Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Current Insight on the Role of Glucokinase and Glucokinase Regulatory Protein in Diabetes.

The glucokinase regulator (GCKR) gene encodes an inhibitor of the glucokinase enzyme (GCK), found only in hepatocytes and responsible for glucose metabolism. A common GCKR coding variation has been linked to various metabolic traits in genome-wide association studies. Rare GCKR polymorphisms influence GKRP activity, expression, and localization. Despite not being the cause, these variations are linked to hypertriglyceridemia. Because of their crystal structures, we now better understand the molecular interactions between GKRP and the GCK. Finally, small molecules that specifically bind to GKRP and decrease blood sugar levels in diabetic models have been identified. GCKR allelic spectrum changes affect lipid and glucose homeostasis. GKRP dysfunction has been linked to a variety of molecular causes, according to functional analysis. Numerous studies have shown that GKRP dysfunction is not the only cause of hypertriglyceridemia, implying that type 2 diabetes could be treated by activating liver-specific GCK via small molecule GKRP inhibition. The review emphasizes current discoveries concerning the characteristic roles of glucokinase and GKRP in hepatic glucose metabolism and diabetes. This information has influenced the growth of directed molecular therapies for diabetes, which has improved our understanding of lipid and glucose physiology.

Glucokinase regulatory protein: a balancing act between glucose and lipid metabolism in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease worldwide, affected by both genetics and environment. Type 2 diabetes (T2D) stands as an independent environmental risk factor that precipitates the onset of hepatic steatosis and accelerates its progression to severe stages of liver damage. Furthermore, the coexistence of T2D and NAFLD magnifies the risk of cardiovascular disease synergistically. However, the association between genetic susceptibility and metabolic risk factors in NAFLD remains incompletely understood. The glucokinase regulator gene (GCKR), responsible for encoding the glucokinase regulatory protein (GKRP), acts as a regulator and protector of the glucose-metabolizing enzyme glucokinase (GK) in the liver. Two common variants (rs1260326 and rs780094) within the GCKR gene have been associated with a lower risk for T2D but a higher risk for NAFLD. Recent studies underscore that T2D presence significantly amplifies the effect of the GCKR gene, thereby increasing the risk of NASH and fibrosis in NAFLD patients. In this review, we focus on the critical roles of GKRP in T2D and NAFLD, drawing upon insights from genetic and biological studies. Notably, prior attempts at drug development targeting GK with glucokinase activators (GKAs) have shown potential risks of augmented plasma triglycerides or NAFLD. Conversely, overexpression of GKRP in diabetic rats improved glucose tolerance without causing NAFLD, suggesting the crucial regulatory role of GKRP in maintaining hepatic glucose and lipid metabolism balance. Collectively, this review sheds new light on the complex interaction between genes and environment in NAFLD, focusing on the GCKR gene. By integrating evidence from genetics, biology, and drug development, we reassess the therapeutic potential of targeting GK or GKRP for metabolic disease treatment. Emerging evidence suggests that selectively activating GK or enhancing GK-GKRP binding may represent a holistic strategy for restoring glucose and lipid metabolic balance.

Contribution of Rs780094 and Rs1260326 Polymorphisms in GCKR Gene to Non-alcoholic Fatty Liver Disease: A Meta-Analysis Involving 26,552 Participants.

BACKGROUND: Many published studies attempted to elucidate the implication of glucokinase regulator gene (GCKR) polymorphisms in the susceptibility to non-alcoholic fatty liver disease (NAFLD), but the results among them were still controversial. OBJECTIVE: This meta-analysis aims to precisely assess the relationship between the GCKR polymorphisms and the risk of NAFLD. METHODS: Systematic computerized searches in six databases were performed and updated on April 6, 2020. Meta-analyses were conducted by calling the R programs based on accumulated epidemiological data. Odds ratio (OR) and 95% confidential interval (CI) were calculated to summarize the effect estimates. RESULTS: In total, 25 studies including 6,598 cases and 19,954 controls were included. The pooled estimates indicated that the T allele carrier of the GCKR rs780094 polymorphism has predisposition to NAFLD (allele model: OR: 1.20, 95% CI: 1.11~1.29; homozygote model: OR: 1.38, 95% CI: 1.15~1.67; heterozygote model: OR: 1.25, 95% CI: 1.12~1.39; dominant model: OR: 1.29, 95% CI: 1.13~1.47; recessive model: OR: 1.18, 95% CI: 1.06~1.31), and the same as the rs1260326 polymorphism (allele model: OR: 1.32, 95% CI: 1.22~1.42; homozygote model: OR: 1.65, 95% CI: 1.40~1.94; heterozygote model: OR: 1.24, 95% CI: 1.07~1.43; dominant model: OR: 1.39, 95% CI: 1.21~1.59; recessive model: OR: 1.44, 95% CI: 1.28~1.62). Further stratified analyses according to age and ethnicity confirmed the statistical existence in most subgroups. CONCLUSION: This meta-analysis suggested that both of the GCKR rs780094 and rs1260326 polymorphisms are significantly associated with the increased risk of NAFLD.

Blood transcriptome profiling as potential biomarkers of suboptimal health status: potential utility of novel biomarkers for predictive, preventive, and personalized medicine strategy.

The early identification of Suboptimal Health Status (SHS) creates a window opportunity for the predictive, preventive, and personalized medicine (PPPM) in chronic diseases. Previous studies have observed the alterations in several mRNA levels in SHS individuals. As a promising "omics" technology offering comprehension of genome structure and function at RNA level, transcriptome profiling can provide innovative molecular biomarkers for the predictive identification and targeted prevention of SHS. To explore the potential biomarkers, biological functions, and signalling pathways involved in SHS, an RNA sequencing (RNA-Seq)-based transcriptome analysis was firstly conducted on buffy coat samples collected from 30 participants with SHS and 30 age- and sex-matched healthy controls. Transcriptome analysis identified a total of 46 differentially expressed genes (DEGs), in which 22 transcripts were significantly increased and 24 transcripts were decreased in the SHS group. A total of 23 transcripts were selected as candidate predictive biomarkers for SHS. Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that several biological processes were related to SHS, such as ATP-binding cassette (ABC) transporter and neurodegeneration. Protein-protein interaction (PPI) network analysis identified 10 hub genes related to SHS, including GJA1, TWIST2, KRT1, TUBB3, AMHR2, BMP10, MT3, BMPER, NTM, and TMEM98. A transcriptome predictive model can distinguish SHS individuals from the healthy controls with a sensitivity of 83.3% (95% confidence interval (CI): 73.9-92.7%), a specificity of 90.0% (95% CI: 82.4-97.6%), and an area under the receiver operating characteristic curve of 0.938 (95% CI: 0.882-0.994). In the present study, we demonstrated that blood (buffy coat) samples appear to be a very promising and easily accessible biological material for the transcriptomic analyses focused on the objective identification of SHS by using our transcriptome predictive model. The pattern of particularly determined DEGs can be used as predictive transcriptomic biomarkers for the identification of SHS in an individual who may, subjectively, feel healthy, but at the level of subcellular mechanisms, the changes can provide early information about potential health problems in this person. Our findings also indicate the potential therapeutic targets in dealing with chronic diseases related to SHS, such as T2DM and CVD, and an early onset of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, as well as the findings suggest the targets for personalized interventions as promoted in PPPM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13167-021-00238-1.

Genetic predisposition similarities between NASH and ASH: Identification of new therapeutic targets.

Fatty liver disease can be triggered by a combination of excess alcohol, dysmetabolism and other environmental cues, which can lead to steatohepatitis and can evolve to acute/chronic liver failure and hepatocellular carcinoma, especially in the presence of shared inherited determinants. The recent identification of the genetic causes of steatohepatitis is revealing new avenues for more effective risk stratification. Discovery of the mechanisms underpinning the detrimental effect of causal mutations has led to some breakthroughs in the comprehension of the pathophysiology of steatohepatitis. Thanks to this approach, hepatocellular fat accumulation, altered lipid droplet remodelling and lipotoxicity have now taken centre stage, while the role of adiposity and gut-liver axis alterations have been independently validated. This process could ignite a virtuous research cycle that, starting from human genomics, through omics approaches, molecular genetics and disease models, may lead to the development of new therapeutics targeted to patients at higher risk. Herein, we also review how this knowledge has been applied to: a) the study of the main PNPLA3 I148M risk variant, up to the stage of the first in-human therapeutic trials; b) highlight a role of MBOAT7 downregulation and lysophosphatidyl-inositol in steatohepatitis; c) identify IL-32 as a candidate mediator linking lipotoxicity to inflammation and liver disease. Although this precision medicine drug discovery pipeline is mainly being applied to non-alcoholic steatohepatitis, there is hope that successful products could be repurposed to treat alcohol-related liver disease as well.

3D disorganization and rearrangement of genome provide insights into pathogenesis of NAFLD by integrated Hi-C, Nanopore, and RNA sequencing.

The three-dimensional (3D) conformation of chromatin is integral to the precise regulation of gene expression. The 3D genome and genomic variations in non-alcoholic fatty liver disease (NAFLD) are largely unknown, despite their key roles in cellular function and physiological processes. High-throughput chromosome conformation capture (Hi-C), Nanopore sequencing, and RNA-sequencing (RNA-seq) assays were performed on the liver of normal and NAFLD mice. A high-resolution 3D chromatin interaction map was generated to examine different 3D genome hierarchies including A/B compartments, topologically associated domains (TADs), and chromatin loops by Hi-C, and whole genome sequencing identifying structural variations (SVs) and copy number variations (CNVs) by Nanopore sequencing. We identified variations in thousands of regions across the genome with respect to 3D chromatin organization and genomic rearrangements, between normal and NAFLD mice, and revealed gene dysregulation frequently accompanied by these variations. Candidate target genes were identified in NAFLD, impacted by genetic rearrangements and spatial organization disruption. Our data provide a high-resolution 3D genome interaction resource for NAFLD investigations, revealed the relationship among genetic rearrangements, spatial organization disruption, and gene regulation, and identified candidate genes associated with these variations implicated in the pathogenesis of NAFLD. The newly findings offer insights into novel mechanisms of NAFLD pathogenesis and can provide a new conceptual framework for NAFLD therapy.

GCKR rs780094 Polymorphism as A Genetic Variant Involved in Physical Exercise.

Exercise performance is influenced by genetics. However, there is a lack of knowledge about the role played by genetic variability in the frequency of physical exercise practice. The objective was to identify genetic variants that modulate the commitment of people to perform physical exercise and to detect those subjects with a lower frequency practice. A total of 451 subjects were genotyped for 64 genetic variants related to inflammation, circadian rhythms, vascular function as well as energy, lipid and carbohydrate metabolism. Physical exercise frequency question and a Minnesota Leisure Time Physical Activity Questionnaire (MLTPAQ) were used to qualitatively and quantitatively measure the average amount of physical exercise. Dietary intake and energy expenditure due to physical activity were also studied. Differences between genotypes were analyzed using linear and logistic models adjusted for Bonferroni. A significant association between GCKR rs780094 and the times the individuals performed physical exercise was observed (p = 0.004). The carriers of the minor allele showed a greater frequency of physical exercise in comparison to the major homozygous genotype carriers (OR: 1.86, 95% CI: 1.36-2.56). The analysis of the GCKR rs780094 variant suggests a possible association with the subjects that present lower frequency of physical exercise. Nevertheless, future studies are needed to confirm these findings.

A case-control study and meta-analysis confirm glucokinase regulatory gene rs780094 is a risk factor for gestational diabetes mellitus.

BACKGROUND: Although the influence of a common variant in the glucokinase regulatory gene (GCKR rs780094) in type 2 diabetes mellitus has been well documented, less data however, is available of its role in gestational diabetes mellitus (GDM). We carried out a case control study to assess the association between GCKR rs780094 and GDM in the Asian, and also a meta-analysis to further assess the strength of the association. METHODS: Demographic, clinical and genotype data were determined for 1122 women (267 cases and 855 controls) recruited from the University of Malaya Medical Centre in the Klang Valley, Kuala Lumpur. Relevant articles were identified from Pubmed, Embase, MEDLINE, and Web of Science. Extraction of data was carried out and summary estimates of the association between rs780094 and GDM were examined. RESULTS: The frequency of risk allele C was significantly higher in the cases than controls (OR 1.34, 95% CI 1.09-1.66, P = 0.006). The C allele was also associated with increased level of random 2-hour fasting plasma glucose and pregravid body mass index. Meta-analysis further confirmed the association of the GCKR rs780094 with GDM (OR 1.32, 95% CI 1.14-1.52, P = 0.0001). CONCLUSION: This study strongly suggests that GCKR rs780094-C is associated with increased risk of GDM.

The genetic backgrounds in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD, can lead to hepatocellular carcinoma (HCC) and hepatic failure. The development and progression of NAFLD are determined by environmental and genetic factors. The effect of genetic factors has been demonstrated by familial studies, twin studies and several cross-sectional studies. In the past 10 years, genome-wide association studies have revealed several single nucleotide polymorphisms (SNPs) associated with the pathology of NAFLD. Among them, the Patatin-like phospholipase domain-containing 3 (PNPLA3) gene variant I148M showed a strong relationship with the development and progression of NAFLD, NASH, and NAFLD-related HCC. The transmembrane 6 superfamily member 2 (TM6SF2) gene variant E167 K was also associated with NAFLD, and it has a relationship with cardiovascular disease. Furthermore, several genes have been proposed as candidate genes to be associated with NAFLD based on case-control studies. We conducted a comprehensive literature search and review on the genetic background of NAFLD.

Polymorphisms in the GCKR are associated with serum lipid traits, the risk of coronary artery disease and ischemic stroke.

The present study was to determine the association of two single nucleotide polymorphisms (SNPs) in the glucokinase regulator gene (GCKR) and serum lipid levels, and the risk of coronary artery disease (CAD) and ischemic stroke (IS). Genotypes of the GCKR rs1260326 and rs8179206 in 1736 unrelated subjects (CAD, 584; IS, 555; and healthy controls; 597) were determined by the Snapshot technology platform. The genotypic and allelic frequencies of rs1260326 and rs8179206 were not different among the three groups (P > 0.05). The subjects with rs1260326TT genotype had higher serum low-density lipoprotein cholesterol (LDL-C) levels in controls, and higher triglyceride (TG) levels in CAD patients than the subjects with CC and CT genotypes after adjustment for age, sex, body mass index, blood pressure, alcohol consumption, and cigarette smoking (P < 0.05). The rs1260326TT genotype was also associated with decreased risk of IS in females (OR = 0.37, 95% CI: 0.18-0.76, P = 0.007). The present study shows that the GCKR rs1260326TT genotype is associated with high LDL-C in controls, high TG levels in CAD patients, and a decreased risk of IS in females.