Interplay of Mediterranean-diet adherence, genetic factors, and metabolic dysfunction-associated steatotic liver disease risk in Korea.

BACKGROUNDS: Metabolic dysfunction-associated steatotic liver disease (MASLD) has gained attention owing to its severe complications. This study aimed to explore the interaction between Mediterranean-diet (MD) adherence, genetic factors, and MASLD risk in a Korean population. METHODS: In total, 33,133 individuals aged 40 years and older from the Korean Genome and Epidemiology Study (KoGES) were analyzed. Participants were assessed for MASLD based on criteria and MD adherence measured by the Korean version of the Mediterranean-Diet Adherence Screener (K-MEDAS). Individuals were categorized into two groups based on their MD adherence: high adherence (K-MEDAS > 6) and low adherence (K-MEDAS < 5). Single nucleotide polymorphism (SNP) genotypes were obtained using the Korea Biobank array. Logistic regression was used to examine the single-marker variants for genetic associations with MASLD prevalence. RESULTS: Individuals were categorized into MASLD (10,018 [30.2%]) and non-MASLD (23,115 [69.8%]) groups. A significant interaction was observed between the rs780094 glucokinase regulatory protein (GCKR) gene and K-MEDAS on MASLD (p < 10 - 2 ). Of individuals with K-MEDAS > 6, those carrying the minor allele (C) of the GCKR gene rs780094 exhibited a lower risk of MASLD compared to those without the allele (odds ratio [OR] = 0.88 [0.85-0.91], p-value = 5.54e-13). CONCLUSION: The study identified a significant interaction involving the rs780094 variant near the GCKR gene, with carriers of the minor allele exhibiting a lower MASLD risk among those adhering well to the MD. Dietary habits influence the MASLD risk associated with the rs780094 allele, emphasizing the need for personalized nutrition recommendations.

Impaired GK-GKRP interaction rather than direct GK activation worsens lipid profiles and contributes to long-term complications: a Mendelian randomization study.

BACKGROUND: Glucokinase (GK) plays a key role in glucose metabolism. In the liver, GK is regulated by GK regulatory protein (GKRP) with nuclear sequestration at low plasma glucose level. Some GK activators (GKAs) disrupt GK-GKRP interaction which increases hepatic cytoplasmic GK level. Excess hepatic GK activity may exceed the capacity of glycogen synthesis with excess triglyceride formation. It remains uncertain whether hypertriglyceridemia associated with some GKAs in previous clinical trials was due to direct GK activation or impaired GK-GKRP interaction. METHODS: Using publicly available genome-wide association study summary statistics, we selected independent genetic variants of GCKR and GCK associated with fasting plasma glucose (FPG) as instrumental variables, to mimic the effects of impaired GK-GKRP interaction and direct GK activation, respectively. We applied two-sample Mendelian Randomization (MR) framework to assess their causal associations with lipid-related traits, risks of metabolic dysfunction-associated steatotic liver disease (MASLD) and cardiovascular diseases. We verified these findings in one-sample MR analysis using individual-level statistics from the Hong Kong Diabetes Register (HKDR). RESULTS: Genetically-proxied impaired GK-GKRP interaction increased plasma triglycerides, low-density lipoprotein cholesterol and apolipoprotein B levels with increased odds ratio (OR) of 14.6 (95% CI 4.57-46.4) per 1 mmol/L lower FPG for MASLD and OR of 2.92 (95% CI 1.78-4.81) for coronary artery disease (CAD). Genetically-proxied GK activation was associated with decreased risk of CAD (OR 0.69, 95% CI 0.54-0.88) and not with dyslipidemia. One-sample MR validation in HKDR showed consistent results. CONCLUSIONS: Impaired GK-GKRP interaction, rather than direct GK activation, may worsen lipid profiles and increase risks of MASLD and CAD. Development of future GKAs should avoid interfering with GK-GKRP interaction.

An insight into the mechanistic role of (-)-Ampelopsin F from Vatica chinensis L. in inducing insulin secretion in pancreatic beta cells.

Resveratrol oligomers, ranging from dimers to octamers, are formed through regioselective synthesis involving the phenoxy radical coupling of resveratrol building blocks, exhibiting remarkable therapeutic potential, including antidiabetic properties. In this study, we elucidate the mechanistic insights into the insulin secretion potential of a resveratrol dimer, (-)-Ampelopsin F (AmF), isolated from the acetone extract of Vatica chinensis L. stem bark in Pancreatic Beta-TC-6 cell lines. The AmF (50 µM) treated cells exhibited a 3.5-fold increase in insulin secretion potential as compared to unstimulated cells, which was achieved through the enhancement of mitochondrial membrane hyperpolarization, elevation of intracellular calcium concentration, and upregulation of GLUT2 and glucokinase expression in pancreatic Beta-TC-6 cell lines. Furthermore, AmF effectively inhibited the activity of DPP4, showcasing a 2.5-fold decrease compared to the control and a significant 6.5-fold reduction compared to the positive control. These findings emphasize AmF as a potential lead for the management of diabetes mellitus and point to its possible application in the next therapeutic initiatives.

Tetrahydropalmatine from medicinal plants activates human glucokinase to regulate glucose homeostasis.

Many synthetic glucokinase activators (GKAs), modulating glucokinase (GK), an important therapeutic target in diabetes have failed to clear clinical trials. In this study, an in silico structural similarity search with differing scaffolds of reference GKAs have been used to identify derivatives from natural product databases. Ten molecules with good binding score and similar interactions to that in the co-crystallized GK as well good activation against recombinant human GK experimentally were identified. Tetrahydropalmatine, an alkaloid present in formulations and drugs from medicinal plants, has not been explored as an antidiabetic agent and no information regarding its mechanism of action or GK activation exists. Tetrahydropalmatine activates GK with EC50 value of 71.7 ± 17.9 µM while lowering the S0.5 (7.1 mM) and increasing Vmax (9.22 µM/min) as compared to control without activator (S0.5 = 10.37 mM; Vmax = 4.8 µM/min). Kinetic data (α and ß values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, Kd values of 3.8 µM suggests a good affinity for GK. In HepG2 cell line, the compound potentiated the uptake of glucose and maintained glucose homeostasis by increasing the expression of GK, glycogen synthase, and insulin receptor genes and lowering the expression of glucokinase regulatory protein (GKRP) and glucagon. Tetrahydropalmatine at low concentrations could elicit a good response by reducing expression of GKRP, increasing expression of GK while also activating it. Thus, it could be used alone or in combination as therapeutic drug as it could effectively modulate GK and alter glucose homeostasis.

New-Generation Glucokinase Activators: Potential Game-Changers in Type 2 Diabetes Treatment.

Achieving glycemic control and sustaining functional pancreatic ß-cell activity remains an unmet medical need in the treatment of type 2 diabetes mellitus (T2DM). Glucokinase activators (GKAs) constitute a class of anti-diabetic drugs designed to regulate blood sugar levels and enhance ß-cell function in patients with diabetes. A significant progression in GKA development is underway to address the limitations of earlier generations. Dorzagliatin, a dual-acting GKA, targets both the liver and pancreas and has successfully completed two phase III trials, demonstrating favorable results in diabetes treatment. The hepato-selective GKA, TTP399, emerges as a strong contender, displaying clinically noteworthy outcomes with minimal adverse effects. This paper seeks to review the current literature, delve into the mechanisms of action of these new-generation GKAs, and assess their efficacy and safety in treating T2DM based on published preclinical studies and recent clinical trials.

Discovery of Strong 3-Nitro-2-Phenyl-2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Bringing precision medicine to the management of pregnancy in women with glucokinase-MODY: a study of diagnostic accuracy and feasibility of non-invasive prenatal testing.

AIMS/HYPOTHESIS: In pregnancies where the mother has glucokinase-MODY (GCK-MODY), fetal growth is determined by fetal genotype. When the fetus inherits a maternal pathogenic GCK variant, normal fetal growth is anticipated, and insulin treatment of maternal hyperglycaemia is not recommended. At present, fetal genotype is estimated from measurement of fetal abdominal circumference on ultrasound. Non-invasive prenatal testing of fetal GCK genotype (NIPT-GCK) using cell-free DNA in maternal blood has recently been developed. We aimed to compare the diagnostic accuracy of NIPT-GCK with that of ultrasound, and determine the feasibility of using NIPT-GCK to guide pregnancy management. METHODS: We studied an international cohort of pregnant women with hyperglycaemia due to GCK-MODY. We compared the diagnostic accuracy of NIPT-GCK with that of measurement of fetal abdominal circumference at 28 weeks' gestation (n=38) using a directly genotyped offspring sample as the reference standard. In a feasibility study, we assessed the time to result given to clinicians in 43 consecutive pregnancies affected by GCK-MODY between July 2019 and September 2021. RESULTS: In terms of diagnostic accuracy, NIPT-GCK was more sensitive and specific than ultrasound in predicting fetal genotype (sensitivity 100% and specificity 96% for NIPT-GCK vs sensitivity 53% and specificity 61% for fetal abdominal circumference 75th percentile). In terms of feasibility, a valid NIPT-GCK fetal genotype (≥95% probability) was reported in all 38 pregnancies with an amenable variant and repeated samples when needed. The median time to report was 5 weeks (IQR 3-8 weeks). For the 25 samples received before 20 weeks' gestation, results were reported at a median gestational age of 20 weeks (IQR 18-24), with 23/25 (92%) reported before 28 weeks. CONCLUSIONS/INTERPRETATION: Non-invasive prenatal testing of fetal genotype in GCK-MODY pregnancies is highly accurate and is capable of providing a result before the last trimester for most patients. This means that non-invasive prenatal testing of fetal genotype is the optimal approach to management of GCK-MODY pregnancies.

Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties.

Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form. The truncated form revealed no significant impact on the kinetic parameters, showing only a slight increase in the Vmax value, higher metal promiscuity, and the same nucleotide specificity as the full-length enzyme. hADP-GK presents an ordered sequential kinetic mechanism in which MgADP is the first substrate to bind and AMP is the last product released, being the same mechanism described for archaeal ADP-dependent sugar kinases, in agreement with the protein topology. Substrate inhibition by glucose was observed due to sugar binding to nonproductive species. Although Mg2+ is an essential component for kinase activity, it also behaves as a partial mixed-type inhibitor for hADP-GK, mainly by decreasing the MgADP affinity. Regarding its distribution, phylogenetic analysis shows that ADP-GK's are present in a wide diversity of eukaryotic organisms although it is not ubiquitous. Eukaryotic ADP-GKs sequences cluster into two main groups, showing differences in the highly conserved sugar-binding motif reported for archaeal enzymes [NX(N)XD] where a cysteine residue is found instead of asparagine in a significant number of enzymes. Site directed mutagenesis of the cysteine residue by asparagine produces a 6-fold decrease in Vmax, suggesting a role for this residue in the catalytic process, probably by facilitating the proper orientation of the substrate to be phosphorylated.

Sex Dimorphic Glucose Transporter-2 Regulation of Hypothalamic Astrocyte Glucose and Energy Sensor Expression and Glycogen Metabolism.

The plasma membrane glucose transporter-2 (GLUT2) monitors brain cell uptake of the critical nutrient glucose, and functions within astrocytes of as-yet-unknown location to control glucose counter-regulation. Hypothalamic astrocyte-neuron metabolic coupling provides vital cues to the neural glucostatic network. Current research utilized an established hypothalamic primary astrocyte culture model along with gene knockdown tools to investigate whether GLUT2 imposes sex-specific regulation of glucose/energy sensor function and glycogen metabolism in this cell population. Data show that GLUT2 stimulates or inhibits glucokinase (GCK) expression in glucose-supplied versus -deprived male astrocytes, but does not control this protein in female. Astrocyte 5'-AMP-activated protein kinaseα1/2 (AMPK) protein is augmented by GLUT2 in each sex, but phosphoAMPKα1/2 is coincidently up- (male) or down- (female) regulated. GLUT2 effects on glycogen synthase (GS) diverges in the two sexes, but direction of this control is reversed by glucoprivation in each sex. GLUT2 increases (male) or decreases (female) glycogen phosphorylase-brain type (GPbb) protein during glucoprivation, yet simultaneously inhibits (male) or stimulates (female) GP-muscle type (GPmm) expression. Astrocyte glycogen accumulation is restrained by GLUT2 when glucose is present (male) or absent (both sexes). Outcomes disclose sex-dependent GLUT2 control of the astrocyte glycolytic pathway sensor GCK. Data show that glucose status determines GLUT2 regulation of GS (both sexes), GPbb (female), and GPmm (male), and that GLUT2 imposes opposite control of GS, GPbb, and GPmm profiles between sexes during glucoprivation. Ongoing studies aim to investigate molecular mechanisms underlying sex-dimorphic GLUT2 regulation of hypothalamic astrocyte metabolic-sensory and glycogen metabolic proteins, and to characterize effects of sex-specific astrocyte target protein responses to GLUT2 on glucose regulation.

A multicentre, randomized, double-blind, parallel, active- and placebo-controlled Phase 3 clinical study of the glucokinase activator PB-201 in treatment-naive patients with type 2 diabetes mellitus: A study protocol.

AIM: To report the rationale for using PB-201, a partial glucokinase activator (GKA), for a Phase 3 study (NCT05102149) assessing its efficacy and safety in a Chinese population and to describe the design of this GKA Phase 3 trial, the first to involve both an active control and a placebo control arm. MATERIALS AND METHODS: This is an ongoing, multicentre, randomized, double-blind, three-arm placebo and active control study to be carried out among 672 Chinese treatment-naive participants with type 2 diabetes mellitus (T2DM) to assess the efficacy and safety of PB-201 for approximately 60 weeks, including a screening period and a safety follow-up period. RESULTS: The primary objective of this study was to monitor change in glycated haemoglobin levels with PB-201 in treatment-naive T2DM participants from baseline to 24 weeks in comparison with vildagliptin and placebo. The key secondary objective was to assess the efficacy and safety of PB-201 following treatment for a time period of 52 weeks. CONCLUSION: This pivotal study will offer critical information regarding the efficacy and safety of PB-201 in Chinese treatment-naive T2DM participants that would help to establish robust evidence for the benefit-risk evaluation of this drug.

Diabetes remission in drug-naïve patients with type 2 diabetes after dorzagliatin treatment: A prospective cohort study.

AIM: To investigate the post-treatment effect of dorzagliatin in drug-naïve patients with type 2 diabetes (T2D) regarding the achievement of stable glycaemic control and drug-free diabetes remission. MATERIALS AND METHODS: Patients who completed dorzagliatin treatment in the SEED trial and achieved stable glycaemic control were enrolled in this 52-week study without any antidiabetic medication. The primary endpoint was the diabetes remission probability at week 52 using the Kaplan-Meier method. The potential factors that contribute to stable glycaemic control and diabetes remission based on the characteristics of patients before and after treatment with dorzagliatin were analysed. A post hoc sensitivity analysis of diabetes remission probability using the American Diabetes Association (ADA) definition was conducted. RESULTS: The Kaplan-Meier remission probability was 65.2% (95% CI: 52.0%, 75.6%) at week 52. Based on the ADA definition, the remission probability was 52.0% (95% CI: 31.2%, 69.2%) at week 12. The significant improvements in the insulin secretion index ΔC30/ΔG30 (41.46 ± 77.68, P = .0238), disposition index (1.22 ± 1.65, P = .0030), and steady-state variables of HOMA2-ß (11.49 ± 14.58, P < .0001) and HOMA2-IR (-0.16 ± 0.36, P = .0130) during the SEED trial were important factors in achieving drug-free remission. A significant improvement in time in range (TIR), a measure of glucose homeostasis, in the SEED trial from 60% to more than 80% (estimated treatment difference, 23.8%; 95% CI: 7.3%, 40.2%; P = .0084) was observed. CONCLUSIONS: In drug-naïve patients with T2D, dorzagliatin treatment leads to stable glycaemic control and drug-free diabetes remission. Improvements in ß-cell function and TIR in these patients are important contributors to diabetes remission.

Impact of patient characteristic factors on the dynamics of liver glucose metabolism: Evaluation of multiparametric imaging with dynamic whole-body 18 F-fluorodeoxyglucose-positron emission tomography.

AIMS: To assess the impact of various patient characteristics on the dynamics of liver glucose metabolism using automated multiparametric imaging with whole-body dynamic 18 F-fluorodeoxyglucose (FDG)-positron emission tomography (PET). MATERIALS AND METHODS: We retrospectively enrolled 540 patients who underwent whole-body dynamic FDG-PET. Three quantitative indices representing hepatic glucose metabolism [mean standardized uptake value normalized by lean body mass (SULmean), metabolic glucose rate (kinetic index) and distribution volume (DV)] were measured from multiparametric PET images produced automatically based on the Patlak plot model. Patient characteristics including age, sex, body mass index, fasting time, blood glucose level, and the presence of diabetes mellitus (DM) or hepatic steatosis (HS) were collected. We examined the correlations between the characteristic factors and three quantitative indices using multiple regression analysis. RESULTS: The success rate of kinetic analysis using multiparametric PET imaging was 93.3% (504/540). Hepatic SULmean was significantly correlated with age (p < .001), sex (p < .001) and blood glucose level (p = .002). DV was significantly correlated with age (p = .033), sex (p < .001), body mass index (p = .002), fasting time (p = .043) and the presence of HS (p = .002). The kinetic index was significantly correlated with age (p < .001) and sex (p = .004). In the comparison of the healthy, DM and HS groups, patients with DM had a significantly increased SULmean, whereas patients with HS had a significantly decreased DV. CONCLUSIONS: Our results showed that liver glucose metabolism was influenced by various patient characteristic factors. Multiparametric FDG-PET imaging can be used to analyse the kinetics of liver glucose metabolism in routine clinical practice.

A novel pathogenic variant in the glucokinase gene found in two Japanese siblings with maturity-onset diabetes of the young 2.

Glucokinase is a glycolytic enzyme that catalyzes the phosphorylation of glucose to glucose-6-phospate in the first step of the glycolytic pathway. It also regulates the threshold for insulin secretion from pancreatic beta cells by catalyzing the phosphorylation of glucose and plays an important role as a glucose sensor. Pathogenic variants in the glucokinase gene (GCK) cause non-progressive but persistent mild fasting hyperglycemia, also recognized as maturity-onset diabetes of the young 2 (MODY2). This report presents the case of two Japanese siblings with MODY2, who were initially diagnosed with impaired glucose intolerance at 20 and 17 years of age, and later developed diabetes mellitus. They had no history of obesity, were negative for islet-related autoantibodies and their serum C-peptide level were within the normal range. Diabetic complications were not observed. Next-generation sequencing revealed a novel heterozygous variant in GCK (NM_000162.5: c.1088A>G, p.Asp363Gly) in both siblings. This variant has not been reported previously. In silico functional analyses, using SIFT and MutationTaster, suggested that the variant was damaging. To confirm the functional impact of the mutated GCK, the HiBiT-tagged p.Asp363Gly variant and the wild-type GCK were transiently expressed in HEK293T cells. The cells expressing the variant GCK exhibited 79% less bioluminescence, compared to those expressing the wild-type GCK, suggesting that the pathophysiology of the variant was a result of haploinsufficiency.

BMAL1 Regulates Glucokinase Expression Through E-Box Elements In Vitro.

The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

"Pesto" Mutation: Phenotypic and Genotypic Characteristics of Eight GCK/MODY Ligurian Patients.

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes mellitus (DM) that accounts for around 2-5% of all types of diabetes. Autosomal dominant inheritance in pathogenic variations of 14 genes related to ß-cell functions can lead to monogenic types of diabetes. In Italy, GCK/MODY is the most frequent form and it is caused by mutations of the glucokinase (GCK). Patients with GCK/MODY usually have stable mild fasting hyperglycaemia with mildly elevated HbA1c levels and rarely need pharmacological treatment. Molecular analysis of the GCK coding exons was carried out by Sanger sequencing in eight Italian patients. All the probands were found to be heterozygous carriers of a pathogenic gross insertion/deletion c.1279_1358delinsTTACA; p.Ser426_Ala454delinsLeuGln. It was previously described for the first time by our group in a large cohort of Italian GCK/MODY patients. The higher levels of HbA1c (6.57% vs. 6.1%), and the higher percentage of patients requiring insulin therapy (25% vs. 2%) compared to the previously studied Italian patients with GCK/MODY, suggest that the mutation discovered could be responsible for a clinically worse form of GCK/MODY. Moreover, as all the patients carrying this variant share an origin from the same geographic area (Liguria), we postulate a possible founder effect and we propose to name it the "pesto" mutation.

Glucokinase Inactivation Ameliorates Lipid Accumulation and Exerts Favorable Effects on Lipid Metabolism in Hepatocytes.

Glucokinase-maturity onset diabetes of the young (GCK-MODY) is a kind of rare diabetes with low incidence of vascular complications caused by GCK gene inactivation. This study aimed to investigate the effects of GCK inactivation on hepatic lipid metabolism and inflammation, providing evidence for the cardioprotective mechanism in GCK-MODY. We enrolled GCK-MODY, type 1 and 2 diabetes patients to analyze their lipid profiles, and found that GCK-MODY individuals exhibited cardioprotective lipid profile with lower triacylglycerol and elevated HDL-c. To further explore the effects of GCK inactivation on hepatic lipid metabolism, GCK knockdown HepG2 and AML-12 cell models were established, and in vitro studies showed that GCK knockdown alleviated lipid accumulation and decreased the expression of inflammation-related genes under fatty acid treatment. Lipidomic analysis indicated that the partial inhibition of GCK altered the levels of several lipid species with decreased saturated fatty acids and glycerolipids including triacylglycerol and diacylglycerol, and increased phosphatidylcholine in HepG2 cells. The hepatic lipid metabolism altered by GCK inactivation was regulated by the enzymes involved in de novo lipogenesis, lipolysis, fatty acid ß-oxidation and the Kennedy pathway. Finally, we concluded that partial inactivation of GCK exhibited beneficial effects in hepatic lipid metabolism and inflammation, which potentially underlies the protective lipid profile and low cardiovascular risks in GCK-MODY patients.

Glucokinase Variant Proteins Are Resistant to Fasting-Induced Uridine Diphosphate Glucose-Dependent Degradation in Maturity-Onset Diabetes of the Young Type 2 Patients.

We previously reported that glucokinase undergoes ubiquitination and subsequent degradation, a process mediated by cereblon, particularly in the presence of uridine diphosphate glucose (UDP-glucose). In this context, we hereby present evidence showcasing the resilience of variant glucokinase proteins of maturity-onset diabetes of the young type 2 (MODY2) against degradation and, concomitantly, their influence on insulin secretion, both in cell lines and in the afflicted MODY2 patient. Hence, glucose-1-phodphate promotes UDP-glucose production by UDP-glucose pyrophosphorylase 2; consequently, UDP-glucose-dependent glucokinase degradation may occur during fasting. Next, we analyzed glucokinase variant proteins from MODY2 or persistent hyperinsulinemic hypoglycemia in infancy (PHHI). Among the eleven MODY2 glucokinase-mutated proteins tested, those with a lower glucose-binding affinity exhibited resistance to UDP-glucose-dependent degradation. Conversely, the glucokinaseA456V-mutated protein from PHHI had a higher glucose affinity and was sensitive to UDP-glucose-dependent degradation. Furthermore, in vitro studies involving UDP-glucose-dependent glucokinase variant proteins and insulin secretion during fasting in Japanese MODY2 patients revealed a strong correlation and a higher coefficient of determination. This suggests that UDP-glucose-dependent glucokinase degradation plays a significant role in the pathogenesis of glucose-homeostasis-related hereditary diseases, such as MODY2 and PHHI.

Profiling the Antidiabetic Potential of Compounds Identified from Fractionated Extracts of Entada africana toward Glucokinase Stimulation: Computational Insight.

Glucokinase plays an important role in regulating the blood glucose level and serves as an essential therapeutic target in type 2 diabetes management. Entada africana is a medicinal plant and highly rich source of bioactive ligands with the potency to develop new target drugs for glucokinase such as diabetes and obesity. Therefore, the study explored a computational approach to predict identified compounds from Entada africana following its intermolecular interactions with the allosteric binding site of the enzymes. We retrieved the three-dimensional (3D) crystal structure of glucokinase (PDB ID: 4L3Q) from the online protein data bank and prepared it using the Maestro 13.5, Schrödinger Suite 2022-3. The compounds identified were subjected to ADME, docking analysis, pharmacophore modeling, and molecular simulation. The results show the binding potential of the identified ligands to the amino acid residues, thereby suggesting an interaction of the amino acids with the ligand at the binding site of the glucokinase activator through conventional chemical bonds such as hydrogen bonds and hydrophobic interactions. The compatibility of the molecules was highly observed when compared with the standard ligand, thereby leading to structural and functional changes. Therefore, the bioactive components from Entada africana could be a good driver of glucokinase, thereby paving the way for the discovery of therapeutic drugs for the treatment of diabetes and its related complications.

Resveratrol Mitigates Bisphenol A-Induced Metabolic Disruptions: Insights from Experimental Studies.

The aim of this study was to investigate the disruptions of metabolic pathways induced by bisphenol A (BPA) and explore the potential therapeutic intervention provided by resveratrol (RSV) in mitigating these disruptions through the modulation of biochemical pathways. Wistar albino rats were divided into three groups: group 1 served as the control, group 2 received 70 mg/Kg of BPA, and group 3 received 70 mg/kg of BPA along with 100 mg/Kg of RSV. After the treatment period, various biomarkers and gene expressions were measured to assess the effects of BPA and the potential protective effects of RSV. The results revealed that BPA exposure significantly increased the serum levels of α-amylase, α-glucosidase, G6PC, insulin, HbA1c, HMG-CoA reductase, FFAs, TGs, DPP-4, MDA, and proinflammatory cytokines such as TNF-α and IL-6. Concurrently, BPA exposure led to a reduction in the levels of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), as well as GLUT4 and HDL cholesterol. However, the administration of RSV along with BPA significantly ameliorated these alterations in the biomarker levels induced through BPA exposure. RSV treatment effectively reduced the elevated levels of α-amylase, α-glucosidase, G6PC, insulin, HbA1c, HMG-CoA reductase, FFAs, TGs, DPP-4, MDA, and proinflammatory cytokines, while increasing the levels of antioxidant enzymes, GLUT4, and HDL cholesterol. Furthermore, BPA exposure suppressed the mRNA expression of glucokinase (GCK), insulin-like growth factor 1 (IGF-1), and glucose transporter 2 (GLUT2) and up-regulated the mRNA expression of uncoupling protein 2 (UCP2), which are all critical biomarkers involved in glucose metabolism and insulin regulation. In contrast, RSV treatment effectively restored the altered mRNA expressions of these biomarkers, indicating its potential to modulate transcriptional pathways and restore normal metabolic function. In conclusion, the findings of this study strongly suggest that RSV holds promise as a therapeutic intervention for BPA-induced metabolic disorders. By mitigating the disruptions in various metabolic pathways and modulating gene expressions related to glucose metabolism and insulin regulation, RSV shows potential in restoring normal metabolic function and counteracting the adverse effects induced by BPA exposure. However, further research is necessary to fully understand the underlying mechanisms and optimize the dosage and duration of RSV treatment for maximum therapeutic benefits.