Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Reversal of high-glucose-induced transcriptional and epigenetic memories through NRF2 pathway activation.

Diabetes complications such as nephropathy, retinopathy, or cardiovascular disease arise from vascular dysfunction. In this context, it has been observed that past hyperglycemic events can induce long-lasting alterations, a phenomenon termed "metabolic memory." In this study, we evaluated the genome-wide gene expression and chromatin accessibility alterations caused by transient high-glucose exposure in human endothelial cells (ECs) in vitro. We found that cells exposed to high glucose exhibited substantial gene expression changes in pathways known to be impaired in diabetes, many of which persist after glucose normalization. Chromatin accessibility analysis also revealed that transient hyperglycemia induces persistent alterations, mainly in non-promoter regions identified as enhancers with neighboring genes showing lasting alterations. Notably, activation of the NRF2 pathway through NRF2 overexpression or supplementation with the plant-derived compound sulforaphane, effectively reverses the glucose-induced transcriptional and chromatin accessibility memories in ECs. These findings underscore the enduring impact of transient hyperglycemia on ECs' transcriptomic and chromatin accessibility profiles, emphasizing the potential utility of pharmacological NRF2 pathway activation in mitigating and reversing the high-glucose-induced transcriptional and epigenetic alterations.

Contributions of elevated CRP, hyperglycaemia, and type 2 diabetes to cardiovascular risk in the general population: observational and Mendelian randomization studies.

OBJECTIVE: To investigate the contributions of low-grade inflammation measured by C-reactive protein (CRP), hyperglycaemia, and type 2 diabetes to risk of ischemic heart disease (IHD) and cardiovascular disease (CVD) death in the general population, and whether hyperglycaemia and high CRP are causally related. RESEARCH DESIGN AND METHODS: Observational and bidirectional, one-sample Mendelian randomization (MR) analyses in 112,815 individuals from the Copenhagen General Population Study and the Copenhagen City Heart Study, and bidirectional, two-sample MR with summary level data from two publicly available consortia, CHARGE and MAGIC. RESULTS: Observationally, higher plasma CRP was associated with stepwise higher risk of IHD and CVD death, with hazard ratios and 95% confidence intervals (95%CI) of 1.50 (1.38, 1.62) and 2.44 (1.93, 3.10) in individuals with the 20% highest CRP concentrations. The corresponding hazard ratios for elevated plasma glucose were 1.10 (1.02, 1.18) and 1.22 (1.01, 1.49), respectively. Cumulative incidences of IHD and CVD death were 365% and 592% higher, respectively, in individuals with both type 2 diabetes and plasma CRP ≥ 2 mg/L compared to individuals without either. Plasma CRP and glucose were observationally associated (ß-coefficient: 0.02 (0.02, 0.03), p = 3 × 10- 20); however, one- and two-sample MR did not support a causal effect of CRP on glucose (-0.04 (-0.12, 0.32) and - 0.03 (-0.13, 0.06)), nor of glucose on CRP (-0.01 (-0.08, 0.07) and - 0.00 (-0.14, 0.13)). CONCLUSIONS: Elevated concentrations of plasma CRP and glucose are predictors of IHD and CVD death in the general population. We found no genetic association between CRP and glucose, or vice versa, suggesting that lowering glucose pharmacologically does not have a direct effect on low-grade inflammation.

Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics.

This study aims to elucidate the roles of Phosphoglycerate Mutase Family Member 5 (Pgam5) and Prohibitin 2 (Phb2) in the context of hyperglycemia-induced myocardial dysfunction, a critical aspect of diabetic cardiomyopathy. The research employed primary cardiomyocytes, which were then subjected to hyperglycemia treatment to mimic diabetic conditions. We used siRNA transfection to knock down Pgam5 and overexpressed Phb2 using adenovirus transfection to assess their individual and combined effects on cardiomyocyte health. Mitochondrial function was evaluated through measurements of mitochondrial membrane potential using the JC-1 probe, and levels of mitochondrial reactive oxygen species (ROS) were assessed. Additionally, the study involved qPCR analysis to quantify the transcriptional changes in genes related to mitochondrial fission and mitophagy. Our findings indicate that hyperglycemia significantly reduces cardiomyocyte viability and impairs mitochondrial function, as evidenced by decreased mitochondrial membrane potential and increased ROS levels. Pgam5 knockdown was observed to mitigate these adverse effects, preserving mitochondrial function and cardiomyocyte viability. On the molecular level, Pgam5 was found to regulate genes associated with mitochondrial fission (such as Drp1, Mff, and Fis1) and mitophagy (including Parkin, Bnip3, and Fundc1). Furthermore, overexpression of Phb2 countered the hyperglycemia-induced mitochondrial dysfunction and normalized the levels of key mitochondrial antioxidant enzymes. The combined data suggest a protective role for both Pgam5 knockdown and Phb2 overexpression against hyperglycemia-induced cellular and mitochondrial damage. The study elucidates the critical roles of Pgam5 and Phb2 in regulating mitochondrial dynamics in the setting of hyperglycemia-induced myocardial dysfunction. By modulating mitochondrial fission and mitophagy, Pgam5 and Phb2 emerge as key players in preserving mitochondrial integrity and cardiomyocyte health under diabetic conditions. These findings contribute significantly to our understanding of the molecular mechanisms underlying diabetic cardiomyopathy and suggest potential therapeutic targets for mitigating myocardial dysfunction in diabetes.

Rephrasing the 'David-Goliath' story in the field of diabetes.

Diabetes Mellitus has become a serious threat to public health. This non-communicable disease is spreading like wildfire to shape in the form of a global pandemic. It affects several organs during silent progression in the human body. The pathophysiological fallouts associate dysregulation of numerous cellular pathways. MicroRNAs have emerged as potent gene expression regulators by post-transcriptional mechanisms in the last two decades or so. Many microRNAs display differential expression patterns under hyperglycemia affecting coupled cellular signaling cascades. The present article attempts to unfold the involvement of microRNAs as biomarkers in diabetic conditions in current scenarios identifying their therapeutic significance.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium.

BACKGROUND: Histone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium. METHODS: We studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2-) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice. RESULTS: Incubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2- generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice. CONCLUSIONS: EZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.

Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia.

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague-Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current-voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

RNA-seq analysis-based study on the effects of gestational diabetes mellitus on macrosomia.

Background: Both the mother and the infant are negatively impacted by macrosomia. Macrosomia is three times as common in hyperglycemic mothers as in normal mothers. This study sought to determine why hyperglycemic mothers experienced higher macrosomia. Methods: Hematoxylin and Eosin staining was used to detect the placental structure of normal mother(NN), mothers who gave birth to macrosomia(NM), and mothers who gave birth to macrosomia and had hyperglycemia (DM). The gene expressions of different groups were detected by RNA-seq. The differentially expressed genes (DEGs) were screened with DESeq2 R software and verified by qRT-PCR. The STRING database was used to build protein-protein interaction networks of DEGs. The Cytoscape was used to screen the Hub genes of the different group. Results: The NN group's placental weight differed significantly from that of the other groups. The structure of NN group's placenta is different from that of the other group, too. 614 and 3207 DEGs of NM and DM, respectively, were examined in comparison to the NN group. Additionally, 394 DEGs of DM were examined in comparison to NM. qRT-PCR verified the results of RNA-seq. Nucleolar stress appears to be an important factor in macrosomia, according on the results of KEGG and GO analyses. The results revealed 74 overlapped DEGs that acted as links between hyperglycemia and macrosomia, and 10 of these, known as Hub genes, were key players in this process. Additionally, this analysis believes that due of their close connections, non-overlapping Hubs shouldn't be discounted. Conclusion: In diabetic mother, ten Hub genes (RPL36, RPS29, RPL8 and so on) are key factors in the increased macrosomia in hyperglycemia. Hyperglycemia and macrosomia are linked by 74 overlapping DEGs. Additionally, this approach contends that non-overlapping Hubs shouldn't be ignored because of their tight relationships.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result in end-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Sex-Specific Alterations in Placental Proteomics Induced by Intrauterine Hyperglycemia.

Gestational diabetes mellitus (GDM) with intrauterine hyperglycemia induces a series of changes in the placenta, which have adverse effects on both the mother and the fetus. The aim of this study was to investigate the changes in the placenta in GDM and its gender differences. In this study, we established an intrauterine hyperglycemia model using ICR mice. We collected placental specimens from mice before birth for histological observation, along with tandem mass tag (TMT)-labeled proteomic analysis, which was stratified by sex. When the analysis was not segregated by sex, the GDM group showed 208 upregulated and 225 downregulated proteins in the placenta, primarily within the extracellular matrix and mitochondria. Altered biological processes included cholesterol metabolism and oxidative stress responses. After stratification by sex, the male subgroup showed a heightened tendency for immune-related pathway alterations, whereas the female subgroup manifested changes in branched-chain amino acid metabolism. Our study suggests that the observed sex differences in placental protein expression may explain the differential impact of GDM on offspring.

Appropriate glycemic management protects the germline but not the uterine environment in hyperglycemia.

Emerging evidence indicates that parental diseases can impact the health of subsequent generations through epigenetic inheritance. Recently, it was shown that maternal diabetes alters the metaphase II oocyte transcriptome, causing metabolic dysfunction in offspring. However, type 1 diabetes (T1D) mouse models frequently utilized in previous studies may be subject to several confounding factors due to severe hyperglycemia. This limits clinical translatability given improvements in glycemic control for T1D subjects. Here, we optimize a T1D mouse model to investigate the effects of appropriately managed maternal glycemic levels on oocytes and intrauterine development. We show that diabetic mice with appropriate glycemic control exhibit better long-term health, including maintenance of the oocyte transcriptome and chromatin accessibility. We further show that human oocytes undergoing in vitro maturation challenged with mildly increased levels of glucose, reflecting appropriate glycemic management, also retain their transcriptome. However, fetal growth and placental function are affected in mice despite appropriate glycemic control, suggesting the uterine environment rather than the germline as a pathological factor in developmental programming in appropriately managed diabetes.

Genetic Determinants of Thiazide-Induced Hyperuricemia, Hyperglycemia, and Urinary Electrolyte Disturbances - A Genome-Wide Evaluation of the UK Biobank.

Thiazide diuretics, widely used in hypertension, cause a variety of adverse reactions, including hyperglycemia, hyperuricemia, and electrolyte abnormalities. In this study, we aimed to identify genetic variants that interact with thiazide-use to increase the risk of these adverse reactions. Using UK Biobank data, we first performed genomewide variance quantitative trait locus (vQTL) analysis of ~ 6.2 million SNPs on 95,493 unrelated hypertensive White British participants (24,313 on self-reported bendroflumethiazide treatment at recruitment) for 2 blood (glucose and urate) and 2 urine (potassium and sodium) biomarkers. Second, we conducted direct gene-environment interaction (GEI) tests on the significant (P < 2.5 × 10-9) vQTLs, included a second UK Biobank cohort comprising 13,647 unrelated hypertensive White British participants (3,478 on thiazides other than bendroflumethiazide) and set significance at P = 0.05 divided by the number of vQTL SNPs tested for GEIs. The vQTL analysis identified eight statistically significant SNPs for blood glucose (5 SNPs) and serum urate (3 SNPs), with none being identified for the urinary biomarkers. Two of the SNPs (1 glucose SNP: CDKAL1 intron rs35612982, GEI P = 6.24 × 10-3; and 1 serum urate SNP: SLC2A9 intron rs938564, GEI P = 4.51 × 10-4) demonstrated significant GEI effects in the first, but not the second, cohort. Both genes are biologically plausible candidates, with the SLC2A9-mediated interaction having been previously reported. In conclusion, we used a two-stage approach to detect two biologically plausible genetic loci that can interact with thiazides to increase the risk of thiazide-associated biochemical abnormalities. Understanding how environmental exposures (including medications such as thiazides) and genetics interact, is an important step toward precision medicine and improved patient outcomes.

ROS in diabetic atria regulate SK2 degradation by Atrogin-1 through the NF-κB signaling pathway.

One of the independent risk factors for atrial fibrillation is diabetes mellitus (DM); however, the underlying mechanisms causing atrial fibrillation in DM are unknown. The underlying mechanism of Atrogin-1-mediated SK2 degradation and associated signaling pathways are unclear. The aim of this study was to elucidate the relationship among reactive oxygen species (ROS), the NF-κB signaling pathway, and Atrogin-1 protein expression in the atrial myocardia of DM mice. We found that SK2 expression was downregulated comitant with increased ROS generation and enhanced NF-κB signaling activation in the atrial cardiomyocytes of DM mice. These observations were mimicked by exogenously applicating H2O2 and by high glucose culture conditions in HL-1 cells. Inhibition of ROS production by diphenyleneiodonium chloride or silencing of NF-κB by siRNA decreased the protein expression of NF-κB and Atrogin-1 and increased that of SK2 in HL-1 cells with high glucose culture. Moreover, chromatin immunoprecipitation assay demonstrated that NF-κB/p65 directly binds to the promoter of the FBXO32 gene (encoding Atrogin-1), regulating the FBXO32 transcription. Finally, we evaluated the therapeutic effects of curcumin, known as a NF-κB inhibitor, on Atrogin-1 and SK2 expression in DM mice and confirmed that oral administration of curcumin for 4 weeks significantly suppressed Atrogin-1 expression and protected SK2 expression against hyperglycemia. In summary, the results from this study indicated that the ROS/NF-κB signaling pathway participates in Atrogin-1-mediated SK2 regulation in the atria of streptozotocin-induced DM mice.

Beta cell specific cannabinoid 1 receptor deletion counteracts progression to hyperglycemia in non-obese diabetic mice.

OBJECTIVE: Type 1 diabetes (T1D) occurs because of islet infiltration by autoreactive immune cells leading to destruction of beta cells and it is becoming evident that beta cell dysfunction partakes in this process. We previously reported that genetic deletion and pharmacological antagonism of the cannabinoid 1 receptor (CB1) in mice improves insulin synthesis and secretion, upregulates glucose sensing machinery, favors beta cell survival by reducing apoptosis, and enhances beta cell proliferation. Moreover, beta cell specific deletion of CB1 protected mice fed a high fat high sugar diet against islet inflammation and beta cell dysfunction. Therefore, we hypothesized that it would mitigate the dysfunction of beta cells in the precipitating events leading to T1D. METHODS: We genetically deleted CB1 specifically from beta cells in non-obese diabetic (NOD; NOD RIP Cre+ Cnr1fl/fl) mice. We evaluated female NOD RIP Cre+ Cnr1fl/fl mice and their NOD RIP Cre-Cnr1fl/fl and NOD RIP Cre+ Cnr1Wt/Wt littermates for onset of hyperglycemia over 26 weeks. We also examined islet morphology, islet infiltration by immune cells and beta cell function and proliferation. RESULTS: Beta cell specific deletion of CB1 in NOD mice significantly reduced the incidence of hyperglycemia by preserving beta cell function and mass. Deletion also prevented beta cell apoptosis and aggressive insulitis in NOD RIP Cre+ Cnr1fl/fl mice compared to wild-type littermates. NOD RIP Cre+ Cnr1fl/fl islets maintained normal morphology with no evidence of beta cell dedifferentiation or appearance of extra islet beta cells, indicating that protection from autoimmunity is inherent to genetic deletion of beta cell CB1. Pancreatic lymph node Treg cells were significantly higher in NOD RIP Cre+ Cnr1fl/flvs NOD RIP Cre-Cnr1fl/fl. CONCLUSIONS: Collectively these data demonstrate how protection of beta cells from metabolic stress during the active phase of T1D can ameliorate destructive insulitis and provides evidence for CB1 as a potential pharmacologic target in T1D.

Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

LOX-1 regulation of H-type vascular endothelial cell regeneration in hyperglycemia.

AIMS: Diabetic osteoporosis (DOP) is the most common secondary form of osteoporosis. Diabetes mellitus affects bone metabolism; however, the underlying pathophysiological mechanisms remain unclear. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression is upregulated in conditions characterized by vascular injury, such as atherosclerosis, hypertension, and diabetes. Additionally, Notch, HIF-1α, and VEGF are involved in angiogenesis and bone formation. Therefore, we aimed to investigate the expression of Notch, HIF-1α, and VEGF in the LOX-1 silencing state. METHODS: Rat bone H-type vascular endothelial cells (THVECs) were isolated and cultured in vitro. Cell identification was performed using immunofluorescent co-expression of CD31 and Emcn. Lentiviral silencing vector (LV-LOX-1) targeting LOX-1 was constructed using genetic recombination technology and transfected into the cells. The experimental groups included the following: NC group, HG group, LV-LOX-1 group, LV-CON group, HG + LV-LOX-1 group, HG + LV-CON group, HG + LV-LOX-1 + FLI-06 group, HG + LV-CON + FLI-06 group, HG + LV-LOX-1 + LW6 group, and HG + LV-CON + LW6 group. The levels of LOX-1, Notch, Hif-1α, and VEGF were detected using PCR and WB techniques to investigate whether the expression of LOX-1 under high glucose conditions has a regulatory effect on downstream molecules at the gene and protein levels, as well as the specific molecular mechanisms involved. RESULTS: High glucose (HG) conditions led to a significant increase in LOX-1 expression, leading to inhibition of angiogenesis, whereas silencing LOX-1 can reverse this phenomenon. Further analysis reveals that changes in LOX-1 will promote changes in Notch/HIF-1α and VEGF. Moreover, Notch mediates the activation of HIF-1α and VEGF. CONCLUSIONS: The activation of LOX-1 and the inhibition of Notch/HIF-1α/VEGF in THVECs are the main causes of DOP. These findings contribute to our understanding of the pathogenesis of DOP and offer a novel approach for preventing and treating osteoporosis.