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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.

Background Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms underlying variant effects in human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. Results Using a yeast growth-based assay, we score the abundance of 95% of GCK missense and nonsense variants. When combining the abundance scores with our previously determined activity scores, we find that 43% of hypoactive variants also decrease cellular protein abundance. The low-abundance variants are enriched in the large domain, while residues in the small domain are tolerant to mutations with respect to abundance. Instead, many variants in the small domain perturb GCK conformational dynamics which are essential for appropriate activity. Conclusions In this study, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis.

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.

HMS5552, a novel fourth-generation glucokinase (GK) activator, has demonstrated promising effects on glycemic control in preclinical models of type 2 diabetes. This single ascending dose study was conducted to investigate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of HMS5552 during its first-in-human exposure. Sixty healthy subjects were enrolled. In each of six dose-cohorts (5, 10, 15, 25, 35, and 50 mg), ten subjects were randomized with eight subjects receiving the same cohort-dose of HMS5552 and two receiving placebo. Plasma HMS5552 exposure, glucose, and insulin were measured repeatedly during fasting and after a standardized meal. Assessment included safety, PK, and PD endpoints. HMS5552 showed dose-proportional increases in area under the curve 0 to the last quantifiable concentration (AUC0-t) and maximum plasma concentration (Cmax). Slopes estimated by linear regression for AUC0-t and Cmax were ~1.0 (0.932 and 0.933, respectively). Geometric mean elimination half-life ranged from 4.48 to 7.51 hours and apparent clearance ranged from 11.5 to 13.1 L/h across all doses. No significant sex effect was observed in PK parameters. HMS5552 also demonstrated dose-related PD responses in terms of maximum glucose change from baseline (%) and mean glucose area under effect curve 0-4 hours change from baseline (%) (P<0.001). Fifteen adverse events were reported by nine subjects (ten with HMS5552 and five with the placebo). All adverse events were mild in intensity and resolved without any treatment. This first-in-human single ascending dose study provided predicted PK of HMS5552 with dose-proportional increases in AUC0-t and Cmax, as well as dose-related glucose-lowering effects over the range of 5-50 mg in healthy subjects. HMS5552 at doses up to 50 mg in healthy subjects was safe and well-tolerated.

Background Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. Result Here, we exploit a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlate with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants are concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. Conclusion Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.

Type 2 diabetes (T2D) is characterized by a progressive deterioration of β-cell function with a continuous decline in insulin secretion. Glucokinase (GCK) facilitates the rate-limiting step of glycolysis in pancreatic β-cells, to acquire the proper glucose-stimulated insulin secretion. Multiple glucokinase activators (GKAs) have been developed and clinically tested. However, the dynamic change of human pancreatic GCK expression during T2D progression has not been investigated. We evaluated GCK expression by measuring the average immunoreactivity of GCK in insulin or glucagon cells from pancreatic sections of 11 nondiabetic subjects (ND), 10 subjects with impaired fasting glucose (IFG), 9 with well-controlled T2D (wT2D), and 5 individuals with poorly controlled T2D (uT2D). We also assessed the relationship between GCK expression and adaptive unfolded protein response (UPR) in human diabetic β-cells. We did not detect changes of GCK expression in IFG islets. However, we found β-cell GCK levels were significantly increased in T2D with adequate glucose control (wT2D) but not in T2D with poor glucose control (uT2D). Furthermore, there was a strong positive correlation between GCK expression and adaptive UPR (spliced X-box binding protein 1 [XBP1s] and activating transcription factor 4 [ATF4]), as well as functional maturity marker (urocortin-3 [UCN3]) in human diabetic β-cells. Our study demonstrates that inductions of GCK enhanced adaptive UPR and UCN3 in human β-cells, which might be an adaptive mechanism during T2D progression. This finding provides a rationale for exploring novel molecules that activate β-cell GCK and thereby improve pharmacological treatment of T2D.

SY-004, a dual-acting full glucokinase activator, is under development to provide a dose-dependent improvement of glucose control. This study aimed to assess the tolerability, safety, and pharmacokinetic and pharmacodynamic properties of SY-004 in healthy Chinese adults. Two study participants were administered 2 mg of SY-004 in the 2-mg cohort, whereas 6 study participants were randomized with 4 study participants receiving SY-004 and 2 receiving placebo in the 20-mg cohort. In each of other 3 dose cohorts (40, 80, and 120 mg), 12 participants were randomized in a 10:2 ratio to receive single oral SY-004 capsules or placebos. Drug concentrations, glucose and insulin levels, and safety data were assessed and analyzed. Noncompartmental analysis was used to determine SY-004 pharmacokinetic parameters. SY-004 was generally well tolerated. Nine of the 44 study participants reported 17 treatment-related adverse events, and most treatment-related adverse events were mild. SY-004 had approximately dose-proportional increases in systemic exposure. The mean t½ ranged from 37.6 to 49.9 hours, and CL/F values ranged from 67.1 to 110 L/h across all doses. The cumulative amounts of the unchanged drug excreted in urine were very low, accounting for no more than 1.53% of the given doses. No significant difference in sex was observed in pharmacokinetic parameters. The pharmacodynamic response appeared to slightly correlate with dose. SY-004, a new potential glucokinase activator, had favorable safety profiles and good PK characteristics. The glucose-lowering effects were slightly dose related. The SY-004 data in healthy Chinese adults supports further development. ClinicalTrials.gov identifier: NCT03171623.

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.

Increased hepatic glucose output, the primary liver dysregulation associated with Type 2 diabetes mellitus (T2DM), is not directly or effectively targeted by the currently available classes of glucose-lowering medications except metformin. This unmet need might be addressed through activation of a specific enzyme-member of the hexokinase family, namely glucokinase (GK). GK serves as a “glucose-sensor” or “glucose receptor” in pancreatic cells, eliciting glucose-stimulated insulin secretion, and as glucose “gate-keeper” in hepatocytes, promoting hepatic glucose uptake and glycogen synthesis and storage. GK activation by small molecules present an alternative approach to restore/improve glycaemic control in patients with T2DM. GK activators (GKAs) may increase insulin secretion from the pancreas and promote glycogen synthesis in the liver, and hence reduce hepatic glucose output. Despite several setbacks in their development, interest in the GKA class has been renewed, particularly since the introduction of a novel, dual-acting full GKA, dorzagliatin, and a novel hepatoselective molecule, TTP399. In this article we provide an overview of the role, efficacy, safety and future developments of GKAs in the management of T2DM.

Glucagon secretion by pancreatic α-cells is triggered by hypoglycemia and suppressed by high glucose levels; impaired suppression of glucagon secretion is a hallmark of both type 1 and type 2 diabetes. Here, we show that α-cell glucokinase ( Gck ) plays a role in the control of glucagon secretion. Using mice with α-cell-specific inactivation of Gck ( αGckKO mice), we find that glucokinase is required for the glucose-dependent increase in intracellular ATP/ADP ratio and the closure of K ATP channels in α-cells and the suppression of glucagon secretion at euglycemic and hyperglycemic levels. αGckKO mice display hyperglucagonemia in the fed state, which is associated with increased hepatic gluconeogenic gene expression and hepatic glucose output capacity. In adult mice, fed hyperglucagonemia is further increased and glucose intolerance develops. Thus, glucokinase governs an α-cell metabolic pathway that suppresses secretion at or above normoglycemic levels; abnormal suppression of glucagon secretion deregulates hepatic glucose metabolism and, over time, induces a pre-diabetic phenotype. Glucagon secretion is promoted during hypoglycemia and inhibited by increased glucose levels. Here, Basco et al. show that glucokinase suppresses glucose-regulated glucagon secretion by modulating the intracellular ATP/ADP ratio and the closure of K ATP channels in α-cells.