Time-restricted feeding improves cognitive function in Alzheimer's mice through GCK/NPY/apoptosis pathway.

Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant.

Although allosteric regulation has been pointed out as one of the cornerstones of biological function, it has been a scarcely studied phenomenon in Archaea carbohydrate metabolism. Given its central role in metabolism, we experimentally investigated how allosteric regulation and its underlying kinetic mechanism evolved along evolutionary pathways within the archaeal ADP-dependent kinase family. Using ancestral sequence reconstruction, we resurrected key ancestors of this family and show that AMP regulation is an ancestral feature retained exclusively in lineages encoding bifunctional ADP-dependent phosphofructokinase (PFK)/glucokinase (GK) enzymes, which are restricted to methanogenic organisms, whereas it is lost in lineage-specific PFK enzymes. Notably, although AMP-dependent allosteric regulation is conserved among bifunctional ADP-PFK/GK enzymes, the kinetic mechanisms underlying activation are not. Instead, we observed a diversity of activation mechanisms (increased affinity for substrates, enhanced catalytic efficiency, or a combination of both), distributed along a 2-billion-year evolutionary trajectory, and that persists across different temperatures studied, both in extant and ancestral enzymes. These results highlight that the structural scaffold of this protein family is evolutionarily robust, preserving function while allowing substantial diversification of the underlying activation mechanisms under sequence variation. Based on these findings, we propose the concept of mechanistic drift, in which evolutionary pressures primarily act on adaptive functional traits that confer an adaptive advantage, rather than on the specific molecular mechanisms by which they are achieved. This framework has broad implications for macromolecular evolution, illustrating how long-term functional conservation can coexist with extensive physicochemical mechanistic diversity.

Recombinant glucokinase (GLK), an important enzyme for blood glucose diagnostics, is commonly prepared in Escherichia coli (E.coli )for laboratory research and related biosynthetic applications. In small-scale laboratory cultivation, complex media are often used because of their convenience and strong growth-supporting capacity, but their ill-defined composition and batch-to-batch variability can compromise reproducibility and increase medium-related costs. Although minimal defined medium (e.g., M9 medium) are generally preferred in scalable E. coli bioprocesses because of their compositional consistency and process controllability, their relatively simple nutrient composition may limit recombinant protein expression performance under laboratory-scale conditions. Therefore, developing an improved chemically defined medium represents a practical strategy to balance expression efficiency, compositional control, and medium cost for recombinant GLK production. To address this, we developed a chemically defined medium including amino acids using high-throughput respiration activity monitoring system (RAMOS) and a high-throughput growth curve analysis system (Growth Profiler 960, GP960), alongside a machine learning-driven iterative optimization system (Machine-learning guided Experimental Trials for Improvement of Systems, METIS). The optimized medium enhanced soluble GLK expression by 33 fold compared to the pre-optimization medium, reaching 74% of the titer obtained in complex media, while reducing medium costs by 74%. Moreover, soluble protein expression per cell increased by 80% compared to the complex medium, lowering the medium usage cost per unit of bacterial protein by 85%. Validation in a 7.5L bioreactor demonstrated that the optimized medium supported continuous growth to an optical density at 600 nm (OD600) of 103, achieving 4-fold and 5.4-fold higher biomass than Luria-Bertina (LB) and M9 media, respectively, with soluble GLK titer reaching 920 mg L- 1, which exceeded conventional media by more than 10-fold. This study tackles the challenge of suboptimal media in GLK fermentation, offering a cost-effective alternative and a transferable strategy for optimizing fermentation processes for other high-value enzymes.

Glucokinase (GCK) mutations are well-known causes of maturity-onset diabetes of the young type 2 (MODY2) and hyperinsulinaemic hypoglycaemia type 3 (HH3). The GCK c.781G>A (p.Gly261Arg) variant has been exclusively reported in MODY2. We describe a term male infant with severe refractory neonatal hypoglycaemia and inappropriately elevated insulin. Genetic analysis identified a heterozygous GCK c.781G>A variant inherited from his father. Protein modelling supported a gain-of-function effect. The infant responded well to diazoxide and discontinued treatment two weeks after discharge. This is the first report of GCK c.781G>A presenting as neonatal HH3, expanding the known phenotypic spectrum of this variant.

Glucokinase (GCK) catalyzes the first step of glycolysis in pancreatic β-cells, where it functions as the body's primary glucose sensor. GCK is extremely sensitive to oxidative inactivation, both in vivo and in vitro . This characteristic provides a mechanism to regulate GCK activity via alterations in the cellular redox environment. To understand the molecular and evolutionary origins of redox regulation, we characterized the sensitivity to oxidative inactivation of four extant GCKs and five ancestral GCKs produced from a recent phylogenetic analysis of the vertebrate family. We find that two invertebrate GCKs are significantly less sensitive to oxidative inactivation compared to their vertebrate counterparts. We also demonstrate that an ancestral GCK from chordates (cGCK) is insensitive to oxidative inactivation, whereas an ancestral GCK from early vertebrates (vGCK) displays a degree of redox responsiveness comparable to the extant human enzyme. The redox insensitive cGCK ancestor lacks cysteine residues at two positions, Cys230 and Cys461, that are conserved in all redox sensitive ancestral and extant enzymes. We find that installation of cysteines at these positions is insufficient to install redox sensitivity into cGCK. Our data demonstrate that the appearance of redox responsiveness in GCKs coincides with an expansion in the conformational landscape of the protein that occurred during early vertebrate evolution. These observations support a model in which the emergence of redox sensitivity required the ability to sample a unique super-open conformation, an event that also facilitated the emergence of two orthogonal GCK regulatory strategies, allosteric regulation by substrate glucose and an inhibitory interaction with the glucokinase regulatory protein.

The glucokinase regulatory protein (GKRP) derives from an ancestral etherase. Despite existing as a single locus in the metazoans, GKRP evolved multiple novel functions unrelated to etherase activity. In jawed vertebrates, a protein-protein interaction (PPI) emerged that inhibits glucokinase (GCK) activity in the liver. This PPI is critical to maintaining glucose homeostasis. In mammals, GKRP is allosterically regulated by carbohydrates, with 6-phospharylated sugars promoting inhibition of GCK by GKRP, while 1-phosphorylated sugars relieve inhibition. Here, we use a vertical evolutionary approach to identify the genetic, biochemical, and biophysical mechanisms underlying the emergence of small-molecule allostery in GKRP. We pinpointed a single leucine to valine substitution in the N-terminus of GKRP from the ancestor of the euarchontoglires that, when introduced into the non-regulated placental mammal GKRP ancestor, installed sensitivity to sorbitol-6-phosphate (S6P). Interestingly, GKRP's inhibitory activity in the absence of S6P was reduced but unchanged in its presence. The mutation enabled co-optation of the ancestral etherase active site, which also existed as an ambiguous phosphorylated carbohydrate binding site in unregulated GKRPs. This substitution likely introduced an alternative conformation of the N-terminus causing apo-GKRP to sample a binding incompetent state prior to GCK binding. Our results suggest a simple model of the evolution of protein functional novelty where a single mutation can cause a large functional shift via co-optation of pre-existing structural features. Importantly, in contrast to many models of protein evolution, ours does not require the addition of new genetic material to realize a novel function such as small-molecule allosteric regulation.

Glucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzyme's conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface - a structural spandrel resulting from prior conformational expansion - was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.

Objective:Glucokinase Regulatory Protein (GKRP) controls the activity of Glucokinase (GCK) to regulate liver glucose uptake and storage. Coding variants in GCKR, the gene encoding GKRP, strongly associate with fatty liver disease, hypertriglyceridemia, and hypercholesterolemia. Here, we sought to investigate the mechanisms by which a common GKRP variant affects hepatic lipid and cholesterol metabolism.Methods:We developed mouse models to examine how the human GKRP P446L variant influences liver and systemic metabolism. Endogenous Gckr expression was ablated in adult mouse hepatocytes, together with re-expression of either human GKRP P446L or the reference GKRP protein. We assessed body weight, adiposity, systemic glucose homeostasis, and hepatic metabolites in mice expressing reference GKRP or GKRP P446L under multiple metabolic conditions. To determine whether the effects of GKRP P446L may result from reduced GCK activity, we analyzed mice with liver-specific deletion of Gck.Results:Hepatic expression of GKRP P446L resulted in reduced GKRP and GCK protein levels and elevated serum cholesterol. Hepatic deletion of Gck in mice recapitulated several effects of GKRP P446L, including increased hepatic cholesterol and triglyceride content. The elevated cholesterol was associated with increased cholesterogenic gene expression and cholesterol synthesis. Hepatic expression of an alternative hexokinase (HKII) normalized the effects of GCK-deficiency, suggesting that impaired glucose phosphorylation underlies the phenotype.Conclusions:The GKRP P446L variant reduced GKRP protein abundance, and diminished GCK activity while increasing cholesterol levels. Loss of GCK elevated cholesterol and hepatic triglyceride levels. Collectively, these findings demonstrate that GCK suppresses hepatic cholesterol synthesis and lipid accumulation, suggesting that reduced GCK activity underlies the metabolic abnormalities associated with the GKRP P446L variant.

Cyclophosphamide (CTX) is limited by hepatotoxicity, whose underlying mechanism remains obscure. Since the liver is the central organ for metabolism, we thereby aimed to investigate the effect of CTX on the metabolism of the primary mouse hepatocytes. The cells were divided into the normal and CTX groups, following the cell counting kit-8 (CCK8), Annexin V and enzyme-linked immunosorbent assay (ELISA) were employed. Our results indicated that the level of 4-hydroxycyclophosphamide elevated with prolonged incubation. This accumulation was accompanied by marked cytotoxic effects on primary mouse hepatocytes. CTX treatment significantly inhibited cell viability, impaired the morphology, reduced the numbers and accelerated the early apoptotic rate of the primary mouse hepatocytes. For lipid metabolism, CTX significantly increased glycerol kinase (GK) and decreased triglycerides (TG) levels; Acetyl-CoA was markedly elevated. For amino acid metabolism, aspartate aminotransferase (AST) was significantly reduced. In glucose metabolism, glycolysis was enhanced by CTX treatment, as evidenced by significantly elevated lactic acid (LA) levels and markedly increased pyruvic acid (PA) levels. Glycogen phosphorylase (GP) was increased, and glycogen (GN) was decreased. Although glucokinase (GCK) was significantly upregulated by CTX treatment, intracellular glucose (Glu) levels were significantly reduced. G6PC levels were significantly increased alongside a paradoxical decrease in fructose-1,6-diphosphate (FDP). 6-phosphogluconate dehydrogenase (G6PD) was increased, and malic acid was elevated. Collectively, these findings demonstrate that CTX drives hepatocytes into a “high consumption, low storage” stress-adapted metabolic phenotype by inducing mitochondrial dysfunction and oxidative stress, disrupting the integrated metabolic network of lipids, amino acids and glucose. The accumulation of 4-hydroxycyclophosphamide might serve as the upstream driver of this metabolic disruption.

IntroductionGlucokinase activators (GKAs) enhance glucose phosphorylation by activating glucokinase (GK) expressed in key metabolic organs (such as the liver, pancreas and intestine), thereby initiating cellular glucose metabolism and contributing to improved glycemic control. Among various GKAs under development, dorzagliatin and cadisegliatin (TTP399) are currently the most promising. The gut microbiota plays a critical role in the pathogenesis and progression of metabolic disorders, including obesity and type 2 diabetes. Substantial evidence indicates that long-term administration of oral glucose-lowering agents, such as metformin, can modulate the composition and function of the gut microbiota. Nevertheless, whether GKAs-as emerging oral hypoglycemic agents-also influence gut microbial homeostasis remains unexplored. This study aims to investigate the effects of oral GKAs on intestinal barrier integrity and gut microbiota composition in high-fat diet (HFD)-induced obese/type 2 diabetic mice. In addition, we compare the differential impacts of distinct GKA agents on glycemic regulation and gut microbial communities.MethodsMice were randomly assigned to receive oral gavage of either a vehicle, dorzagliatin, or TTP399 for four consecutive weeks following 5 weeks of HFD feeding. Throughout the study, changes in key metabolic parameters, intestinal barrier integrity, and inflammatory markers were evaluated. Additionally, fecal samples were collected and subjected to 16S-rRNA gene sequencing for analysis of the gut microbiota composition.ResultsBoth dorzagliatin and TTP399 exerted beneficial hypoglycemic effects in HFD mice. Furthermore, results also showed that both dorzagliatin and TTP399 regulated the gut microbiota structure in HFD mice, specifically increasing the relative abundance of short-chain fatty acids-producing and anti-inflammatory bacteria. Notably, under the conditions of this study, neither activator exhibited significant effects on intestinal barrier integrity or inflammatory markers.DiscussionDorzagliatin and TTP399 are associated with alterations in gut microbiota composition at the genus level in HFD-fed mice, with a concomitant increase in the abundance of beneficial genera, and no significant association with changes in intestinal barrier integrity or inflammation. Further investigation is warranted to elucidate the association between long-term GKAs treatment and microbial communities, as well as the potential relationship between microbial changes and hypoglycemic effects.

Glucose is a potent reinforcer of intake, yet most foods contain complex saccharides that do not yield free glucose until after digestion. How the oral sensory system rapidly evaluates the potential metabolic value of food remains unclear. Here, we identify an oral enzymatic–metabolic sensing mechanism that enables detection of glucose-yielding carbohydrates independent of canonical sweet taste receptors. Using genetic, virogenetic, molecular, and behavioral approaches in mice, we show that glucokinase (GCK) in taste cells is necessary for the attraction to glucose-containing sugars. We further demonstrate that maltase glucoamylase (MGAM), a glycosidic enzyme expressed on and near taste cells, facilitates rapid oral sugar sensing. Disruption of either GCK or MGAM in the major taste fields selectively attenuates the attraction to maltose and a carbohydrate-rich mixed diet, establishing both as intermediaries in the initial transduction pathway for complex saccharides that ultimately give rise to nutrient reward. Molecular profiling of taste papillae further revealed that deficient sweet sensing was accompanied by a compensatory increase in lingual MGAM, highlighting an adaptive mechanism for maintaining oral carbohydrate sensitivity. Together, these findings reveal that the oral epithelium actively preprocesses and metabolically evaluates dietary carbohydrates, providing a mechanism for rapid estimation of energetic value prior to ingestion.

Partial inactivation of glucokinase (GCK) is typically characterized by mild hyperglycemia and a favorable lipid profile compared to type 2 diabetes. Previous studies have shown that GCK activity influences serum lipid profiles in a diet-dependent manner; however, its role in hepatic lipid metabolism in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains unclear. To address this, we utilized a newly established heterozygous GCK mutation knock-in mouse model (GCKMut) fed either a normal diet (ND) or a high-fat diet (HFD). Under ND conditions, GCKMut mice developed mild hyperglycemia without overt hepatic injury but displayed reduced hepatic glycogen storage, likely due to decreased energy flux. Metabolomic analyses further revealed substantial reprogramming of hepatic amino acid and lipid metabolism in GCKMut mice. Notably, levels of lysophosphatidylcholines (LPCs)-bioactive metabolites implicated in lipotoxicity and the pathogenesis of MASLD-were significantly reduced, as confirmed by ELISA. Under HFD conditions, GCK inactivation markedly attenuated hepatic lipid accumulation, as demonstrated by biochemical quantification and histological analysis. This protective effect was associated with downregulation of genes involved in de novo lipogenesis and fatty acid uptake, as revealed by transcriptomic analyses of primary hepatocytes. Moreover, both the expression of phospholipase A2 (PLA2) and its product LPC were significantly reduced in GCKMut mice, whereas pharmacologic activation of GCK increased hepatic LPC accumulation. These findings suggest that partial GCK inactivation reprograms hepatic metabolism and mitigates lipid-induced hepatic stress, highlighting reduced hepatic GCK activity as a potential therapeutic strategy for early intervention in MASLD.

Duration-dependent alterations of lipid profiles and microvascular complications in GCK-MODY.

The structural flexibility of enzymes plays an essential role in determining their catalytic efficiency and thermal stability. Cold-adapted enzymes are typically highly flexible, resulting in high catalytic activity but low stability. Glucokinase (GK) consists of the large substrates binding domain, small catalytic domain, and hinge region that undergoes conformational changes upon substrates binding. We recently reported that the psychrophilic GK from Pseudoalteromonas sp. AS-131 (PsGK) exhibits both high catalytic efficiency and remarkable thermal stability compared to the mesophilic GK from Escherichia coli (EcGK). We also found that a disulfide bond connecting the N- and C-termini in PsGK contributes to its unusual thermal stability. However, cold adaptation mechanism of cold-adapted PsGK has remained unclear. To clarify how PsGK acquires high activity, we utilized site-directed spin labeling electron spin resonance (SDSL-ESR) spectroscopy for PsGK and EcGK in the absence and presence of substrates in the wide range of temperatures. PsGK without substrates was more flexible than EcGK. Particularly, the small domain and hinge region of PsGK were highly flexible while its large domain was relatively rigid. In contrast, EcGK showed lower entire flexibility and did not exhibit domain dependent differences. When the substrates were bound, both enzymes became more rigid, but the small domain and hinge region of PsGK was still flexible whereas its large domain was considerably rigid. These results suggest that enhancing catalytic activity requires increasing flexibility only in proper sites rather than in the entire enzyme. These findings provide insight into how cold-adapted enzymes balance activity and stability.

Structure-guided discovery of marine natural products as glucokinase activators for type 2 diabetes mellitus: A computational perspective.

Objective: β-cell dysfunction and loss are major pathological determinants of impaired islet function and hyperglycemia in diabetes. Given the inability of current therapies to restore β-cell viability or glucose-responsive insulin secretion, this study aimed to investigate whether a cell-permeable PBX1 fusion protein (TAT-PBX1) could rescue streptozotocin (STZ)-induced β-cell injury and restore β-cell functional integrity. Methods: A TAT-PBX1 recombinant fusion protein was produced using a prokaryotic expression system. Its protective effects were assessed in STZ-treated MIN6 β cells and in a mouse model of STZ-induced diabetes, with the glucokinase (GK) activator dorzagliatin included as a positive control. We evaluated β-cell apoptosis, DNA damage, ATP and NAD+/NADH levels, insulin signaling (IRS1/PI3K/Akt), and the expression of PDX1 and GK. Glucose-stimulated insulin secretion (GSIS), glucose tolerance, islet morphology, and β-cell proliferation were also examined in vivo. Results: TAT-PBX1 was detectable and significantly enriched in pancreatic tissue and mitigated STZ-induced cytotoxicity by reducing DNA damage, PARP1-associated energy depletion, and β-cell apoptosis. It restored intracellular ATP and NAD+/NADH ratios and reactivated IRS1/PI3K/Akt signaling. TAT-PBX1 further enhanced PDX1 protein levels and upregulated GK, resulting in improved glucose uptake and GSIS. In addition, it increased Ki67+ β-cell proliferation. In diabetic mice, TAT-PBX1 improved glucose tolerance, preserved islet morphology and number, and improved insulin signaling responsiveness. Conclusions: TAT-PBX1 restores β-cell function through coordinated protection of cellular metabolism and insulin signaling, leading to improved β-cell survival, glucose responsiveness, and regenerative capacity. These findings support TAT-PBX1 as a promising molecular strategy for β-cell-protective and β-cell-restorative diabetes therapy.

BackgroundMaturity-onset diabetes of the young type 2 (GCK-MODY), caused by heterozygous inactivating mutations in the glucokinase (GCK) gene, is generally considered a mild and stable form of diabetes with a relatively low risk of chronic complications. However, whether GCK deficiency predisposes to retinal microvascular injury under metabolic stress remains unclear.MethodsA GCK-Q26L knock-in mouse model (GCKMut) was used to evaluate age- and diet-dependent alterations in retinal morphology and molecular pathology under normal diet (ND) and high-fat diet (HFD) conditions at 28, 40, and 60 weeks. Retinal structure and vasculature were examined by H&E staining and trypsin digestion. Oxidative stress, inflammation, and apoptosis were assessed using dihydroethidium fluorescence, Western blotting, and immunohistochemistry. Correlation analyses were performed to determine the relationship between NOX2 expression and inflammatory/apoptotic markers.ResultsUnder ND, retinal morphology and microvasculature were comparable between GCKMut and WT mice at 28, 40, and 60 weeks. In contrast, after prolonged HFD exposure, 60-week-old GCKMut mice exhibited clear microvascular injury, characterized by increased acellular capillaries and pronounced pericyte loss. At this late stage, retinal ROS levels were elevated, accompanied by NOX2 upregulation and increased expression of IL-1β and TNF-α. Apoptotic signaling was concurrently enhanced, as reflected by increased cleaved caspase-3 and a higher Bax/Bcl-2 ratio. Consistently, NOX2 protein levels correlated positively with inflammatory and apoptotic markers.ConclusionsThis study demonstrates that GCK inactivation can predispose to retinal microvascular injury under prolonged metabolic stress. These findings support a NOX2-centered oxidative stress–linked inflammatory and apoptotic axis in late-stage retinal injury and highlight potential therapeutic targets for risk reappraisal in GCK-MODY.

Precision medicine starts with a precision diagnosis. Yet up to 80% of cases of monogenic diabetes, a form of diabetes characterized by mutations in a single gene, are either overlooked or misdiagnosed. A genetic test for monogenic diabetes does not always lead to a precise diagnosis, as novel variants are often classified as variants of unknown significance. Variant interpretation requires collation of a framework of evidence, including population, computational, and segregation data, and can be assisted by functional analysis. The inclusion of functional data can be challenging, depending on the number of benign and pathogenic variants available for benchmarking assays. Glucokinase is the rate-limiting step for glucose metabolism in the pancreatic beta-cell and governs the threshold for glucose-stimulated insulin release. Loss-of-function alleles in the glucokinase (GCK) gene are a cause of stable fasting hyperglycemia from birth and/or diabetes. In this study, we functionally characterized 25 variants identified during diagnostic testing or in exome sequencing studies. We assessed their kinetic characteristics, stability, and interaction with pharmacological and physiological regulators. We integrated our functional data with existing data from the ClinGen Monogenic Diabetes Variant Curation Expert Review panel using a gene-specific framework to assist variant classification. We show how functional evidence can aid variant classification, thus enabling diagnostic certainty.

Obstructive Sleep Apnea, Resting Heart Rate, and Glycemic Variability in Adults With Maturity-Onset Diabetes of the Young