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Hypoxia-inducible factor (HIF) mediates protection via hypoxic preconditioning in both, in vitro and in vivo ischemia models. However, the underlying mechanism remains largely unknown. Prolyl hydroxylase domain proteins serve as the main HIF regulator via hydroxylation of HIFα leading to its degradation. At present, prolyl hydroxylase inhibitors including enarodustat are under clinical trials for the treatment of renal anemia. In an in vitro model of ischemia produced by oxygen-glucose deprivation of renal proximal tubule cells in culture, enarodustat treatment and siRNA knockdown of prolyl hydroxylase 2, but not of prolyl hydroxylase 1 or prolyl hydroxylase 3, significantly increased the cell viability and reduced the levels of reactive oxygen species. These effects were offset by the simultaneous knockdown of HIF1α. In another in vitro ischemia model induced by the blockade of oxidative phosphorylation with rotenone/antimycin A, enarodustat-enhanced glycogen storage prolonged glycolysis and delayed ATP depletion. Although autophagy is another possible mechanism of prolyl hydroxylase inhibition-induced cytoprotection, gene knockout of a key autophagy associated protein, Atg5, did not affect the protection. Enarodustat increased the expression of several enzymes involved in glycogen synthesis, including phosphoglucomutase 1, glycogen synthase 1, and 1,4-α glucan branching enzyme. Increased glycogen served as substrate for ATP and NADP production and augmented reduction of glutathione. Inhibition of glycogen synthase 1 and glutathione reductase nullified enarodustat's protective effect. Enarodustat also protected the kidneys in a rat ischemia reperfusion injury model and the protection was partially abrogated by inhibiting glycogenolysis. Thus, prolyl hydroxylase inhibition protects the kidney from ischemia via upregulation of glycogen synthesis.

IntroductionThis study aimed to identify the associations between lifestyle factors and intrapancreatic fat deposition in patients with type 2 diabetes.Research design and methodsThe participants were 185 patients with type 2 diabetes who were hospitalized at Osaka University Hospital between 2008 and 2020 and underwent abdominal CT during hospitalization. Information regarding lifestyle factors, including the number of meals consumed per day, snacking habits, exercise habits, exercise at work, smoking habits, alcohol intake, insomnia, sleep apnea syndrome, and night-shift working, was acquired from self-administered questionnaires or medical records. We measured the mean CT values for the pancreas (P), liver (L), and spleen (S), and the visceral fat area (VFA), and quantified intrapancreatic and liver ectopic fat accumulation as P−S and L−S, respectively.ResultsAfter adjustment for age, sex, hemoglobin A1c, and body mass index (BMI), participants who consumed two meals per day had significantly lower P−S (higher intrapancreatic fat deposition, p=0.02) than those who consumed three meals per day. There were no significant associations between the number of meals consumed and liver ectopic fat accumulation and VFA (p=0.73 and p=0.67, respectively).ConclusionsPatients with diabetes who consumed two meals per day showed greater intrapancreatic fat deposition than those who consumed three meals per day, even after adjustment for BMI. These findings support the current guideline for diabetes treatment that skipping meals should be avoided.

Hyperinsulinemia is often observed in obese subjects because of insulin resistance, but it may occur in nonobese subjects with unknown etiology. A 72-year-old man was admitted to our hospital for the examination of hyperinsulinemia, reactive hypoglycemia, and liver dysfunction. The patient’s body mass index was 23.7 kg/m2, but he had an elevated visceral fat area (125 cm2). His laboratory data showed mildly elevated liver enzymes, whereas plasma fasting glucose and serum insulin levels were 91 mg/dL and 52.3 μU/mL, respectively. In a 75-g oral glucose tolerance test, the serum insulin level reached the highest value of 1124 μU/mL at 180 minutes. There was no obvious etiology except for mild liver steatosis shown by liver biopsy. We suspected genetic abnormalities related to hyperinsulinemia. We performed whole-exome sequencing (WES) analyses and identified a heterozygous nonsense variant p.R924X in the insulin receptor (INSR) gene, a novel heterozygous missense variant p.V416M in the AKT1 gene, and a novel hemizygous missense variant p.R310Q in the PHKA2 gene, which is the causative gene of hepatic injury as glycogen storage disease type IX. It was speculated that the INSR gene variant, in addition to visceral fat accumulation, was the main cause of hyperinsulinemia and reactive hypoglycemia, and the remaining 2 variants were also partly responsible for hyperinsulinemia. WES analysis revealed candidate gene variants of hyperinsulinemia and hepatic-type glycogenosis. Thus, WES analysis may be a useful tool for clarifying the etiology when unexplained genetic pathophysiological conditions are suspected.

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

We previously reported several factors that cross-sectionally correlate with treatment satisfaction in Japanese patients with type 2 diabetes visiting diabetes clinics. The aim of this study is to identify factors associated with longitudinal changes in treatment satisfaction in patients with type 2 diabetes.The study included 649 patients with type 2 diabetes treated with oral glucose-lowering agents who completed the first questionnaire in 2016. The collected data included scores from the Diabetes Treatment Satisfaction Questionnaire (DTSQ) and other parameters regarding diabetes treatment. We analyzed 1-year longitudinal changes in DTSQ scores and investigated factors associated with these changes.Univariate linear regression analyses showed that changes in body weight, adherence to diet therapy, adherence to exercise therapy, cost burden, motivation for treatment, regularity of mealtimes, and perceived hypoglycemia correlated with changes in DTSQ scores. On the basis of multiple linear regression analyses, a decrease in hypoglycemia (β ± SE = - 0.394 ± 0.134, p = 0.0034), cost burden (β ± SE = - 0.934 ± 0.389, p = 0.017), and an increase in treatment motivation (β ± SE = 1.621 ± 0.606, p = 0.0077) correlated with DTSQ score increases, suggesting that motivation for treatment had the strongest impact on score increases. Subgroup analyses revealed that an increase in motivation for treatment most significantly correlated with a DTSQ score increase in obese and poor glycemic control groups, regardless of age.This is the first longitudinal study clarifying that an increase in motivation for treatment most strongly correlates with an increase in DTSQ score in patients with type 2 diabetes.

Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that JTZ-951, a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. JTZ-951 also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that JTZ-951 has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of JTZ-951, have potential as TIF biomarkers.

Mutations of mismatch repair MutL homologs are causative of a hereditary cancer, Lynch syndrome. Investigation of MutL facilitates genetic diagnoses essential for cancer risk managements and therapies. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although existence of a general acid catalyst had not been conceived in the mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is accelerated by general acid-base catalysis. Analyses of mutant aqMutLs showed that Lys79 is the general acid, and the corresponding residues were confirmed to be critical for activities of human homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5–15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3–5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

IntroductionChronic inflammation is observed in type 2 diabetes islets, and fat deposition in the pancreas affects insulin secretion and glucose tolerance. However, the relationship between this inflammation and pancreatic fat deposition has not been elucidated.Research design and methodsWe examined pancreatic sections from 60 Japanese patients obtained by pancreatectomy. We evaluated pancreatic fat-cell area (%) and CD68-positive (CD68+) cells per islet histologically and examined the relationships between these histological findings and various clinical parameters.ResultsThe number of CD68+ cells per islet in the diabetes group was significantly higher than that in the normal glucose tolerance group (p=0.026). Moreover, CD68+ cells per islet were significantly correlated with body mass index (r=0.33, p=0.0080), fasting C-peptide immunoreactivity (r=0.46, p=0.0042), homeostasis model assessment insulin resistance (r=0.38, p=0.016), C-peptide index (r=0.38, p=0.018), the area under the glucose concentration curve (AUCglucose) at the 75 g oral glucose tolerance test (r=0.49, p=0.0065) and fat-cell area (r=0.51, p=0.0027) and AUCglucose (β=0.453, p=0.0042) were the independent and significant determinants of CD68+ cells per islet.ConclusionThe inflammation of islets is associated with pancreatic fatty infiltration and hyperglycemia, which may further aggravate glucose tolerance.

Background and Aims Hypoxia inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors (also known as HIF stabilizers) increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. Considering that HIF induces the expression of various genes, HIF stabilizers might have pleiotropic effects on the progression of kidney diseases as well as improvement in anemia. Interestingly, HIF induces the metabolic reprogramming from tricarboxylic acid (TCA) cycle to glycolysis as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies have shown accumulation of glucose and TCA cycle metabolites in diabetic renal tissue, which might be related to the progression of diabetic kidney disease (DKD). We hypothesized that HIF stabilization might reverse these metabolism alterations and conducted a proof-of-concept study using enarodustat (JTZ-951), an oral HIF-PH inhibitor. Method We utilized the streptozotocin-induced diabetic rat and alloxan-induced diabetic mouse models. Animals were divided into three groups: Control (normal animals eating normal food), DKD (diabetic animals eating normal food) and DKD+enarodustat (diabetic animals eating food mixed with enarodustat). Blood, urine and kidney samples were collected two weeks after grouping. Metabolism status in renal tissue were compared between groups from transcriptome and metabolome perspectives. Results Although plasma creatinine levels were not different between groups, enarodustat tended to reverse diabetic renal changes such as urinary albumin excretion, glomerulomegaly and glomerular basement membrane thickening (Figure 1). Transcriptome analysis has revealed that enarodustat counteracts the diabetic renal metabolism alterations; fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, while glucose metabolism was upregulated by enarodustat. These symmetric metabolism alterations were confirmed by metabolome analysis (Figure 2); glycolysis and TCA cycle metabolites were accumulated, and amino acids were reduced in diabetic renal tissue, while these metabolism alterations were mitigated by enarodustat. Moreover, enarodustat alleviated the accumulation of glutathione disulfide (GSSG) in diabetic renal tissue and thus showed higher glutathione (GSH)/GSSG ratio, which suggested that enarodustat relieved oxidative stress in DKD. Conclusion HIF stabilization counteracts the renal energy metabolism alterations in the early stages of DKD, in association with the improvement in urinary albumin excretion and renal pathological abnormalities. Our study suggests that HIF stabilization may serve as a potential intervention targeting dysregulated energy metabolism of diabetic kidneys.

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69–1.73 A. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

Objective This study aimed to clarify the clinical significance of the maximum body mass index (BMI) before the onset of type 2 diabetes (MBBO) for predicting pancreatic beta-cell function. Methods This was a cross-sectional observational study. Of 1304 consecutively admitted patients with type 2 diabetes, we enrolled 410 patients satisfying the criteria in this study. The correlations between the C-peptide index (CPI), which is one of the parameters that reflects beta-cell function, and various clinical parameters, including MBBO and duration of diabetes, were analyzed in multiple linear regression analyses. Results The analyses revealed that MBBO was correlated with CPI independently after adjustment for age, sex, HbA1c, and duration of diabetes. When we divided the subjects into three subgroups by MBBO (MBBO < 25 kg/m2; 25 kg/m2 ≤ MBBO < 30 kg/m2; MBBO ≥ 30 kg/m2), CPI was negatively correlated with duration of diabetes in each subgroup, while the rates of CPI based on the duration of diabetes were not different among the three MBBO subgroups. In contrast, the declining rates of CPI were higher in the BMI ≥ 25 kg/m2 group on admission than in the BMI < 25 kg/m2 group on admission. Conclusions MBBO may be an independent factor correlating with beta-cell function and may predict insulin secretion capacity at diagnosis, but it does not seem to affect the rate of decline in insulin secretion capacity after diagnosis. It is important to preserve beta-cell function by decreasing a patient’s BMI during treatment after diagnosis regardless of MBBO.

In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY-YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA-specific DNA-binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch-binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1-dependent suppression of homeologous recombination.

Peculiar findings of orofacial actinomycosis mimicking the clinical appearance of a tumor of the upper gingiva are reported. An 83-year-old man with bleeding of the gingiva visited our hospital. The clinical diagnosis was a benign gingival tumor, and the lesion was surgically removed. Histologically, the excised specimens showed an ulcerative granuloma lesion covered by bacterial colonies consisting of club-shaped filaments. DNA samples were extracted from paraffin sections and examined by polymerase chain reaction (PCR) for Actinomyces species. The PCR products examined by direct DNA sequencing demonstrated the presence of Actinomyces israelii. Finally, a pathological diagnosis was made of a pyogenic granuloma associated with actinomycosis. The PCR method aided the early and exact diagnosis of the paraffin-embedded sample of oral mucosal infectious diseases including actinomycosis. Keywords: Actinomyces israelii, actinomycosis, DNA sequence, nested PCR, pyogenic granuloma

Inhibition of hypoxia inducible factor prolyl hydroxylase (PHD) represents a promising strategy for the discovery of a next generation treatment for renal anemia. We identified several 5,6-fused ring systems as novel scaffolds of the PHD inhibitor on the basis of pharmacophore analysis. In particular, triazolopyridine derivatives showed potent PHD2 inhibitory activities. Examination of the predominance of the triazolopyridines in potency by electrostatic calculations suggested favorable π–π stacking interactions with Tyr310. Lead optimization to improve the efficacy of erythropoietin release in cells and in vivo by improving cell permeability led to the discovery of JTZ-951 (compound 14), with a 5-phenethyl substituent on the triazolopyridine group, which increased hemoglobin levels with daily oral dosing in rats. Compound 14 was rapidly absorbed after oral administration and disappeared shortly thereafter, which could be advantageous in terms of safety. Compound 14 was selected as a clinical candidate.

Context: Hyperinsulinemia is often observed in obese people, owing to their insulin resistance accompanied by visceral fat accumulation, but the frequency of hyperinsulinemia in nonobese people is not well known. Mutations in the insulin receptor gene are known to cause insulin resistance and hyperinsulinemia in type A insulin resistance syndrome, Rabson-Mendenhall syndrome, and Donohue syndrome. However, insulin receptor gene abnormalities have not been investigated in asymptomatic hyperinsulinemic subjects. Purpose: The aim of the current study was to investigate the prevalence of hyperinsulinemia in nonobese Japanese subjects and to examine the involvement of insulin receptor gene mutations. Methods: We enrolled 11,046 subjects who received health checkups. From these, we extracted nonobese subjects (body mass index G (p.Y702X) and c.2779-2780 GC>A]. The prevalence of insulin receptor gene mutations was 18.2% (2/11). Conclusions: To our knowledge, this is the first report of the prevalence of hyperinsulinemia in nonobese healthy subjects. We identified two novel mutations in the insulin receptor gene. These findings indicate that mutations in the insulin receptor gene may be related to fasting hyperinsulinemia, and insulin receptor gene screening may be useful for determining the cause of unexplained hyperinsulinemia., In nonobese healthy subjects, the prevalence of hyperinsulinemia without apparent diabetes is 0.4%. The prevalence of insulin receptor gene mutations in 11 subjects with hyperinsulinemia is 18.2%.

Psychological stress induces many diseases including post-traumatic stress disorder (PTSD); however, the causal relationship between stress and brain atrophy has not been clarified. Applying single-prolonged stress (SPS) to explore the global effect of severe stress, we performed brain magnetic resonance imaging (MRI) acquisition and Voxel-based morphometry (VBM). Significant atrophy was detected in the bilateral thalamus and right visual cortex. Fluorescent immunohistochemistry for Iba-1 as the marker of activated microglia indicates regional microglial activation as stress-reaction in these atrophic areas. These data certify the impact of severe psychological stress on the atrophy of the visual cortex and the thalamus. Unexpectedly, these results are similar to chronic neuropathic pain rather than PTSD clinical research. We believe that some sensitisation mechanism from severe stress-induced atrophy in the visual cortex and thalamus, and the functional defect of the visual system may be a potential therapeutic target for stress-related diseases.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ.

Aims/Introduction The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine-induced apoptotic reactions of β-like insulin-producing cells differentiated from the iPSCs. Materials and Methods iPSCs were generated from fibroblasts of patients with fulminant type 1 diabetes by inducing six reprogramming factors. Insulin-producing cells were differentiated from the iPSCs in vitro. The proportion of cleaved caspase-3-positive or terminal deoxynucleotidyl transferase 2′-deoxyuridine, 5′-triphosphate nick end labeling-positive cells among insulin (INS)-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines was evaluated under treatment with tumor necrosis factor-α, interleukin-1β and interferon-γ. Ribonucleic acid sequencing was carried out to compare gene expressions in INS-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines. Results Two iPSC clones were established from each of three patients with fulminant type 1 diabetes. The differentiation of insulin-producing cells from fulminant type 1 diabetes iPSC was confirmed by immunofluorescence analysis and KCl-induced C-peptide secretion. After treatment with pro-inflammatory cytokines, these INS-positive cells showed higher expression of cleaved caspase-3 than those derived from control human iPSCs. Altered expression levels of several apoptosis-related genes were observed in INS-positive cells derived from the fulminant type 1 diabetes iPSCs by ribonucleic acid sequencing. Conclusions We generated iPSCs from patients with fulminant type 1 diabetes and differentiated them into insulin-producing cells. This in vitro disease model can be used to elucidate the disease mechanisms of fulminant type 1 diabetes.

Context:Fulminant type 1 diabetes mellitus (T1DM) is thought to be partly caused by virus infection.Objective:This study investigated the mechanism of β cell destruction in fulminant T1DM after drug-induced hypersensitivity syndrome (DIHS).Methods:We determined the localization of human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and Epstein-Barr virus (EBV) and the expression of interferon regulatory factor 3 (IRF3) and viral receptors of Z-DNA binding protein 1 (ZBP1) and retinoic acid-inducible gene I (RIG-I), together with inflammatory cells, by immunohistochemistry of the autopsy pancreas of a patient with fulminant T1DM with DIHS and in seven subjects with normal glucose tolerance who underwent pancreatectomy.Results:HCMV-positive cells were detected in islets and exocrine areas in the patient with fulminant T1DM. Greater numbers of macrophages and CD4+ and CD8+ T lymphocytes had infiltrated into HCMV-positive islets than into HCMV-negative islets, and 52.6% of HCMV-positive cells were also positive for IRF3. α Cells expressed IRF3, ZBP1, or RIG-I. No HCMV-positive cells were detected in the control subjects. HHV-6−positive, but not EBV-positive, cells were present in the patient and the control subjects.Conclusions:These findings indicate that the immunoresponse caused by HCMV infection was associated with β cell injury.

Cardiovascular disease (CVD) is the main cause of death in patients with kidney disease. Hypoxia plays a crucial role in the progression of chronic kidney disease (CKD) and cardiovascular disease, which is associated with fibrosis, inflammation, and oxidative injury. Previous studies have indicated that prolyl hydroxylase (PHD) inhibitors, stabilizers of hypoxia-inducible factors (HIFs), can be used to treat acute organ injuries such as renal ischemia-reperfusion, myocardial infarction, and, in some contexts, CKD. However, the effects of PHD inhibitors on cardiovascular complications in CKD remain unknown. In the present study, we investigated whether HIF activation has a beneficial effect on kidney and cardiovascular outcomes in the remnant kidney model. We used the 5/6 nephrectomy model with the nitric oxide synthase inhibitor Nω-nitro-l-arginine (20 mg/L in the drinking water). Rats received diet with 0.005% enarodustat (PHD inhibitor) or vehicle for 8 wk starting 2 wk before 5/6 nephrectomy. Activation of HIF by the PHD inhibitor reduced cardiac hypertrophy and ameliorated myocardial fibrosis in association with restored capillary density and improvement in mitochondrial morphology. With regard to kidneys, enarodustat ameliorated fibrosis in association with reduced proinflammatory cytokine expression, reduced apoptosis, and restored capillary density, even though renal endpoints such as proteinuria and serum creatinine levels were not significantly affected by enarodustat, except for blood urea nitrogen levels at 4 wk. In addition, cardiac hypertrophy marker genes, including atrial natriuretic peptide, were suppressed in P19CL6 cells treated with enarodustat. These findings suggest that PHD inhibitors might show beneficial effects in cardiovascular complications caused by CKD.

Background Prolyl hydroxylase domain (PHD) inhibitors, which stimulate erythropoietin production through the activation of hypoxia-inducible factor (HIF), are novel therapeutic agents used for treating renal anemia. Several PHD inhibitors, including enarodustat, are currently undergoing phase 2 or phase 3 clinical trials. Because HIF regulates a broad spectrum of genes, PHD inhibitors are expected to have other effects in addition to erythropoiesis, such as protection against metabolic disorders. However, whether such beneficial effects would extend to metabolic disorder-related kidney disease is largely unknown. Methods We administered enarodustat or vehicle without enarodustat in feed to diabetic black and tan brachyury (BTBR) ob/ob mice from 4 to 22 weeks of age. To elucidate molecular changes induced by enarodustat, we performed transcriptome analysis of isolated glomeruli and in vitro experiments using murine mesangial cells. Results Compared with BTBR ob/ob mice that received only vehicle, BTBR ob/ob mice treated with enarodustat displayed lower body weight, reduced blood glucose levels with improved insulin sensitivity, lower total cholesterol levels, higher adiponectin levels, and less adipose tissue, as well as a tendency for lower macrophage infiltration. Enarodustat-treated mice also exhibited reduced albuminuria and amelioration of glomerular epithelial and endothelial damage. Transcriptome analysis of isolated glomeruli revealed reduced expression of C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) in enarodustat-treated mice compared with the vehicle-only group, accompanied by reduced glomerular macrophage infiltration. In vitro experiments demonstrated that both local HIF-1 activation and restoration of adiponectin by enarodustat contributed to CCL2/MCP-1 reduction in mesangial cells. Conclusions These results indicate that the PHD inhibitor enarodustat has potential renoprotective effects in addition to its potential to protect against metabolic disorders.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.

The aim of this study was to determine the efficacy of insulin degludec (IDeg) relative to insulin glargine (IGlar) or insulin detemir (IDet) in glycemic control, as evaluated by continuous glucose monitoring (CGM) in insulin-deficient patients with type 1 diabetes.We studied 28 outpatients treated with IGlar or IDet (IGlar/IDet). Basal insulin was switched to IDeg when glycemic control was considered unstable, as judged by the dawn phenomenon or nocturnal hypoglycemia. Whole-day CGM data were also divided into daytime and nighttime data.The dawn phenomenon or nocturnal hypoglycemia under IGlar/IDet treatment was observed in all patients. Among 26 patients who completed the study, there were no significant differences in parameters representing glycemic variability, hyperglycemia, mean glycemic control, and HbA1c or insulin therapy-related quality of life at the night score. Measures of hypoglycemia [whole-day %Low and area under the curve (AUC) below 70] were significantly lower under IDeg treatment than under IGlar/IDet treatment (%Low, 9.6 ± 11.5 vs. 14.7 ± 14.9%,Patients with unstable glycemic control under IGlar/IDet treatment did not improve glycemic control upon switching to IDeg, but the frequency and the degree of hypoglycemia was reduced in insulin-deficient outpatients with type 1 diabetes, especially in those suffering from severe hypoglycemia.

In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on β-clamp.