Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet ß cells in various mouse models of diabetes with distinct age of initiation.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the ß cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet ß cells. Cre-LoxP systems were used to generate islet cell type-specific (α, ß, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto ß-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled ß cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of ß cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. ß cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into ß cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of ß cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing ß cells could also re-trans-differentiate into insulin-producing ß cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of ß cells. Our findings shed light on how islet ß cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants.

IMPORTANCE: A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

Restored UBE2C expression in islets promotes ß-cell regeneration in mice by ubiquitinating PER1.

Insulin deficiency may be due to the reduced proliferation capacity of islet ß-cell, contributing to the onset of diabetes. It is therefore imperative to investigate the mechanism of the ß-cell regeneration in the islets. NKX6.1, one of the critical ß-cell transcription factors, is a pivotal element in ß-cell proliferation. The ubiquitin-binding enzyme 2C (UBE2C) was previously reported as one of the downstream molecules of NKX6.1 though the exact function and mechanism of UBE2C in ß-cell remain to be elucidated. Here, we determined a subpopulation of islet ß-cells highly expressing UBE2C, which proliferate actively. We also discovered that ß-cell compensatory proliferation was induced by UBE2C via the cell cycle renewal pathway in weaning and high-fat diet (HFD)-fed mice. Moreover, the reduction of ß-cell proliferation led to insulin deficiency in ßUbe2cKO mice and, therefore, developed type 2 diabetes. UBE2C was found to regulate PER1 degradation through the ubiquitin-proteasome pathway via its association with a ubiquitin ligase, CUL1. PER1 inhibition rescues UBE2C knockout-induced ß-cell growth inhibition both in vivo and in vitro. Notably, overexpression of UBE2C via lentiviral transduction in pancreatic islets was able to relaunch ß-cell proliferation in STZ-induced diabetic mice and therefore partially alleviated hyperglycaemia and glucose intolerance. This study indicates that UBE2C positively regulates ß-cell proliferation by promoting ubiquitination and degradation of the biological clock suppressor PER1. The beneficial effect of UBE2C on islet ß-cell regeneration suggests a promising application in treating diabetic patients with ß-cell deficiency.

A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite.

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 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 are 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 RNAs 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 RNA3, 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 fungal satellite.

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

AIMS/HYPOTHESIS: 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). METHODS: 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. RESULTS: 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 Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. CONCLUSIONS/INTERPRETATION: We 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. DATA AVAILABILITY: The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

The reversible effects of free fatty acids on sulfonylurea-stimulated insulin secretion are related to the expression and dynamin-mediated endocytosis of KATP channels in pancreatic ß cells.

Objective: Lipotoxicity-induced pancreatic ß cell-dysfunction results in decreased insulin secretion in response to multiple stimulus. In this study, we investigated the reversible effects of palmitate (PA) or oleate (OA) on insulin secretion and the relationship with pancreatic ß-cell ATP-sensitive potassium (KATP) channels. Methods: MIN6 cells were treated with PA and OA for 48 h and then washed out for 24 h to determine the changes in expression and endocytosis of the KATP channels and glucose-stimulated insulin secretion (GSIS) and sulfonylurea-stimulated insulin secretion (SU-SIS). Results: MIN6 cells exposed to PA or OA showed both impaired GSIS and SU-SIS; the former was not restorable, while the latter was reversible with washout of PA or OA. Decreased expressions of both total and surface Kir6.2 and SUR1 and endocytosis of KATP channels were observed, which were also recoverable after washout. When MIN6 cells exposed to free fatty acids (FFAs) were cotreated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or dynasore, we found that endocytosis of KATP channels did not change significantly by AICAR but was almost completely blocked by dynasore. Meanwhile, the inhibition of endocytosis of KATP channels after washout could be activated by PIP2. The recovery of SU-SIS after washout was significantly weakened by PIP2, but the decrease of SU-SIS induced by FFAs was not alleviated by dynasore. Conclusions: FFAs can cause reversible impairment of SU-SIS on pancreatic ß cells. The reversibility of the effects is partial because of the changes of expression and endocytosis of Kir6.2 and SUR1 which was mediated by dynamin.

Amplicon Sequencing Reveals Novel Fungal Species Responsible for a Controversial Tea Disease.

Amplicon sequencing is a powerful tool for analyzing the fungal composition inside plants, whereas its application for the identification of etiology for plant diseases remains undetermined. Here, we utilize this strategy to clarify the etiology responsible for tea leaf brown-black spot disease (LBSD), a noticeable disease infecting tea plants etiology that remains controversial. Based on the ITS-based amplicon sequencing analysis, Didymella species were identified as separate from Pestalotiopsis spp. and Cercospora sp., which are concluded as the etiological agents. This was further confirmed by the fungal isolation and their specific pathogenicity on diverse tea varieties. Based on the morphologies and phylogenetic analysis constructed with multi-loci (ITS, LSU, tub2, and rpb2), two novel Didymella species-tentatively named D. theae and D. theifolia as reference to their host plants-were proposed and characterized. Here, we present an integrated approach of ITS-based amplicon sequencing in combination with fungal isolation and fulfillment of Koch's postulates for etiological identification of tea plant disease, revealing new etiology for LBSD. This contributes useful information for further etiological identification of plant disease based on amplicon sequencing, as well as understanding, prevention, and management of this economically important disease.

Phosphoproteome reveals molecular mechanisms of aberrant rhythm in neurotransmitter-mediated islet hormone secretion in diabetic mice.

BACKGROUND: Acetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear. METHODS AND RESULTS: Immunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase-specific siRNAs in different islet cell lines (αTC1-6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co-staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb). CONCLUSIONS: Abnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.

The common rs13266634 C > T variant in SLC30A8 contributes to the heterogeneity of phenotype and clinical features of both type 1 and type 2 diabetic subtypes.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Differences of Circulating CD25hi Bregs and Their Correlations with CD4 Effector and Regulatory T Cells in Autoantibody-Positive T1D Compared with Age-Matched Healthy Individuals.

Circulating CD25hi B cells, a subset of regulatory B cells in humans, are closely related to inflammation and autoimmune diseases. This study is aimed at investigating the alternation of CD25hi Bregs and their correlation with CD4 effector and regulatory T cells in T1D individuals. We included 68 autoantibody-positive T1D and 68 age-matched healthy individuals with peripheral blood mononuclear cells (PBMCs) and assessed them with CD25hi Bregs and CD4 effector or regulatory T cells by flow cytometry. Here, we demonstrate that the frequency of CD25hi Bregs was significantly decreased in T1D subjects (P = 0.0016), but they were not affected by disease status (age at T1D diagnosis or duration) or T1D risk loci (rs2104286 or rs12251307) in IL2RA (all P > 0.05). Moreover, higher IgD (P = 0.043) and lower CD27 (P = 0.0003) expression was observed in CD25hi Bregs of T1D individuals, but not the expression of IgM, CD24, or CD38 (all P > 0.05). Although there was no correlation between CD25hi Bregs and CD4 effector T cell subsets in either T1D or healthy individuals (all P > 0.05), we found a positive correlation between CD25hi Bregs and CD4 Tregs in healthy controls (Sp. r = 0.3544, P = 0.0249), which disappeared in T1D subjects (Sp. r = 0.137, P = 0.401). In conclusion, our results suggest that decreased CD25hi Bregs and alternation of their phenotypes are features of T1D regardless of disease duration and T1D genetic risk loci, and an impaired balance between CD25hi Bregs and CD4 Tregs might contribute to the pathogenesis of T1D.

Circulating microRNA­135a­3p in serum extracellular vesicles as a potential biological marker of non­alcoholic fatty liver disease.

Non­alcoholic fatty liver disease (NAFLD) is a widespread threat to human health. However, the present screening methods for NAFLD are time­consuming 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 12­week high­fat 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 transcription­quantitative 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 miR­135a­3p, miR­129b­5p and miR­504­3p, and an increase in miR­122­5p expression levels in circulating EVs in mice treated with HFD and patients with NAFLD. There were also similar miR­135a­3p and miR­122­5p expression patterns in the serum. ROC analysis demonstrated that miR­135a­3p in circulating EVs was highly accurate in diagnosing NAFLD, with the area under the curve value being 0.849 (95% CI, 0.777­0.921; P<0.0001). Bioinformatics analysis indicated that dysregulated miR­135a­3p was associated with 'platelet­derived growth factor receptor signaling pathway' and 'AMP­activated protein kinase signaling pathway'. In summary, circulating miR­135a­3p in EVs may serve as a potential non­invasive biomarker to diagnose NAFLD. This miRNA was a more sensitive and specific biological marker for NAFLD compared with ALT.

Temporal metabolic and transcriptomic characteristics crossing islets and liver reveal dynamic pathophysiology in diet-induced diabetes.

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, ß, 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 ß-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.

CHL1 promotes insulin secretion and negatively regulates the proliferation of pancreatic ß cells.

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 ß cells of patients with type 2 diabetes (T2DM). However, the detailed role of CHL1 in ß 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 granules <50 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 ß 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 ß cell compensatory hyperplasia in obesity-induced pre-diabetes.

The associations between three genome-wide risk variants for serum C-peptide of T1D and autoantibody-positive T1D risk, and clinical characteristics in Chinese population.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Altered expression of glucose metabolism associated genes in a tacrolimus­induced post­transplantation diabetes mellitus in rat model.

Post­transplantation 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 Tac­induced PTDM, the involvement of insulin resistance in PTDM remains unknown. In the present study, expression levels of adipocytokines, glucose metabolism associated genes and peroxisome proliferator­activated receptor (PPAR)­Î³ 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 PPAR­Î³ activator. Adipose, muscle and liver tissues were obtained on day 15 after induction and the results demonstrated that expression levels of adipocytokines, PPAR­Î³ 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 PPAR­Î³ signaling may serve important roles in the pathogenesis of Tac­induced PTDM, which may provide a promising application in the treatment of PTDM in the future.

Hepatocytes derived extracellular vesicles from high-fat diet induced obese mice modulate genes expression and proliferation of islet ß cells.

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 ß cells during obesity. Compared with standard chow diet (CD), hepatocellular EVs derived from high-fat diet (HFD) induced obese mice promoted proliferation of ß 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.

Aberrant activation of Notch-1 signaling inhibits podocyte restoration after islet transplantation in a rat model of diabetic nephropathy.

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.

Reversal of Early Diabetic Nephropathy by Islet Transplantation under the Kidney Capsule in a Rat Model.

Objective. Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus, and insulin therapy has many side effects in the treatment of DN. Islet transplantation has emerged as a promising therapy for diabetic patients. This study was established to investigate its advantageous effects in a rat model of early DN. Methods. Streptozotocin was administered to the rats to induce diabetes. Twelve weeks later, the diabetic rats were divided into 3 groups: the islet-transplanted group (IT group), the insulin-treated group (IN group), and the untreated group (DN group). Renal injury and kidney structure were assessed by urinalysis and transmission electron microscopy (TEM) detection. Immunohistochemical staining and western blotting were performed to assess renal fibrosis levels. Results. The early DN features were reversed and the glomerular filtration barrier and basement membrane structures were improved at 4 weeks after islet transplantation. The urine microalbumin-to-creatinine ratio (ACR), protein-to-creatinine ratio, and mean thickness of the glomerular basement membrane (GBM) were significantly decreased in the IT group. The expression of renal fibrotic factors was also significantly decreased. Conclusions. These data suggest that early DN can be reversed after islet transplantation, and they may facilitate the development of a clinical therapeutic strategy for human diabetes mellitus.

Combined Microencapsulated Islet Transplantation and Revascularization of Aortorenal Bypass in a Diabetic Nephropathy Rat Model.

OBJECTIVE: Revascularization of aortorenal bypass is a preferred technique for renal artery stenosis (RAS) in diabetic nephropathy (DN) patients. Restenosis of graft vessels also should be considered in patients lacking good control of blood glucose. In this study, we explored a combined strategy to prevent the recurrence of RAS in the DN rat model. METHODS: A model of DN was established by intraperitoneal injection of streptozotocin. Rats were divided into 4 groups: SR group, MIT group, Com group, and the untreated group. The levels of blood glucose and urine protein were measured, and changes in renal pathology were observed. The expression of monocyte chemoattractant protein-1 (MCP-1) in graft vessels was assessed by immunohistochemical staining. Histopathological staining was performed to assess the pathological changes of glomeruli and tubules. RESULTS: The levels of urine protein and the expression of MCP-1 in graft vessels were decreased after islet transplantation. The injury of glomerular basement membrane and podocytes was significantly ameliorated. CONCLUSIONS: The combined strategy of revascularization and microencapsulated islet transplantation had multiple protective effects on diabetic nephropathy, including preventing atherosclerosis in the graft vessels and alleviating injury to the glomerular filtration barrier. This combined strategy may be helpful for DN patients with RAS.

Lycopene Ameliorates Transplant Arteriosclerosis in Vascular Allograft Transplantation by Regulating the NO/cGMP Pathways and Rho-Associated Kinases Expression.

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.