Stimulatory effect of imeglimin on incretin secretion.

AIMS/INTRODUCTION: Imeglimin is a new antidiabetic drug structurally related to metformin. Despite this structural similarity, only imeglimin augments glucose-stimulated insulin secretion (GSIS), with the mechanism underlying this effect remaining unclear. Given that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) also enhance GSIS, we examined whether these incretin hormones might contribute to the pharmacological actions of imeglimin. MATERIALS AND METHODS: Blood glucose and plasma insulin, GIP, and GLP-1 concentrations were measured during an oral glucose tolerance test (OGTT) performed in C57BL/6JJcl (C57BL/6) or KK-Ay/TaJcl (KK-Ay) mice after administration of a single dose of imeglimin with or without the dipeptidyl peptidase-4 inhibitor sitagliptin or the GLP-1 receptor antagonist exendin-9. The effects of imeglimin, with or without GIP or GLP-1, on GSIS were examined in C57BL/6 mouse islets. RESULTS: Imeglimin lowered blood glucose and increased plasma insulin levels during an OGTT in both C57BL/6 and KK-Ay mice, whereas it also increased the plasma levels of GIP and GLP-1 in KK-Ay mice and the GLP-1 levels in C57BL/6 mice. The combination of imeglimin and sitagliptin increased plasma insulin and GLP-1 levels during the OGTT in KK-Ay mice to a markedly greater extent than did either drug alone. Imeglimin enhanced GSIS in an additive manner with GLP-1, but not with GIP, in mouse islets. Exendin-9 had only a minor inhibitory effect on the glucose-lowering action of imeglimin during the OGTT in KK-Ay mice. CONCLUSIONS: Our data suggest that the imeglimin-induced increase in plasma GLP-1 levels likely contributes at least in part to its stimulatory effect on insulin secretion.

A novel view of the insulin signaling pathway based on prediction of protein structure by the AI platform AlphaFold.

The predicted structures of major proteins involved in the insulin signaling pathway obtained from the AlphaFold Protein Structure Database.

Effects of imeglimin on mitochondrial function, AMPK activity, and gene expression in hepatocytes.

Imeglimin is a recently launched antidiabetic drug structurally related to metformin. To provide insight into the pharmacological properties of imeglimin, we investigated its effects on hepatocytes and compared them with those of metformin. The effects of imeglimin on mitochondrial function in HepG2 cells or mouse primary hepatocytes were examined with an extracellular flux analyzer and on gene expression in HepG2 cells by comprehensive RNA-sequencing analysis. The effects of the drug on AMPK activity in HepG2 cells, mouse primary hepatocytes, and mouse liver were also examined. Treatment of HepG2 cells or mouse primary hepatocytes with imeglimin reduced the oxygen consumption rate coupled to ATP production. Imeglimin activated AMPK in these cells whereas the potency was smaller than metformin. Bolus administration of imeglimin in mice also activated AMPK in the liver. Whereas the effects of imeglimin and metformin on gene expression in HepG2 cells were similar overall, the expression of genes encoding proteins of mitochondrial respiratory complex III and complex I was upregulated by imeglimin but not by metformin. Our results suggest that imeglimin and metformin exert similar pharmacological effects on mitochondrial respiration, AMPK activity, and gene expression in cultured hepatocytes, whereas the two drugs differ in their effects on the expression of certain genes related to mitochondrial function.

New mechanism of metformin action mediated by lysosomal presenilin enhancer 2.

Formation of the PEN2-ATP6AP1 complex induced by the binding of metformin to PEN2 results in the inhibition of v-ATPase activity and in the recruitment of AXIN/LKB1 to lysosomes, which in turn results in the phosphorylation and activation of AMPK.

A case of α-chain variant hemoglobin (Hb Chad) with falsely high HbA1c levels measured by immunoassay.

Variant hemoglobin is often detected during the diagnosis and treatment of diabetes mellitus. We here describe a case of α2-chain variant hemoglobin (Hb Chad) that was identified as a result of differences in HbA1cs values determined by different assays. HbA1c measured by immunoassay was thus falsely high, whereas that measured by high-performance liquid chromatography (HPLC) was slightly low. Sequencing analysis revealed a heterozygous GAG (glutamic acid) → AAG (lysine) mutation at amino acid position 23 of the α2-globin gene. This residue is located at the surface of the α-chain in the crystal structure of hemoglobin. The high HbA1c value determined by immunoassay might have been the result of increased antigenicity of the variant hemoglobin, whereas the low value measured by HPLC reflected differential fractionation of the variant relative to the wild-type protein. Hb Chad has been reported in only three cases to date, and HbA1c was measured for the first time. This is the first case where falsely high HbA1c measured by immunoassay due to increased antigenicity in α-chain variant hemoglobin. This case highlights the importance of comparison with other parameters related to plasma glucose such as glycated albumin if an HbA1c abnormality is suspected. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00529-y.

Structure Elucidation of Calyxoside B, a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement.

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

The gut microbial metabolite imidazole propionate inhibits metformin action.

Imidazole propionate inhibits metformin action in a manner dependent on a p38γ-Akt-AMPK axis.

Dose-dependent accumulation of glucose in the intestinal wall and lumen induced by metformin as revealed by 18 F-labelled fluorodeoxyglucose positron emission tomography-MRI.

AIM: To investigate the relationships between various clinical variables and the metformin-induced accumulation of fluorodeoxyglucose (FDG) in the intestine, with distinction between the intestinal wall and lumen, in individuals with type 2 diabetes who were receiving metformin treatment and underwent 18 F-labelled FDG ([18 F]FDG) positron emission tomography (PET)-MRI. MATERIALS AND METHODS: We evaluated intestinal accumulation of [18 F]FDG with both subjective (a five-point visual scale determined by two experienced radiologists) and objective analyses (measurement of the maximum standardized uptake value [SUVmax ]) in 26 individuals with type 2 diabetes who were receiving metformin and underwent [18 F]FDG PET-MRI. [18 F]FDG accumulation within the intestinal wall was discriminated from that in the lumen on the basis of SUVmax . RESULTS: SUVmax for the large intestine was correlated with blood glucose level (BG) and metformin dose, but not with age, body mass index, HbA1c level or estimated glomerular filtration rate (eGFR). SUVmax for the small intestine was not correlated with any of these variables. Visual scale analysis yielded essentially similar results. Metformin dose and eGFR were correlated with SUVmax for the wall and lumen of the large intestine, whereas BG was correlated with that for the wall. Multivariable analysis identified metformin dose as an explanatory factor for SUVmax in the wall and lumen of the large intestine after adjustment for potential confounders including BG and eGFR. CONCLUSIONS: Metformin dose is an independent determinant of [18 F]FDG accumulation in the wall and lumen of the large intestine in individuals treated with this drug.

Enhanced Release of Glucose Into the Intraluminal Space of the Intestine Associated With Metformin Treatment as Revealed by [18F]Fluorodeoxyglucose PET-MRI.

OBJECTIVE: Positron emission tomography (PET)-computed tomography has revealed that metformin promotes the intestinal accumulation of [18F]fluorodeoxyglucose (FDG), a nonmetabolizable glucose derivative. It has remained unknown, however, whether this accumulation occurs in the wall or intraluminal space of the intestine. We here addressed this question with the use of [18F]FDG PET-MRI, a recently developed imaging method with increased accuracy of registration and high soft-tissue contrast. RESEARCH DESIGN AND METHODS: Among 244 individuals with type 2 diabetes who underwent PET-MRI, we extracted 24 pairs of subjects matched for age, BMI, and HbA1c level who were receiving treatment with metformin (metformin group) or were not (control group). We evaluated accumulation of [18F]FDG in different portions of the intestine with both a visual scale and measurement of maximum standardized uptake value (SUVmax), and such accumulation within the intestinal wall or lumen was discriminated on the basis of SUVmax. RESULTS: SUVmax of the jejunum, ileum, and right or left hemicolon was greater in the metformin group than in the control group. [18F]FDG accumulation in the ileum and right or left hemicolon, as assessed with the visual scale, was also greater in the metformin group. SUVmax for the intraluminal space of the ileum and right or left hemicolon, but not that for the intestinal wall, was greater in the metformin group than in the control group. CONCLUSIONS: Metformin treatment was associated with increased accumulation of [18F]FDG in the intraluminal space of the intestine, suggesting that this drug promotes the transport of glucose from the circulation into stool.

Relationship between metformin use and vitamin B12 status in patients with type 2 diabetes in Japan.

AIMS/INTRODUCTION: Metformin therapy has been associated with vitamin B12 (VB12) deficiency, but information regarding this adverse effect in Asian populations is limited. We have now examined the relationship between metformin use and VB12 status in individuals with type 2 diabetes mellitus in Japan. MATERIALS AND METHODS: This cross-sectional study was carried out with type 2 diabetes mellitus patients treated (Met group, n = 122) or not treated (control group, n = 63) with metformin. The primary end-point was the difference in the serum concentration of homocysteine, a marker of VB12 activity, between the two groups. The serum concentrations of VB12, blood hemoglobin level and mean corpuscular volume were also compared between the groups. Subset analysis was carried out with individuals aged ≥70 years. The potential correlation between the daily dose or duration of metformin treatment and the other measured parameters was also examined. RESULTS: The level of homocysteine, as well as the VB12 level, hemoglobin concentration and mean corpuscular volume, did not differ significantly between the control and treated with metformin groups. The level of homocysteine was positively and that of VB12 negatively correlated with the daily dose of metformin. Among elderly individuals, the hemoglobin level was significantly lower in the treated with metformin group than in the control group, although the mean corpuscular volume was similar in the two groups. CONCLUSIONS: The risk of VB12 deficiency during metformin treatment appears to be low in Japanese type 2 diabetes mellitus patients. However, high doses of metformin might result in a moderate decrease in the circulating VB12 level, as well as in anemia in elderly individuals.

Phenotypic differences and similarities of monozygotic twins with maturity-onset diabetes of the young type 5.

Here, we report phenotypic differences and similarities of monozygotic twins with maturity-onset diabetes of the young type 5 harboring a partial deletion of chromosome 17q12. The proband and her twin sister manifested complete aplasia and marked hypoplasia, respectively, of the body and tail of the pancreas. Whereas both twins showed marked hypoplasia of the right kidney and multiple cysts in both kidneys, only the proband's sister showed hydronephrosis in the left kidney. The proband had profound defects in insulin and glucagon secretion, as well as mild renal dysfunction, whereas her sister had pronounced renal dysfunction accompanied by mild defects in insulin and glucagon secretion. Both twins manifested hypomagnesemia and hyperuricemia, but no apparent liver dysfunction or intellectual disability. The severity of renal and pancreatic defects differed between monozygotic twins with maturity-onset diabetes of the young type 5, suggesting that the phenotypes of this condition are determined not solely by genetic factors.

In silico and in vitro analyses of the pathological relevance of the R258H mutation of hepatocyte nuclear factor 4α identified in maturity-onset diabetes of the young type 1.

Mutations of the hepatocyte nuclear factor 4α (HNF4α) gene give rise to maturity-onset diabetes of the young type 1. Although many such mutations have been identified in affected individuals, part of these mutations has been characterized with regard to their pathological relevance. We here identified a missense mutation (c.773G>A, p.R258H) of HNF4A in a mother and daughter with early-onset diabetes and impaired insulin secretion. In silico simulation and in vitro luciferase reporter analyses showed that the mutation impairs the stability of self-dimerization and the transactivation activity of HNF4α. Although arginine-258 does not appear to participate directly in dimerization, its mutation alters the electrostatic surface potential of the dimer interface. Our results thus suggest that this mutation impairs the function of HNF4α and thereby contributes to the pathogenesis of maturity-onset diabetes of the young type 1.

ß-Cell signalling and insulin secretagogues: A path for improved diabetes therapy.

Insulin secretagogues including sulfonylureas, glinides and incretin-related drugs such as dipeptidyl peptidase 4 (DPP-4) inhibitors and glucagon-like peptide-1 receptor agonists are widely used for treatment of type 2 diabetes. In addition, glucokinase activators and G-protein-coupled receptor 40 (GPR40) agonists also have been developed, although the drugs are not clinically usable. These different drugs exert their effects on insulin secretion by different mechanisms. Recent advances in ß-cell signalling studies have not only deepened our understanding of insulin secretion but also revealed novel mechanisms of insulin secretagogues. Clarification of the signalling mechanisms of the insulin secretagogues will contribute to improved drug therapy for diabetes.

A Novel Diphenylthiosemicarbazide Is a Potential Insulin Secretagogue for Anti-Diabetic Agen.

Insulin secretagogues are used for treatment of type 2 diabetes. We attempted to discover novel small molecules to stimulate insulin secretion by using in silico similarity search using sulfonylureas as query, followed by measurement of insulin secretion. Among 38 compounds selected by in silico similarity search, we found three diphenylsemicarbazides and one quinolone that stimulate insulin secretion. We focused on compound 8 (C8), which had the strongest insulin-secreting effect. Based on the structure-activity relationship of C8-derivatives, we identified diphenylthiosemicarbazide (DSC) 108 as the most potent secretagogue. DSC108 increased the intracellular Ca2+ level in MIN6-K8 cells. Competitive inhibition experiment and electrophysiological analysis revealed sulfonylurea receptor 1 (SUR1) to be the target of DSC108 and that this diphenylthiosemicarbazide directly inhibits ATP-sensitive K+ (KATP) channels. Pharmacokinetic analysis showed that DSC108 has a short half-life in vivo. Oral administration of DSC108 significantly suppressed the rises in blood glucose levels after glucose load in wild-type mice and improved glucose tolerance in the Goto-Kakizaki (GK) rat, a model of type 2 diabetes with impaired insulin secretion. Our data indicate that DSC108 is a novel insulin secretagogue, and is a lead compound for development of a new anti-diabetic agent.

Structure and functional roles of Epac2 (Rapgef4).

Epac (exchange protein activated by cyclic-AMP) 2 is a direct target of 3'-5'-cyclic adenosine monophosphate (cAMP) and is involved in cAMP-mediated signal transduction through activation of the Ras-like small GTPase Rap. Crystallographic analyses revealed that activation of Epac2 by cAMP is accompanied by dynamic structural changes. Epac2 is expressed mainly in brain, neuroendocrine and endocrine tissues, and is involved in diverse cellular functions in the tissues. In this review, we summarize the structure and function of Epac2. We also discuss the physiological and pathophysiological roles of Epac2, and the possibility of Epac2 as a therapeutic target.

Characteristics of 20 Patients with Autochthonous Acute Hepatitis E in Hokkaido, Japan: First Report of Bilateral Facial Palsy Following the Infection with Genotype 4 Hepatitis E Virus.

Autochthonous hepatitis E is increasingly being recognized in industrialized countries, including Japan. Although neurological abnormalities have been sporadically reported as an extrahepatic manifestation of hepatitis E virus (HEV) infection, it is rare and has not been reported in Japan. The present study aimed to characterize a total of 20 patients consecutively diagnosed with sporadic acute hepatitis E at a city hospital in Hokkaido, Japan, during 2001-2014, focusing on a patient complicated with neuropathy. Seventeen patients were infected with genotype 4 HEV, while the remaining three patients were with genotype 3 HEV. Although a 67-year-old male with severe hepatitis did not have predisposing factors associated with the development of neurological disorders, such as diabetes mellitus and the use of immunosuppressive agents, he developed bilateral peripheral facial palsy six days after admission. A neurological examination revealed the inability to smile, frown, close his eyes completely or puff out his cheeks. MRI brain scans were considered to be normal. Although it took 83 days after admission for the total bilirubin levels to normalize, his neurological symptoms resolved gradually within three weeks without any sequelae following conservative therapy. A full-length genomic analysis of the HEV strain (HE-JA30) isolated from the patient belonged to genotype 4 and was closest to that currently circulating in Hokkaido, Japan. This is the first report of HEV-associated neuropathy in Japan. While all of previous reports on HEV-related neuropathy involve genotype 3 HEV, the present report is unique in that genotype 4 HEV is responsible for the neuropathy.

Glutamate acts as a key signal linking glucose metabolism to incretin/cAMP action to amplify insulin secretion.

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting ß cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic ß cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

Comparison of incretin immunoassays with or without plasma extraction: Incretin secretion in Japanese patients with type 2 diabetes.

UNLABELLED: Aims/Introduction: The effectiveness of incretin-based therapies in Asian type 2 diabetes requires investigation of the secretion and metabolism of glucose-dependent insulinotropic polypepide (GIP) and glucagon-like peptide 1 (GLP-1). Plasma extractions have been suggested to reduce variability in intact GLP-1 levels among individuals by removing interference that affects immunoassays, although no direct demonstration of this method has been reported. We have evaluated the effects of ethanol and solid-phase extractions on incretin immunoassays. We determined incretin levels during meal tolerance tests in Japanese patients with type 2 diabetes and characterized predictors for incretin secretion. MATERIALS AND METHODS: Japanese patients with type 2 diabetes (23 anti-diabetic drug-naïve and 18 treated with sulfonylurea [SU] alone) were subjected to meal tolerance tests, and incretin levels were determined by immunoassays with or without extraction. RESULTS: Intact GLP-1 levels determined by an intact GLP-1 immunoassay with ethanol and solid-phase extractions were lower than those determined without extraction. Intact GLP-1 levels determined by the extractions were highly correlated with each other, much more so than the levels with and without extraction. Total GLP-1 was unaffected by extractions, showing that extractions remove interference only in the case of intact GLP-1. Incretin secretion after meal ingestion was similar between drug-naïve and SU-treated patients. Fasting and postprandial GLP-1 levels were correlated positively with fasting free fatty acids and negatively with dipeptidyl peptidase-4 activity. CONCLUSIONS: Ethanol and solid-phase extractions remove interference for intact GLP-1 immunoassay. SU showed little effect on incretin secretion. GLP-1 and GIP secretion were predicted by different factors. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00141.x, 2012).

Predicting efficacy of dipeptidyl peptidase-4 inhibitors in patients with type 2 diabetes: Association of glycated hemoglobin reduction with serum eicosapentaenoic acid and docosahexaenoic acid levels.

This study was initiated to identify clinical and dietary parameters that predict efficacy of dipeptidyl peptidase-4 inhibitors. A total of 72 untreated Japanese patients with type 2 diabetes who received DPP-4 inhibitors (sitagliptin, alogliptin or vildagliptin) for 4 months were examined for changes of glycated hemoglobin (HbA1c) and body mass index (BMI), and self-administered 3-day food records, as well as serum levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). DPP-4 inhibitors significantly reduced HbA1c (before initiation of DPP-4 inhibitors 7.2 ± 0.7%, 4 months after initiation of DPP-4 inhibitors 6.7 ± 0.6% [paired t-test, P < 0.01 vs before]). Multiple regression analysis showed that changes of HbA1c were significantly correlated with baseline HbA1c, as well as estimated intake of fish. Furthermore, changes of HbA1c were significantly correlated with serum levels of EPA (r = -0.624, P < 0.01) and DHA (r = -0.577, P < 0.01). HbA1c reduction by DPP-4 inhibitors is significantly correlated with estimated intake of fish and serum levels of EPA and DHA. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00214.x, 2012).