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Background The limited proliferation capacity of β-cells contributes to insulin insufficiency, which promotes the onset of diabetes. β-cells secrete multiple beneficial substances, except for insulin; therefore, the regeneration mechanism of β-cells requires further study. Previously, ubiquitin-binding enzyme 2C (UBE2C) was reported to be downstream of the key β-cell transcription factor NKX6.1, but the specific function and mechanism of UBE2C in pancreatic islets remain unknown. Methods We performed islet perfusion, immunofluorescence staining, and transmission electron microscopy (TEM) to examine the role of UBE2C in mice with a Cre-loxP-constructed β-cell-specific knockout (βUbe2cKO) or overexpression through lentivirus infusion within the pancreatic ducts. Single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq, tandem mass tag (TMT) quantification proteomics, immunoprecipitation, and immunoblotting were conducted to explore the mechanisms by which UBE2C regulates β-cell proliferation. Results We discovered that ubiquitin-conjugating enzyme E2C (UBE2C) was highly expressed in a β-cell subgroup, which exhibited active proliferation capacity. We confirmed that UBE2C promotes β-cell compensatory proliferation by activating cell cycle renewal during a high-fat diet (HFD) and weaning. Consequently, β-cell-specific Ube2c knockout (βUbe2cKO) mice developed typical type 2 diabetes owing to β-cell loss caused by insulin insufficiency. Mechanistically, UBE2C combines with CUL1 to regulate PER1 degradation through ubiquitination. Notably, restored Ube2c expression in islets activated β-cell regeneration in streptozotocin (STZ)-induced diabetic mice, and alleviated diabetic hyperglycaemia and glucose intolerance. Conclusion This study indicates that UBE2C positively regulates β-cell proliferation by promoting ubiquitination and degradation of the biological clock suppressor PER1. The UBE2C-mediated beneficial effects on β-cells suggest a promising application in the treatment of diabetic patients with β-cell deficiency.

Didymella theifoliaspecifically infects some local varieties ofCamellia sinensisin China, representing a unique fungal species, and characterization of the mycoviruses related to this fungal species is attractive. Three double-stranded RNAs (dsRNAs; dsRNAs 1, 2 and 3 with sizes of 6338, 5910 and 727 bp, respectively) were identified in an avirulent strain CJP4-1 ofD. theifoliaexhibiting normal growth and morphologies. Characterization of the dsRNAs 1 and 2 revealed that they are genomic components of a novel botybirnavirus, tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), and encapsidated in isometric virions with a size of ∼39.8 nm in diameter. It is worth noting that dsRNA3 shares no detectable identity with those sequences deposited in NCBI database, while a high identity (36.58% and 40.93%) with the left regions of dsRNAs 1 and 2, but is not encapsidated in DtBRV1 particles, suggesting it is a unique dsRNA unit that is not a DtBRV1 component or a satellite and its taxonomic classification remains unclear. SDS-polyacrylamide gel electrophoresis in combination with peptide mass fingerprint analysis revealed that DtBRV1 capsid protein consisting of polypeptides encoded by the left regions of both genomic components. DtBRV1 is efficiently vertically transmitted through conidia while difficult in horizontal transmission from strain CJP4-1 to other strains. DtBRV1 has no effects on fungal growth and virulence as accessed with the transfectants of virulent strain JYC1-6 ofD. theifoliainfected by DtBRV1. DtBRV1 with specific molecular traits contributes useful information for a better understanding of the mycoviral community.ImportanceTea plants represent an ancient and unique plant species community cultured in China, while the mycoviruses related to the phytopathogenetic fungi infecting tea remain limited. Here, we characterized a novel botybirnavirus (tentatively named Didymella theifolia botybirnavirus1 (DtBRV1), and a specific dsRNA infectingDidymella theifoliaresponsible for a noticeable disease of tea plants. DtBRV1 contains two dsRNAs (1 and 2) encapsidated in isometric virions in size of ∼39.8 nm, while dsRNA3 is not encapsidated in the viral particles although it has a high identity with the mycoviral genomic components. Additionally, DtBRV1 coat proteins are composed of fused proteins encoded by both dsRNA-coding open reading frames most likely after cleave and fuse processing progress, which is striking unlike most mycoviruses. With some specific molecular traits, DtBRV1 and the related specific dsRNA unit expand our understanding of virus diversity, taxonomy, and evolution.

Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to a newly proposed genus “Alphahypovirus” of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining is heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite dsRNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of dsRNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of satellite.IMPORTANCESatellites in association with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Here, a special satellite-like dsRNA (SatL-dsRNA) together with its helper virus, a novel hypovirus from Pestalotiopsis fici, was identified and characterized. This SatL-dsRNA lacks a substantial open reading frame and a poly (A) tail, which is unlike the known satellite dsRNAs of hypoviruses. It is also unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of the SatL-dsRNA, the helper virus genome was significantly down-regulated, suggesting that it negatively regulates the genomic expression of the helper virus. This special SatL-dsRNA has substantial sequence homology with the host viral genome and is not encapsidated in the coat protein of its helper virus, which represents a novel class of satellite-like nucleic acids, and it broadens the definition of satellite.

Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD).Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression.A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease.The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

Recent meta-genome-wide association studies identified several genetic variants associated with beta-cell function in type 1 diabetes (T1D). The aim of this study was to investigate the associations between these variants and T1D risk, C-peptide levels, islet-specific autoantibodies, and lipid levels in Chinese Han population. A total of 1005 unrelated autoantibody-positive T1D cases and 1417 healthy controls were included, which were genotyped for rs559047, rs9260151, and rs3135002. T1D individuals were measured for both C-peptide and lipid levels. Logistic regression models were used to examine these associations. We found that rs3135002 A allele showed a genome-wide significant association with T1D risk (OR = 0.22, 95% CI = 0.17–0.30; P = 7.49 × 10−27), and significant heterogeneity of effect size was observed between early-onset and later-onset T1D subgroups (I2 = 80% and P = 0.026). Rs559047 had a nominal association with fasting C-peptide levels in newly diagnosed T1D individuals (P = 0.036). Moreover, rs3135002 A allele was significantly associated with GADA positivity (OR = 0.52, 95% CI = 0.30–0.91, P = 0.02). In addition, nominal correlations were observed with HDL levels for rs559047 (P = 0.042), while LDL levels for rs9260151 (P = 0.032) in T1D individuals. Our results indicate that there are both similarities and differences for the associations of genetic variants among T1D development, progression, and related autoimmunity, metabolic traits.

T2D and T1D are phenotypically heterogeneous. This study aims to reveal the relationship between the common SLC30A8 rs13266634 variant and subgroups of T2D and T1D and their clinical characteristics.We included 3158 OGTT-based healthy controls, unrelated 1754 T2D, and 1675 autoantibody-positive T1D individuals. The associations between rs13266634 and subtypes of T2D, T1D, autoantibody status and glycemic-related quantitative traits were performed by binary logistic regression analysis under the additive model and multiple linear regression with appropriate adjustment.We found that the T allele of rs13266634 was protectively associated with lean (OR = 0.810, P = 6.91E-04) but not obese T2D with considerable heterogeneity (P = 0.018). This allele also conferred significant protection with T1D of single (OR = 0.847, P = 9.76E-03), but not multi autoantibodies with substantial heterogeneity (P = 0.005). This variant significantly affected OGTT-related insulin release in lean (P = 2.66E-03, 3.88E-03 for CIR and DI, respectively) but not obese healthy individuals. Furthermore, rs13266634 T allele correlated with the risk of ZnT8A (OR = 1.440, P = 3.31E-05) and IA-2A (OR = 1.219, P = 1.32E-03) positivity, with more effect size in children/adolescents compared with adult-onset T1D subtypes.These suggested that the SLC30A8 rs13266634 variant might be put into genetic risk scores to assess the risk of the subtypes of T1D and T2D and their related clinical features.

Non‑alcoholic fatty liver disease (NAFLD) is a widespread threat to human health. However, the present screening methods for NAFLD are time‑consuming or invasive. The present study aimed to assess the potential of microRNAs (miRNAs/miRs) in serum extracellular vesicles (EVs) as a biomarker of NAFLD. C57BL/6J mice were fed either a 12‑week high‑fat diet (HFD) or standard chow to establish NAFLD and control groups, respectively. Serum samples were obtained from the mouse model of NAFLD, as well as 50 patients with NAFLD and 50 healthy individuals, and EVs were extracted and verified. Using reverse transcription‑quantitative PCR, the mRNA expression level of selected miRNAs in the serum and EVs was analyzed. In order to determine the diagnostic value, receiver operating characteristic (ROC) curves were used. The mice treated with HFD showed notable hepatic steatosis and higher concentrations of serum alanine aminotransferase (ALT). There was also a significant decrease in the expression levels of miR‑135a‑3p, miR‑129b‑5p and miR‑504‑3p, and an increase in miR‑122‑5p expression levels in circulating EVs in mice treated with HFD and patients with NAFLD. There were also similar miR‑135a‑3p and miR‑122‑5p expression patterns in the serum. ROC analysis demonstrated that miR‑135a‑3p in circulating EVs was highly accurate in diagnosing NAFLD, with the area under the curve value being 0.849 (95% CI, 0.777‑0.921; P

ObjectiveHyperinsulinemia and insulin resistance are co-existing characteristics of type 2 diabetes, whereas the molecular mechanism underlying this deleterious cycle remains elusive. The temporal transcriptomic landscape of core organs responsible for insulin secretion (islets) and insulin action (liver) could provide new insights.MethodsThe longitudinal profiling of glucose metabolism, insulin sensitivity, islet architecture and secretion were conducted in C57BL/6N mice fed a high-fat diet (HFD) or chow diet for 24 weeks. RNA-sequencing of islets and liver were performed once every 4 weeks. Weighted gene co-expression network analysis and Ingenuity Pathway Analysis were applied to construct networks and evaluate co-ordinated molecular interactions between islets and liver.ResultsMice exhibited progressively deteriorated glucose homeostasis with hyperinsulinemia but impaired first-phase insulin secretion after 4 weeks on HFD. Insulin, glucagon and somatostatin secretion in response to glucose with or without palmitate gradually deteriorated from dysregulation to failure. Systemic insulin resistance developed over 24 weeks with variable time course in tissue-specific insulin action. Our transcriptomic datasets outlined the impact of HFD on dynamics of islet and liver molecular network at different stages. Correlation analyses revealed that both organs jointly programmed β-cell compensatory adaption via cell proliferation at early phase and irreversible islet dysfunction by inappropriate immune response at later stage. Alternations of T cell subpopulations validated the participation of adaptive immune response through priming and amplification phases in diabetic progression.ConclusionOur data provide a comprehensive landscape of crosstalk between islets and liver in diet-induced diabetes, elucidating the development of islet dysfunction and insulin resistance.HighlightsDiet-induced diabetes is featured by transition from islet dysfunction to failureInsulin resistance develops with variable time course in different tissuesDynamics of islet and liver molecular network interplay at different stagesCell proliferation and improper immune reaction mediated interorgan crosstalkAdaptive immune response participated via priming and amplification phases

Cell adhesion molecule L1-like protein (CHL1) is a member of neural recognition molecules of immunoglobulin superfamily primarily expressing in the nervous system. CHL1 regulates neuronal migration, axonal growth, and dendritic projection. Downregulation of CHL1 has been reported in β cells of patients with type 2 diabetes (T2DM). However, the detailed role of CHL1 in β cells has not been characterized. In this study, Real-Time PCR and Western blot were applied to investigate the tissue/cell distribution and expression of CHL1. Gain- or loss-of function studies were conducted in MIN6 cells to determine the effects of CHL1 on cell proliferation, apoptosis, cell cycle, and insulin secretion. Following silencing of CHL1 in MIN6 cells (si-CHL1), insulin secretion and the number of insulin secretary granules50 nm from the cell membrane decreased in response to 20 mM glucose. Besides, silencing of CHL1 induced cell proliferation, reduced apoptosis, and prolonged S phase and shortened G1 phase of the cell cycle, contrary to overexpressing of CHL1. The inhibitor of ERK1/2MAPK eliminated the effect of CHL1 deficiency on the proliferation of MIN6 cells. In addition, high-fat diet could result in increased islet volume and β cell proliferation, decreased CHL1 expression and activation of ERK pathway in mice islets. Consequently, CHL1 expression was decreased in islets of high-fat induced mice, which resulted in cell proliferation via ERK pathway and regulation of the cell cycle through p53 pathway. These mechanisms may contribute to pancreatic β cell compensatory hyperplasia in obesity-induced pre-diabetes.

Signaling abnormalities play important roles during podocyte injury and have been indicated as crucial events for triggering many glomerular diseases. There is emerging evidence demonstrating significant improvements in preventing renal injury and restoring podocytes after islet transplantation. However, whether signaling abnormalities affect the therapeutic efficacy of islet transplantation remain unclear. This study was established to investigate the impact of Notch-1 signaling activation on renal injury and podocyte restoration after islet transplantation. Experiments were performed in vivo and in vitro under conditions of diabetic nephropathy and high-glucose medium, respectively. Podocyte injury in vitro was induced by high-glucose concentration, and expression levels of genes associated with the Notch-1 pathway were also regulated by Jagged-1/FC and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT). Podocytes were co-cultured with islets to investigate the protective effect of islets in high-glucose conditions. Histopathological staining and transmission electron microscopy were performed to assess pathological changes in podocytes in glomeruli. The results from this study showed that Notch-1 signaling in podocytes was significantly decreased by functional islet cells in vivo and in vitro. Compared with the co-cultured group and transplanted group, highly activated Notch-1 signaling significantly moderated the effect of islets in affecting podocyte restoration and renal injury. Renal damage and podocyte injury were alleviated after DAPT treatment. Furthermore, the balance between apoptosis and autophagy was diverse under different treatments. All the data in this study showed that highly activated Notch-1 signaling could affect the therapeutic efficacy of islet transplantation on renal injury and podocyte restoration in high-glucose conditions. The balance between apoptosis and autophagy was also closely associated with the degree of podocyte restoration. This finding may suggest that the in vivo microenvironment plays a critical role in podocyte restoration after islet transplantation, which provides a promising and individual assessment and targeting treatment for different diabetic nephropathy patients after islet transplantation into the future.

Post‑transplantation diabetes mellitus (PTDM) is a known side effect in transplant recipients administered with immunosuppressant drugs, such as tacrolimus (Tac). Although injury of islet cells is considered a major reason for Tac‑induced PTDM, the involvement of insulin resistance in PTDM remains unknown. In the present study, expression levels of adipocytokines, glucose metabolism associated genes and peroxisome proliferator‑activated receptor (PPAR)‑γ in adipose, muscular and liver tissues from a rat model induced with Tac (1 mg/kg/day) were examined. Rats developed hyperglycemia and glucose intolerance after 10 days of Tac administration. A subgroup of diabetic rats was further treated with rosiglitazone (4 mg/kg), a PPAR‑γ activator. Adipose, muscle and liver tissues were obtained on day 15 after induction and the results demonstrated that expression levels of adipocytokines, PPAR‑γ and proteins in the insulin associated signaling pathway varied in the different groups. Rosiglitazone administration significantly improved hyperglycemia, glucose intolerance and expression levels of proteins associated with insulin signaling, as well as adipocytokines expression. The results of this study demonstrated that adipocytokines and PPAR‑γ signaling may serve important roles in the pathogenesis of Tac‑induced PTDM, which may provide a promising application in the treatment of PTDM in the future.

Recent meta-genome-wide association studies identified several genetic variants associated with beta-cell function in type 1 diabetes (T1D). The aim of this study was to investigate the associations between these variants and T1D risk, C-peptide levels, islet-specific autoantibodies, and lipid levels in Chinese Han population.A total of 1005 unrelated autoantibody-positive T1D cases and 1417 healthy controls were included, which were genotyped for rs559047, rs9260151, and rs3135002. T1D individuals were measured for both C-peptide and lipid levels. Logistic regression models were used to examine these associations.We found that rs3135002 A allele showed a genome-wide significant association with T1D risk (OR = 0.22, 95% CI = 0.17-0.30; P = 7.49 × 10Our results indicate that there are both similarities and differences for the associations of genetic variants among T1D development, progression, and related autoimmunity, metabolic traits.

Liver secretes proliferative factors participating compensatory hyperplasia of islets during obesity and insulin resistance. Extracellular vesicles (EVs) mediate intercellular communication by delivering inner factors to recipient cells. This study explored the biological effects of hepatocellular EVs on islet β cells during obesity. Compared with standard chow diet (CD), hepatocellular EVs derived from high-fat diet (HFD) induced obese mice promoted proliferation of β cell line-MIN6 cells, but didn't influence their insulin secretion. Microarray analysis found 13 miRNAs with significantly differential expression in hepatocellular EVs between HFD with CD group. Meanwhile, RNA-sequencing detected 80 genes with significantly differential expression in MIN6 cells treated with HFD and CD hepatocellular EVs respectively. Six miRNAs and 11 potential target genes were pre-screened by synthesizing TargetScan prediction and RNA-sequencing results. After miRNA mimic transfection and testing the expressions of target genes and cell vitality, miR-7218-5p was verified to affect MIN6 cell proliferation through targeting CD74 gene. SiRNA transfection and dual luciferase reporter assay further confirmed the binding and regulation of miRNA-7218-5p on CD74. These findings suggest HFD induced obesity could change miRNA profiles in hepatocellular EVs, which modulate expression of multiple genes and proliferation of MIN6 cells and maybe mediate compensatory hyperplasia of islets.

Summary To investigate the molecular mechanisms underlying islet dysfunction and insulin resistance in diet-induced diabetes, we conducted temporal RNA sequencing of tissues responsible for insulin secretion (islets) and action (liver) every 4 weeks in mice on high-fat (HFD) or chow diet for 24 weeks, linking to longitudinal profile of metabolic characteristics. The diverse responses of α, β, and δ cells to glucose and palmitate indicated HFD-induced dynamic deterioration of islet function from dysregulation to failure. Insulin resistance developed with variable time course in different tissues. Weighted gene co-expression network analysis and Ingenuity Pathway Analysis implicated islets and liver jointly programmed β-cell compensatory adaption via cell proliferation at early phase and irreversible islet dysfunction by inappropriate immune response at later stage, and identified interconnected molecules including growth differentiation factor 15. Frequencies of T cell subpopulation showed an early decrement in Tregs followed by increases in Th1 and Th17 cells during progression to diabetes., Graphical abstract, Highlights • Diet-induced diabetes is featured by transition from islet dysfunction to failure • Insulin resistance develops with variable time course in different tissues • Dynamics of islet and liver molecular network interplay at different stages • T-cell-mediated immune response participates via priming and amplification phases, Animal Physiology ; Diabetology ; Transcriptomics

Objective.Transplant arteriosclerosis is considered one of the major factors affecting the survival time of grafts after organ transplantation. In this study, we proposed a hypothesis of whether lycopene can protect grafted vessels through regulating key proteins expression involved in arteriosclerosis.Methods.Allogeneic aortic transplantation was performed using Brow-Norway rats as donors and Lewis rats as recipients. After transplantation, the recipients were divided into two groups: the allograft group and the lycopene group. Negative control rats (isograft group) were also established. Histopathological staining was performed to observe the pathological changes, and the expression levels of Ki-67, caspase-3, Rho-associated kinases, intercellular adhesion molecules (ICAM-1), and eNOS were assessed. Western blotting analysis and real-time PCR were also performed for quantitative analysis.Results.The histopathological staining showed that vascular stenosis and intimal thickening were not evident after lycopene treatment. The Ki-67, ROCK1, ROCK2, and ICAM-1 expression levels were significantly decreased. However, eNOS expression in grafted arteries and plasma cGMP concentration were increased after lycopene treatment.Conclusions.Lycopene could alleviate vascular arteriosclerosis in allograft transplantation via downregulating Rho-associated kinases and regulating key factor expression through the NO/cGMP pathways, which may provide a potentially effective method for transplant arteriosclerosis in clinical organ transplantation.