bims-glecem 2023-02-05 papers

Issue of 2023–02–05
four papers selected by
Dipsikha Biswas, Københavns Universitet

Anal Biochem. 2023 Feb 01. pii: S0003-2697(23)00036-2. [Epub ahead of print] 115071

Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase.

Martin Thomassen, Jens Bangsbo, Morten Hostrup.

Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAβ1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

Keywords: Biochemical method; Immuno-assay; Muscle physiology; Type-2 diabetics; Western blotting

DOI: https://doi.org/10.1016/j.ab.2023.115071

J Biomol Struct Dyn. 2023 Jan 30. 1-10

In silico approaches to develop new phenyl-pyrimidines as glycogen synthase kinase 3 (GSK-3) inhibitors with halogen-bonding capabilities: 3D-QSAR CoMFA/CoMSIA, molecular docking and molecular dynamics studies.

David Cabezas, Guido Mellado, Nicolás Espinoza, José Antonio Gárate, César Morales, Alejandro Castro-Alvarez, Maria J Matos, Marco Mellado, Jaime Mella.

Glycogen synthase kinase 3 (GSK-3) is involved in different diseases, such as manic-depressive illness, Alzheimer's disease and cancer. Studies have shown that insulin inhibits GSK-3 to keep glycogen synthase active. Inhibiting GSK-3 may have an indirect pro-insulin effect by favouring glycogen synthesis. Therefore, the development of GSK-3 inhibitors can be a useful alternative for the treatment of type II diabetes. Aminopyrimidine derivatives already proved to be interesting GSK-3 inhibitors. In the current study, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) have been performed on a series of 122 aminopyrimidine derivatives in order to generate a robust model for the rational design of new compounds with promising antidiabetic activity. The q2 values obtained for the best CoMFA and CoMSIA models have been 0.563 and 0.598, respectively. In addition, the r2 values have been 0.823 and 0.925 for CoMFA and CoMSIA, respectively. The models were statistically validated, and from the contour maps analysis, a proposal of 10 new compounds has been generated, with predicted pIC50 higher than 9. The final contribution of our work is that: (a) we provide an extensive structure-activity relationship for GSK-3 inhibitory pyrimidines; and (b) these models may speed up the discovery of GSK-3 inhibitors based on the aminopyrimidine scaffold. Finally, we carried out docking and molecular dynamics studies of the two best candidates, which were shown to establish halogen-bond interactions with the enzyme.Communicated by Ramaswamy H. Sarma.

Keywords: 3D-QSAR; Type II diabetes; glycogen synthase kinase 3; insulin; pyrimidine

DOI: https://doi.org/10.1080/07391102.2023.2172457

Can J Physiol Pharmacol. 2023 Jan 30.

Hepatalin: The missing link in prediabetes, obesity, and type 2 diabetes.

W Wayne Lautt.

Hepatalin is a hormone secreted by the liver in response to pulses of insulin after a mixed nutrient meal, but only if the liver receives two permissive synergistic feeding signals from the stomach. Hepatalin stimulates glucose uptake and storage as glycogen in skeletal muscle, heart, and kidney but not liver, intestines, or adipocytes. Insulin acts primarily on liver and fat. Reduced hepatalin action results in postprandial hyperglycemia, compensatory elevation of insulin secretion, and a resultant shift in partitioning of nutrient energy storage from glycogen in muscle, to fat. Chronic hepatalin suppression leads to a predictable chronology of dysfunctions, first diagnosable as Absence of Meal-induced Insulin Sensitization (AMIS) which progresses to prediabetes, adiposity, and type 2 diabetes. The focus on nutrient partitioning and the role of hepatalin allows AMIS to be diagnosed, prevented, and treated, including through the use of lifestyle interventions.

DOI: https://doi.org/10.1139/cjpp-2022-0332

Arch Microbiol. 2023 Feb 01. 205(2): 78

Function of the mdxR gene encoding a novel regulator for carbohydrate metabolism and sporulation in Bacillus subtilis 168.

Tianshi Wang, Jung-Wan Kim.

The mdxR gene located upstream of mdxD, encoding a maltogenic amylase, has been annotated as a member of LacI-type transcriptional regulator in Bacillus subtilis 168 but its function has not been investigated yet. In this study, expression pattern of the mdxR promoter (PmdxR) and effects of mdxR were investigated to elucidate the function of mdxR. Expression of PmdxR was monitored by the β-galactosidase activity expressed from the PmdxR-lacZ fusion integrated at the amyE locus on the chromosome. The promoter was induced by starch, β-cyclomaltodextrin, or maltose at early exponential phase and kept expressed until late stationary phase. However, it was repressed by glucose, sucrose, or glycerol, suggesting that it was under catabolite repression. Furthermore, interactions of MdxR and Spo0A to the DNA fragment carrying PmdxR or PmdxD were detected by mobility-shift assay, implying that MdxR was a novel transcription regulator for both genes, which were regulated also by Spo0A. The mdxR mutant impaired the expressions of mdxD and malL (encoding an α-glucosidase); degraded accumulated glycogen slower than the wild type and the mdxD mutant. Both of the mdxR and the mdxD mutants formed more endospores (50.95% and 47.10%) than the wild type (23.90%). Enhanced sporulation by these mutations could be of industrial interest where sporulation or endospores of B. subtilis matters. These results indicate that MdxR functions as a transcriptional regulator for mdxR, mdxD, and other genes in the gene cluster that is related to the maltose/maltodextrin metabolism. MdxR and MdxD are also involved in glycogen metabolism and sporulation, tentatively by modulating the net energy balance in the cell.

Keywords: Bacillus subtilis; Glycogen metabolism; Maltogenic amylase; MdxR; Sporulation; Transcriptional regulator

DOI: https://doi.org/10.1007/s00203-023-03423-0