Glucokinase Regulatory Protein Research Articles

Sex-Dimorphic Effects of Hypoglycemia on Metabolic Sensor mRNA Expression in Ventromedial Hypothalamic Nucleus-Dorsomedial Division (VMNdm) Growth Hormone-Releasing Hormone Neurons.

Growth hormone-releasing hormone (Ghrh) neurons in the dorsomedial ventromedial hypothalamic nucleus (VMNdm) express the metabolic transcription factor steroidogenic factor-1 and hypoglycemia-sensitive neurochemicals of diverse chemical structures, transmission modes, and temporal signaling profiles. Ghrh imposes neuromodulatory control of coexpressed transmitters. Multiple metabolic sensory mechanisms are employed in the brain, including screening of the critical nutrient glucose or the energy currency ATP. Here, combinatory laser-catapult-microdissection/single-cell multiplex qPCR tools were used to investigate whether these neurons possess molecular machinery for monitoring cellular metabolic status and if these biomarkers exhibit sex-specific sensitivity to insulin-induced hypoglycemia. Data show that hypoglycemia up- (male) or downregulated (female) Ghrh neuron glucokinase (Gck) mRNA; Ghrh gene silencing decreased baseline and hypoglycemic patterns of Gck gene expression in each sex. Ghrh neuron glucokinase regulatory protein (Gckr) transcript levels were respectively diminished or augmented in hypoglycemic male vs female rats; this mRNA profile was decreased by Ghrh siRNA in both sexes. Gene transcripts encoding catalytic alpha subunits of the energy monitor 5-AMP-activated protein kinase (AMPK), i.e., Prkaa1 and 2, were increased by hypoglycemia in males, yet only the former mRNA was hypoglycemia-sensitive in females. Ghrh siRNA downregulated baseline and hypoglycemia-associated Prkaa subunit mRNAs in males but elicited divergent changes in Prkaa2 transcripts in eu- vs hypoglycemic females. Results provide unique evidence that VMNdm Ghrh neurons express the characterized metabolic sensor biomarkers glucokinase and AMPK and that the corresponding gene profiles exhibit distinctive sex-dimorphic transcriptional responses to hypoglycemia. Data further document Ghrh neuromodulation of baseline and hypoglycemic transcription patterns of these metabolic gene profiles.

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.

The pleiotropic approach to coronavirus disease-19 pathogenesis: The impact of liver diseases associated host genetic variants.

Coronavirus disease-2019 (COVID-19) is a novel multisystemic viral disease caused pandemic. The disease impact involves liver and associated systems. Undoubtedly, host genetic background influences the predisposition and prediction of infection. Variants among human populations might increase susceptibility or protect against severe outcomes. In this manner, rs738409 variant of patatin-like phospholipase domain-containing protein 3 gene appears to be protective in some populations in spite of its aggravating effect on non-alcoholic fatty liver diseases (NAFLDs) and steatohepatitis. DRB1*15:01 allele of human leukocyte antigen is associated with protective effect in European and Japanese populations. DRB1*03:01 contrarily increases the susceptibility of severe COVID-19 infection in European populations. rs1260326 in glucokinase regulatory protein gene, rs112875651 in tribbles homolog 1 gene, rs429358 in apolipoprotein 1, and rs58542926 in transmembrane 6 superfamily 2 alleles are found related with NAFLD and obesity; thus, hypercoagulability and severe COVID-19 outcomes. In chronic or acute liver diseases, comorbid syndromes are the key factors to explain increased severity. There might not be a direct association between the variant and severe COVID-19 infection. As it is concluded, there are genes and variants known and unknown yet to be studied to reveal the association with disease severity.

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±17.9μM while lowering the S0.5 (7.1mM) and increasing Vmax (9.22μM/min) as compared to control without activator (S0.5=10.37mM; Vmax=4.8μM/min). Kinetic data (α and β values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, Kd values of 3.8μM 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.

Prevalence of GCKR rs1260326 Variant in Subjects with Obesity Associated NAFLD and T2DM: A Case-Control Study in South Punjab, Pakistan.

The glucokinase regulatory protein (GCKR) regulates glycogen metabolism and insulin secretion, and the GCKR rs1260326 is a putative single nucleotide polymorphism (SNP) associated with metabolic disorders including nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). This study was conducted to investigate the genetic association of the GCKR rs1260326 in NAFLD and T2DM in our population. NAFLD (n = 103), T2DM (n = 100), and control (n = 100) samples were collected and genotyped for GCKR rs1260326 by tetra-arm PCR. The genetic variant GCKR rs1260326 was significantly linked with NAFLD and T2DM, while the GCKR rs1260326 was significantly associated with the progression of obesity only in NAFLD subjects. The frequency of the C allele (mutant) was higher in both NAFLD (f = 0.69) and T2DM (f = 0.66) subjects as compared to healthy controls of NAFLD (0.52) and T2DM (f = 0.32). The frequency of the C allele was also positively linked with the progression of obesity in both diseases. The frequency of the C allele was 0.66, 0.67, and 0.74 in NAFLD normal weight, overweight, and obese subjects, respectively, while the frequency of the C allele was 0.60, 0.60, and 0.74 in T2DM in normal weight, overweight, and obese subjects, respectively. Homozygous mutant (CC) was 53% in both NAFLD and T2DM subjects, while heterozygous mutant (CT) was 15.53% in NAFLD and 22% in T2DM subjects. Wild-type allele (TT) was 31.06% in NAFLD and 25% in T2DM subjects. In conclusion, the GCKR rs1260326 is a highly prevalent SNP in NAFLD and T2DM subjects, which possibly contributed to obesity, insulin resistance, and metabolic disorders in our population.

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.

Glucokinase regulatory protein (GCKR) in mediating the genetic risk of NAFLD

Glucokinase regulatory protein (GCKR) in mediating the genetic risk of NAFLD

Molecular insights into the inhibitory potential of anthocyanidins on glucokinase regulatory protein.

Computational methods were used to investigate six anthocyanidins exhibiting antidiabetic activity by inhibiting glucokinase regulatory protein (GKRP) activity. Density functional theory was used to optimise the geometry of anthocyanidins and calculate their quantum chemical properties. A blind docking method was employed to conduct a molecular docking study, which revealed that delphinidin (Del), cyanidin (Cya), and pelargonidin (Pel) as potential GKRP inhibitors with the lowest binding free energy of -8.7, -8.6, and -8.6 kcal/mol, corresponding to high binding affinity. The molecular dynamics study further verified the blind docking results by showing high GKRP-F1P complex stability and high binding affinity calculated through the MM/GBSA method, upon the binding of pelargonidin. The lower RMSF values of pivotal GK-interacting residues for GKRP-F1P-Pel compared to GKRP-F1P, as a positive control, indicating pelargonidin ability to maintain the inactive conformation of GKRP through the inhibition of GK binding. The key residues that control the binding of the F1P to GKRP and anthocyanidin to GKRP-F1P were also identified in this study. Altogether, pelargonidin is anthocyanidins-derived natural products that have the most potential to act as inhibitors of GKRP and as antidiabetic nutraceuticals.

In-Silico Prediction of Phytoconstituents from Manilkara Hexandra for Antidiabetic Activity Targeting GKRP (Glucokinase Regulatory Protein)

Objective: A complex metabolic condition known as diabetes mellitus is caused by either inadequate or dysfunctional insulin. Once more, medicinal plants are being researched for the treatment of diabetes. Prototypical compounds found in medicinal plants have been the source of many conventional medications. The part of our investigation was to test the phytoconstituents of Manilkara hexandra for antidiabetic efficacy, in-silico. Methods: The pattern of interaction between the phytoconstituents from the Manilkara hexandra (Roxb.) Dubard, plant and the crystal structure of the antidiabetic proteins is evaluated using molecular docking in Discovery Studio (PDB ID: 4LY9). Later, SwissADME and pkCSM were used to screen for toxicity as well as the pharmacokinetic profile. Results: The docked results suggest that quercetin (-9.3 kcal/mol), kaempferol (-9.1 kcal/mol), p-coumaric acid (-6.4 kcal/mol) and cinnamic acid (-6.3 kcal/mol) for 4LY9 macromolecule has best binding towards antidiabetic activity as compared to the standard drug metformin (-5.0 kcal/mol). According to ADMET tests, pharmacokinetics and toxicity characteristics were also within acceptable bounds. Conclusion: Results from the binding potential of phytoconstituents aimed at antidiabetic activity were encouraging. It promotes the usage of Manilkara hexandra and offers crucial details on pharmaceutical research and clinical care.

The GCKR-P446L gene variant predisposes to raised blood cholesterol and lower blood glucose in the P446L mouse-a model for GCKR rs1260326

ObjectivesThe Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L. MethodsGKRP-deficient hepatocytes were transfected with adenoviral vectors for human or mouse GKRP:446 P or 446 L for cellular comprehensive analysis including transcriptomics consequent to P446 > L. Physiological traits in the diet-challenged P446L mouse were compared with pleiotropic associations at the human rs1260326 locus. Transcriptomics was compared in P446L mouse liver with hepatocytes overexpressing glucokinase or GKRP:446 P/L. Results1. P446 > L substitution in mouse or human GKRP similarly compromises protein expressivity of GKRP:446 L, nuclear sequestration of glucokinase and counter-regulation of gene expression. 2. The P446L knockin mouse has lower liver glucokinase and GKRP protein similar to human liver homozygous for rs1260326-446 L. 3. The diet-challenged P446L mouse has lower blood glucose, raised blood cholesterol and altered hepatic cholesterol homeostasis consistent with relative glucokinase-to-GKRP excess, but not raised blood triglycerides. ConclusionsMouse GKRP phenocopies the human GKRP:P446 > L substitution despite the higher affinity for glucokinase of human GKRP. The diet-challenged P446L mouse replicates several traits found in association with the rs1260326 locus on chromosome 2 including raised blood cholesterol, lower blood glucose and lower liver glucokinase and GKRP protein but not raised blood triglycerides.

GlucoKinaseDB: A comprehensive, curated resource of glucokinase modulators for clinical and molecular research

Glucokinase (GK), an isoform of hexokinase expressed predominantly in liver, pancreas and hypothalamus is crucial to blood glucose management. It is a critical component of the glucose-sensing mechanism of the pancreatic islet cells and glycogen regulation in hepatocytes. GK modulators such as allosteric GKAs (glucokinase activators) and GK-GKRP (glucokinase regulatory protein) disruptors have found potential applications as safer antihyperglycemics. Recent studies have also demonstrated the potential of GK modulators as antiparasitic agents. Researchers targeting GK often undertake the time-consuming task of independently collecting and compiling modulator information due to the lack of any dedicated single-platform resource. Towards this, in the present study we demonstrate the design and development of GlucoKinaseDB (GKDB), a comprehensive, curated, online resource of GK modulators. GKDB contains experimentally derived structural and bioactivity information of 1723 modulators along with their detailed molecular descriptors. The web-interface is user-friendly with features such as in-browser visualization, advanced search queries, cross-links to other databases and original reference etc. The bioactivity and descriptor data can be downloaded in bulk (for entire database) or for individual modulators. The 3D structures are also downloadable in multiple formats. GKDB employs a PHP-based web design with Bootstrap styling and a MySQL database backend. GKDB can be utilized for clinical and molecular research via development of pharmacophore hypotheses, QSAR/QSPR models, predictive machine learning models etc. GKDB is freely accessible online at https://glucokinasedb.in.

Association of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) Gene Polymorphisms with Type 2 Diabetes among Malay Ethnics.

Background Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder, and the underlying causes remain unknown and have not been fully elucidated. Several candidate genes have been associated with T2DM in various populations with conflicting results. The variations found in glucokinase ( GCK ), glucokinase regulatory protein ( GCKR ), and glucose-6-phosphatase 2 ( G6PC2 ) genes were not well studied, particularly among Asians. Aims The main objective of this study was to determine the candidate genetic polymorphisms of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) genes in T2DM among Malay ethnics. Methods In this candidate gene association study, a total of 180 T2DM subjects and 180 control subjects were recruited to determine the genotypes using polymerase chain reaction-restriction fragment length polymorphism and Taqman probe assay methods. Genotype and allele frequencies in case and control samples were compared using the chi-squared test to determine a significant difference. Results The body mass index, fasting blood glucose, hemoglobin A1c, systolic and diastolic blood pressure, and total cholesterol were significantly different ( p < 0.05) between T2DM and control subjects. The genotypic and allelic frequencies of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) gene polymorphisms were significantly different between T2DM and controls ( p < 0.05). Conclusion Hence, rs1799884 of GCK gene and rs780094 of GCKR gene and rs560887 of the G6PC2 gene are possible genetic biomarkers in T2DM development among Malay ethnics in Malaysia.

GCKR and GCK polymorphisms are associated with increased risk of end-stage kidney disease in Chinese patients with type 2 diabetes: The Hong Kong Diabetes Register (1995–2019)

AimsGlucokinase (GCK) and glucokinase regulatory protein (GKRP) regulate glucose and lipid metabolism. We investigated the associations of GCKR and GCK polymorphisms with kidney outcomes. MethodsAnalyses were performed in a prospective cohort who were enrolled in the Hong Kong Diabetes Register between 1995 and 2017. The associations of GCKR rs1260326 and GCK rs1799884 polymorphisms with incident end-stage kidney disease (ESKD), albuminuria and rapid eGFR decline were analysed by Cox regression or logistic regression with adjustment. Results6072 patients (baseline mean age 57.4 years; median diabetes duration 6.0 years; 54.5 % female) were included, with a median follow-up of 15.5 years. The GCKR rs1260326 [HR (95 %CI) 1.23 (1.05–1.44) for CT; HR 1.23 (1.02–1.48) for TT] and GCK rs1799884 T alleles [HR 1.73 (1.24–2.40) for TT] were independently associated with increased risk of ESKD versus their respective CC genotypes. GCKR rs1260326 T allele was also associated with albuminuria [OR 1.18 (1.05–1.33) for CT; OR 1.34 (1.16–1.55) for TT] and rapid eGFR decline. ConclusionsIn Chinese patients with type 2 diabetes, T allele carriers of GCKR rs1260326 and GCK rs1799884 were at high risk for ESKD. These genetic markers may be used to identify high risk patients for early intensive management for renoprotection.

Electrochemically induced assembling of isatins, kojic acid, and malonic acid derivatives into substituted spiro[indole‐3,4′‐pyran]‐2(1H)‐one scaffold and predicting potential protein targets

AbstractElectrochemically induced multicomponent assembling of isatins, kojic acid, and malonic acid derivatives in n‐propanol in the presence of sodium iodide as an electrolyte in an undivided cell results in the formation of unsymmetrical spiro(indole‐3,4′‐pyrano[3,2‐b]pyranes) in 86–98% yields. The optimized reaction conditions and a mechanistic rationale for this catalytic electrochemical transformation are presented. This new electrocatalytic multicomponent process is a facile and efficient environmentally benign way to the functionalized unsymmetrical spiro(indole‐3,4′‐pyrano[3,2‐b]pyranes), containing inserted kojic acid fragment, which are perspective compounds for different biomedical applications, among them anticonvulsant, anti‐cancer, anti‐AIDS agents, and anti‐inflammatory remedies. All synthesized compounds were investigated using a molecular docking procedure. The results of prediction were incorporated in one pool of potential targets. The automated method was proposed and performed for the docking procedure. It is distinct from screening by higher accuracy, and it is much more scalable than the classic docking procedure. It was found that the synthesized compounds are potential ligands for 4OHK (glucokinase regulatory protein) and 3V2B (human poly[adp‐ribose] polymerase 15) proteins.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Review on upregulation of glucokinase expression by selected plants and their phytoconstituents

Glucokinase is, an allosteric enzyme involved in glycolysis, is rapidly regulated in the liver through glucokinase regulatory protein and remains essential for blood glucose homeostasis. The present review provides brief information about the effects of a few selected plants and phytochemicals on human glucokinase activation and gene modulation. A thorough and relevant literature search from several scientific databases, comprising Google Scholar, Web of Science, Scopus and PubMed, was carried out. We highlighted the seven plants (Acorus tatariowii Schott, Allium hirtifolium Boiss, Apache red maize, Mulberry species, Pterocarpus marsupium, Sapium ellepticum, Mangifera indica) and their phytoconstituents ( Tatanans A-C, alliogenin , gitogenin , kaempferol , quercetin and shallomin,1-deoxynojirimycin,cyanidin-3-rutinoside,resveratrol,cyanidin-3-glucoside,oxyresveratrol, lupeol, alpha-tocopherol, uteolin-7-glucoside, amentoflavone, and Mangiferin) on human glucokinase enzyme activation and gene modulation. This review concluded that investigation of glucokinase activators of plant origin is the major research focus in the management of type 2 diabetes.

Effects of short-term food deprivation on catecholamine and metabolic-sensory biomarker gene expression in hindbrain A2 noradrenergic neurons projecting to the forebrain rostral preoptic area: Impact of negative versus positive estradiol feedback.

Hindbrain A2 noradrenergic neurons assimilate estrogenic and metabolic cues. In female mammals, negative- versus positive-feedback patterns of estradiol (E) secretion impose divergent regulation of the gonadotropin-releasing hormone (GnRH)-pituitary-gonadal (HPG) neuroendocrine axis. Current research used retrograde tracing, dual-label immunocytochemistry, single-cell laser-microdissection, and multiplex qPCR methods to address the premise that E feedback modes uniquely affect metabolic regulation of A2 neurons involved in HPG control. Ovariectomized female rats were given E replacement to replicate plasma hormone levels characteristic of positive (high-E dose) or negative (low-E dose) feedback. Animals were either full-fed (FF) or subjected to short-term, e.g., 18-h food deprivation (FD). After FF or FD, rostral preoptic area (rPO)-projecting A2 neurons were characterized by the presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunostaining. FD augmented or suppressed mRNAs encoding the catecholamine enzyme dopamine-beta-hydroxylase (DβH) and the metabolic-sensory biomarker glucokinase (GCK), relative to FF controls, in nGKRP-immunoreactive (ir)-positive A2 neurons from low-E or high-E animals, respectively. Yet, these transcript profiles were unaffected by FD in nGKRP-ir-negative A2 neurons at either E dosage level. FD altered estrogen receptor (ER)-alpha and ATP-sensitive potassium channel subunit sulfonylurea receptor-1 gene expression in nGKRP-ir-positive neurons from low-E, but not high-E animals. Results provide novel evidence that distinct hindbrain A2 neuron populations exhibit altered versus unaffected transmission to the rPO during FD-associated metabolic imbalance, and that the direction of change in this noradrenergic input is controlled by E feedback mode. These A2 cell types are correspondingly distinguished by FD-sensitive or -insensitive GCK, which correlates with the presence versus absence of nGKRP-ir. Further studies are needed to determine how E signal volume regulates neurotransmitter and metabolic sensor responses to FD in GKRP-expressing A2 neurons.

1342-P: Reduction of Glucokinase and Glucokinase Regulatory Protein Expression by siRNA Is Associated with the Reduction of Lactate and Pyruvate Export from Canine Hepatocytes

Glucokinase (GCK) mediates hepatic glucose uptake during hyperglycemia and is crucial for the regulation of glucose-responsive glycolysis genes. Mutations in GCK are responsible for maturity-onset diabetes of the young, and glucokinase regulatory protein (GKRP) , the regulator of hepatic GCK, is also a diabetes susceptibility locus. Recently in in vivo experiments, we used hepatic lactate export as a surrogate for GCK activity, which was a measure of “glucose effectiveness.” We hypothesized that in vitro GCK and GKRP suppression in hepatocytes promotes a reduction in lactate and pyruvate export from the liver. We used cryopreserved canine hepatocytes and knocked down GCK and GKRP via lipid delivery of siRNA. A control group did not receive siRNA. We measured lactate dehydrogenase D (LDHD) and pyruvate kinase (PK) gene expression and lactate and pyruvate release into the medium 24 and 48 h after siRNA delivery. Selective blocking of GCK and GKRP did not change cell proliferation but reduced the expression of these genes by 90% and 85%, respectively (P&amp;lt;0.0vs. controls; n=4) . GCK and GKRP knockdown reduced LDHD by 71% and 74%, respectively (P&amp;lt;0.001) ; similarly, PK was reduced by 84% and 82%, respectively (P&amp;lt;0.001) . Lactate exported into the medium was reduced by 36% (P&amp;lt;0.001) and 26% (P&amp;lt;0.05) by GCK and GKRP siRNA, respectively, after 48 h of hepatocyte culture. However, pyruvate was reduced by 17% only after 48 h culture of GCK siRNA (P&amp;lt;0.05) . Thus, we showed that direct suppression of GCK and GKRP genes by siRNA in vitro affects glucose-responsive glycolysis genes, consequently reducing lactate export which can be used as a surrogate for reduced glucokinase activity in hepatocytes. The use of antisense oligonucleotides or CRISPR gene-editing methods in animals will be useful methods to study the glucose-lactate relationship in vivo and will evaluate the role of glucose effectiveness in the pathogenesis of type 2 diabetes. Disclosure M.Kabir: None. M.Ader: None. R.N.Bergman: Consultant; Fractyl Health, Inc., Novo Nordisk, Research Support; AstraZeneca, Janssen Research &amp; Development, LLC. Funding National Institutes of Health R01- DK027619-28

Hindbrain lactate regulation of hypoglycemia-associated patterns of catecholamine and metabolic-sensory biomarker gene expression in A2 noradrenergic neurons innervating the male versus female ventromedial hypothalamic nucleus

Caudal hindbrain A2 noradrenergic neurons provide critical metabolic-sensory input to the brain glucostatic circuitry. In males, insulin-induced hypoglycemia (IIH)-associated patterns of A2 cell dopamine-beta-hydroxylase (DβH) protein expression reflect diminution of the oxidizable fuel L-lactate, yet DβH exhibits sex-dimorphic responses to IIH. Here, retrograde tracing and combinatory single-cell laser-microdissection/multiplex qPCR techniques were used to examine whether lactate imposes sex-specific control of hypoglycemia-associated metabolic-sensory function and noradrenergic neurotransmission in A2 neurons that innervate the ventromedial hypothalamic nucleus (VMN), a key glucose-regulatory structure. VMN-projecting A2 neurons from each sex were characterized by presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunoreactivity (-ir). IIH caused lactate-reversible up- or down-regulation of DβH mRNA in male and female nGKRP-ir-positive A2 neurons, respectively, and stimulated glucokinase (GCK) and sulfonylurea receptor-1 (SUR-1) gene expression in these cells in each sex. Hypoglycemia did not alter DβH, GCK, and SUR-1 transcript profiles in nGKRP-ir-negative male or female A2 neurons innervating the VMN. Estrogen receptor (ER) gene profiles in nGKRP-ir-positive neurons showed sex-specific [ER-alpha; G-protein-coupled estrogen-receptor-1 (GPER)] or sex-monomorphic (ER-beta) transcriptional responses to IIH. Fewer ER gene profiles were affected by IIH in nGKRP-ir-negative A2 neurons from male or female rats. Results show that during IIH, VMN-projecting A2 neurons may deliver altered, sex-dependent (nGKRP-positive) or unaffected (nGKRP-negative) noradrenergic input to the VMN. In each sex, metabolic-sensory gene profiles were reactive to hypoglycemia in nGKRP-ir-positive, not -negative A2 cells. Further studies are needed to elucidate the role of GKRP in transduction of metabolic imbalance into noradrenergic signaling, and to determine if input by one or more ER variants establishes sex differences in DβH transcriptional sensitivity to IIH.

Metabolic Syndrome but Not Fatty Liver-Associated Genetic Variants Correlates with Glomerular Renal Function Decline in Patients with Non-Alcoholic Fatty Liver Disease.

The association between non-alcoholic fatty liver disease (NAFLD) and chronic kidney disease (CKD) has been extensively demonstrated. Recent studies have focused attention on the role of patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism in the association between NAFLD and CKD in non-metabolic adults and children, but the genetic impact on NAFLD-CKD association is still a matter of debate. The aim of the study was to investigate the impact of PNPLA3, transmembrane 6 superfamily member 2 (TM6SF2), membrane-bound O-acyltransferase domain containing 7 (MBOAT7) and glucokinase regulatory protein (GCKR) gene variants rather than metabolic syndrome features on renal function in a large population of NAFLD patients. The present study is a post hoc analysis of the Plinio Study (ClinicalTrials.gov: NCT04036357). PNPLA3, TM6SF2, MBOAT7 and GCKR genes were analyzed by using real-time PCR with TaqMan probes. Glomerular filtration rate (GFR) was estimated with CKD-EPI. We analyzed 538 NAFLD; 47.2% had GFR < 90 mL/min/1.73 m2 while 5.9% had GFR < 60 mL/min/1.73 m2. The distribution of genotypes was superimposable according to GFR cut-offs. Results from the multivariable regression model did not show any correlation between genotypes and renal function. Conversely, metabolic syndrome was highly associated with GFR < 90 mL/min/1.73 m2 (odds ratio (OR): 1.58 [1.10–2.28]) and arterial hypertension with GFR < 60 mL/min/1.73 m2 (OR: 1.50 [1.05–2.14]). In conclusion, the association between NAFLD and CKD might be related to the shared metabolic risk factors rather than the genetic NAFLD background.