate. B) Two-hybrid interaction of GBD-GKRP with GAD-GK or GAD-GK(p.Ala449Thr) mutant. Yeast strain Y187 was used, and fusion proteins were expressed from pGBKT7 and pACTII derivatives. Values are means ± SEM from ß-galactosidase activity of six independent transformants. In control experiments, GBD-GKRP did not interact with GAD and GAD-GK did not interact with GBD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#pone.0030518-Galan1" target="_blank">[22]</a>.</p

Effect of temperature on the stability of the GST-GK(p.Ala449Thr) protein.

<p>Stock enzyme solutions were diluted to 250 µg/ml in buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM dithiothreitol, 200 mM KCl and 50 mM Tris/HCl, pH 8.0. A) The enzyme solutions were incubated for 30 min at different temperatures ranging from 30 to 55°C and then assayed at 30°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030518#s2" target="_blank">Material and Methods</a>. B) The enzyme solutions were incubated for different periods of time from 5 to 60 min at 50°C. Means and SEM of three independent enzyme preparations are shown.</p