The growing landscape of lysine acetylation links metabolism and cell signalling.

Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

AIMS/HYPOTHESIS: Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. METHODS: We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. RESULTS: Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. CONCLUSIONS/INTERPRETATION: Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. DATA AVAILABILITY: Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

Protein acylations induced by a ketogenic diet demonstrate diverse patterns depending on organs and differ between histones and global proteins.

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.

Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter.

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation.

Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target.

Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD(+)-dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5(-/-) mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5(-/-) animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5(-/-) compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.

Genetic ablation of p62/SQSTM1 demonstrates little effect on pancreatic ß-cell function under autophagy deficiency.

Autophagy is known to play an essential role in intracellular quality control through the degradation of damaged organelles and components. We previously demonstrated that ß-cell-specific autophagy deficient mice, which lack Atg7, exhibited impaired glucose tolerance, accompanied by the accumulation of sequestosome 1/p62 (hereafter referred to as p62). Whereas p62 has been reported to play essential roles in regulating cellular homeostasis in the liver and adipose tissue, we previously showed that ß-cell-specific p62 deficiency does not cause any apparent impairment in glucose metabolism. In the present study, we investigated the roles of p62 in ß cells under autophagy-deficient conditions, by simultaneously inactivating both Atg7 and p62 in a ß-cell specific manner. Whereas p62 accumulation was substantially reduced in the islets of Atg7 and p62 double-deficient mice, glucose tolerance and insulin secretion were comparable to Atg7 single-deficient mice. Taken together, these findings suggest that the p62 accumulation appears to have little effect on ß-cell function under conditions of autophagy inhibition.

Cumulative autophagy insufficiency in mice leads to progression of ß-cell failure.

Autophagy is known to play a pivotal role in ß-cell function. While the lifelong inhibition of autophagy through Atg7 deletion in ß cells has been demonstrated to lead to impaired glucose tolerance together with ß-cell dysfunction, the temporal association between autophagy inhibition and ß-cell dysfunction remains unclear. To address such questions, inducible ß-cell-specific Atg7-knockout (ißAtg7KO) mice were generated, and autophagy inhibition was induced for two different time durations. Whereas 2 weeks of Atg7 ablation was sufficient to induce autophagy deficiency, confirmed by the accumulation of p62, ißAtg7KO mice exhibited normal glucose tolerance. In contrast, prolonged autophagy deficiency for 6 weeks resulted in glucose intolerance together with impaired insulin secretion. Direct mRNA sequencing and pathway analysis revealed that the gene set associated with insulin secretion was downregulated only after the 6-week prolonged autophagy inhibition. Furthermore, we identified a novel gene, Sprr1a, which was expressed at more than 50-fold higher levels during both the 2-week and 6-week autophagy inhibition. These findings suggest that autophagy insufficiency cumulatively leads to ß-cell failure after a certain interval, accompanied by stepwise alterations of gene expression patterns.

Higd1a improves respiratory function in the models of mitochondrial disorder.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.

A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2.

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G0/G1 switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FOF1-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

Conversion of pancreatic α cells into insulin-producing cells modulated by ß-cell insufficiency and supplemental insulin administration.

The emergence of bihormonal (BH) cells expressing insulin and glucagon has been reported under diabetic conditions in humans and mice. Whereas lineage tracing studies demonstrated that glucagon-producing α cells can be reprogrammed into BH cells, the underlying dynamics of the conversion process remain poorly understood. In the present study, we investigated the identities of pancreatic endocrine cells by genetic lineage tracing under diabetic conditions. When ß-cell ablation was induced by alloxan (ALX), a time-dependent increase in BH cells was subsequently observed. Lineage tracing experiments demonstrated that BH cells originate from α cells, but not from ß cells, in ALX-induced diabetic mice. Notably, supplemental insulin administration into diabetic mice resulted in a significant increase in α-cell-derived insulin-producing cells that did not express glucagon. Furthermore, lineage tracing in Ins2Akita diabetic mice demonstrated a significant induction of α-to-ß conversion. Thus, adult α cells have plasticity, which enables them to be reprogrammed into insulin-producing cells under diabetic conditions, and this can be modulated by supplemental insulin administration.

Increased risk of cardiovascular mortality by strict glycemic control (pre-procedural HbA1c < 6.5%) in Japanese medically-treated diabetic patients following percutaneous coronary intervention: a 10-year follow-up study.

BACKGROUND: In the secondary prevention of cardiovascular (CV) disease in patients with diabetes, an optimal level of HbA1c, the most widely-used glycemic control indicator, for favorable clinical consequences still remains to be established. This study assessed the association between preprocedural HbA1c level and CV mortality in Japanese diabetic patients undergoing percutaneous coronary intervention (PCI). METHODS: This is a retrospective observational study using a single-center prospective PCI database involving consecutive 4542 patients who underwent PCI between 2000 and 2016. Patients with any antidiabetic medication including insulin at PCI were included in the analysis (n = 1328). We divided the patients into 5 and 2 groups according to HbA1c level; HbA1c: < 6.5% (n = 267), 6.5-7.0% (n = 268), 7.0-7.5% (n = 262), 7.5-8.5% (n = 287) and ≥ 8.5% (n = 244), and 7.0% > and ≤ 7.0%, respectively. The primary outcome was CV mortality including sudden death. The median follow-up duration was 6.2 years. RESULTS: In the follow-up period, CV and sudden death occurred in 81 and 23 patients, respectively. While unadjusted Kaplan-Meier analysis showed no difference in cumulative CV mortality rate between patients binarized by preprocedural HbA1c 7.0%, analysis of the 5 groups of HbA1c showed significantly higher cumulative CV death in patients with HbA1c < 6.5% compared with those with 7.0-7.5% (P = 0.042). Multivariate Cox hazard analysis revealed a U-shaped relationship between preprocedural HbA1c level and risk of CV death, and the lowest risk was in the HbA1c 7.0-7.5% group (Hazard ratio of HbA1c < 6.5% compared to 7.0-7.5%: 2.97, 95% confidence interval: 1.33-7.25, P = 0.007). Similarly, univariate analysis revealed the lowest risk of sudden death was in the HbA1c 7.0-7.5% group. CONCLUSION: The findings indicate an increased risk of CV mortality by strict glycemic control (HbA1c < 6.5%) in the secondary prevention of CV disease in Japanese patients with medically-treated diabetes. Trial registration This study reports the retrospective analysis of a prospective registry database of patients who underwent PCI at Juntendo University Hospital, Tokyo, Japan (Juntendo Physicians' Alliance for Clinical Trials, J-PACT), which is publicly registered (University Medical Information Network Japan-Clinical Trials Registry UMIN-CTR 000035587).

Rubicon in pancreatic beta cells plays a limited role in maintaining glucose homeostasis following increased insulin resistance.

Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase (PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in ßTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (ßKO) mice, by administering tamoxifen to Rubiconflox/flox:MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon ßKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.

Establishment of a system for screening autophagic flux regulators using a modified fluorescent reporter and CRISPR/Cas9.

Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.

Defective autophagy in vascular smooth muscle cells enhances atherosclerotic plaque instability.

Autophagy is considered as an evolutionarily conserved cellular catabolic process. Defective autophagy has been implicated in various human diseases, including cardiovascular diseases. Recently, we and others demonstrated that defective autophagy in vascular smooth muscle cells (SMCs) promotes the progression of atherosclerosis. In this study, we investigated the role of autophagy in SMCs on plaque instability in vivo. We generated mice with a defect atg7in which is an essential gene for autophagy, in SMCs by crossing Atg7f/f mice with transgelin (Tagln) Cre+/0 mice (Atg7cKO). Then, Atg7cKO and apolipoprotein E (apoe)-deficient (apoeKO) mice were crossed to generate Atg7cKO:apoeKO mice. To generate a mouse model of plaque instability, we conducted to form a tandem stenosis in the carotid artery of Atg7cKO:apoeKO mice and their controls (apoeKO mice) at the age of 10 weeks. At 5 weeks after surgery, the percentage of cross-sectional stenosis area in the operated common carotid artery of Atg7cKO:apoeKO mice was significantly higher than that in apoeKO mice. In addition, thrombus, which was not observed in apoeKO mice, was frequently found in Atg7cKO:apoeKO mice. Furthermore, the number of Berlin blue staining-positive areas, which indicated intraplaque hemorrhage, was significantly higher in Atg7cKO:apoeKO mice than in control apoeKO mice. Taken together, our data suggest that defective autophagy in SMCs enhances plaque instability and the risk of plaque rupture.

Heterogeneity of autophagic status in pancreatic ß cells under metabolic stress.

Autophagy in ß cells has been demonstrated to play a pivotal role in cellular homeostasis and the progression of glucose intolerance. Although autophagic activity is affected by metabolic stress both in vivo and in vitro, it remains unclear as to what extent the autophagic status in each ß cell is different from its neighboring cells. To address this question, GFP-LC3 reporter mice, which can visualize the autophagic status of each ß cell as green-fluorescent puncta, were crossed with obese diabetic db/db mice. Imaging of green-fluorescent puncta in the islets of GFP-LC3 mice revealed that ß cells are a heterogeneous population, as the density of GFP-LC3 puncta in each cell was variable. Furthermore, the variability was greater in GFP-LC3; db/db mice than in non-diabetic GFP-LC3; db/+ mice. Furthermore, when GFP-LC3 mice were treated with a low dose of S961, which antagonizes insulin signaling without inducing overt hyperglycemia, the number of ß cells with a high density of GFP puncta was increased, suggesting that insulin resistance affects autophagic status independently of glucose profiles. These results suggest that pancreatic ß cells under metabolic stress are heterogeneous regarding their autophagic status, which provides insights into the cellular dynamics of each ß cell rather than the whole ß-cell population.

Effects of work-matched moderate- and high-intensity warm-up on power output during 2-min supramaximal cycling.

We tested the hypothesis that compared with a moderate-intensity warm-up, a work-matched high-intensity warm-up improves final-sprint power output during the last 30 s of a 120-s supramaximal exercise that mimics the final sprint during events such as the 800-m run, 1,500-m speed skate, or Keirin (cycling race). Nine active young males performed a 120-s supramaximal cycling exercise consisting of 90 s of constant-workload cycling at a workload that corresponds to 110% peak oxygen uptake (VO2peak) followed by 30 s of maximal cycling. This exercise was preceded by 1) no warm-up (control), 2) a 10-min cycling warm-up at a workload of 40% VO2peak (moderate-intensity), or 3) a 5-min cycling warm-up at a workload of 80% VO2peak (high-intensity). Total work was matched between the two warm-up conditions. Both warm-ups increased 5-s peak (observed within 10 s at the beginning of maximal cycling) and 30-s mean power output during the final 30-s maximal cycling compared to no warm-up. Moreover, the high-intensity warm-up provided a greater peak (577±169 vs. 541±175 W, P=0.01) but not mean (482±109 vs. 470±135W, P=1.00) power output than the moderate-intensity warm-up. Both VO2 during the 90-s constant workload cycling and the post-warm-up blood lactate concentration were higher following the high-intensity than moderate-intensity warm-up (all P≤0.05). We show that work-matched moderate- (~40% VO2peak) and high- (~80% VO2peak) intensity warm-ups both improve final sprint (~30 s) performance during the late stage of a 120-s supramaximal exercise bout, and that a high-intensity warm-up provides greater improvement of short-duration (<10 s) maximal sprinting performance.