High Glucose Stress Research Articles

Bruceine A protects against diabetic kidney disease via inhibiting galectin-1

Bruceine A is a natural quassinoid compound extracted from the fruit of the Traditional Chinese Medicine Brucea javanica (L.) Merr. that has various types of various biological activities. However, whether the compound has a protective effect on diabetic kidney disease remains unknown. Galectin-1 is actively involved in a variety of chronic inflammation-relevant human diseases including diabetic kidney disease. Here, we identified Bruceine A as a kidney protective molecule against a model of diabetic kidney disease in db/db mice with potent anti-inflammatory activity both invitro and invivo. Mechanistically, by selectively binding to the conserved carbohydrate-recognition domain of galectin-1 and disrupting the interaction between galectin-1 and the receptor for activated protein C kinase 1, Bruceine A was found to inhibit galectin-1-mediated inflammatory signal transduction under high glucose stress in rat mesangial HBZY-1 cells. Thus, our findings reveal Bruceine A as an unidentified galectin-1 inhibitor affording significant protection against diabetic kidney disease and may provide novel pharmacological therapeutics for the disease.

LXR activation ameliorates high glucose stress-induced aberrant mitochondrial dynamics via downregulation of Calpain1 expression in H9c2 cardiomyoblasts

Liver-X-receptor (LXR) has previously been shown to exert a cardioprotective effect against the development of diabetic cardiomyopathy (DCM) associated with a reduction in mitochondrial dysfunction. However, the underlying mechanism by which LXR activation attenuates the structural and functional mitochondrial impairments caused by high glucose (HG) stress remains unclear. We demonstrate here that LXR activation inhibits HG stress-induced mitochondrial dysfunction and ameliorates aberrant mitochondrial dynamics. Furthermore, LXR activation regulates mitochondrial dynamics by inhibiting HG stress-induced upregulation of Calpain1 expression. These data indicate that amelioration of Calpain1-mediated aberrant mitochondrial dynamics may be at least part of the mechanism underlying the cardioprotective effects of LXR against HG stress. Therefore, LXR is a potentially attractive molecular target for treating cardiac mitochondrial dysfunction in patients with diabetes.

Calcineurin/NFATc2 and PI3K/AKT signaling maintains β-cell identity and function during metabolic and inflammatory stress

SummaryPancreatic islets respond to metabolic and inflammatory stress by producing hormones and other factors that induce adaptive cellular and systemic responses. Here we show that intracellular Ca2+ ([Ca2+]i) and ROS signals generated by high glucose and cytokine-induced ER stress activate calcineurin (CN)/NFATc2 and PI3K/AKT to maintain β-cell identity and function. This was attributed in part by direct induction of the endocrine differentiation gene RFX6 and suppression of several β-cell “disallowed” genes, including MCT1. CN/NFATc2 targeted p300 and HDAC1 to RFX6 and MCT1 promoters to induce and suppress gene transcription, respectively. In contrast, prolonged exposure to stress, hyperstimulated [Ca2+]i, or perturbation of CN/NFATc2 resulted in downregulation of RFX6 and induction of MCT1. These findings reveal that CN/NFATc2 and PI3K/AKT maintain β-cell function during acute stress, but β-cells dedifferentiate to a dysfunctional state upon loss or exhaustion of Ca2+/CN/NFATc2 signaling. They further demonstrate the utility of targeting CN/NFATc2 to restore β-cell function

Adipose-Derived Stem Cells Preincubated with Green Tea EGCG Enhance Pancreatic Tissue Regeneration in Rats with Type 1 Diabetes through ROS/Sirt1 Signaling Regulation.

Type 1 diabetes stem-cell-based therapy is one of the best therapeutic approaches for pancreatic damage treatment due to stem cell tissue regeneration. Epigallocatechin gallate (EGCG) is one of the active components found in green tea. Experimental results suggest that EGCG shows beneficial effects on cell protection. This study explores whether a better pancreatic regeneration therapeutic effect could be found in mesenchymal stem cells pretreated with EGCG compared to stem cells without EGCG pretreatment. A cell model confirmed that adipose-derived stem cells (ADSC) incubated with EGCG increase cell viability under high-glucose (HG) stress. This is due to survival marker p-Akt expression. In an animal model, type 1 diabetes induced the activation of several pathological signals, including islet size reduction, extracellular fibrotic collagen deposition, oxidative stress elevation, survival pathway suppression, apoptosis signaling induction, and Sirt1 antioxidant pathway downregulation. Ordinary ADSC transplantation slightly improved the above pathological signals. Further, EGCG-pretreated ADSC transplantation significantly improved the above pathological conditions. Taken together, EGCG-pretreated ADSCs show clinical potential in the treatment of patients with type 1 diabetes through the regeneration of damaged pancreatic tissues.

Protein kinases Elm1 and Sak1 of Saccharomyces cerevisiae exerted different functions under high-glucose and heat shock stresses.

Phosphorylation catalyzed by protein kinases is the most common and important regulatory pathway in the adaptive physiological responses to the changes in nutrition and environment of yeast. This study focused on the functions of Elm1, Sak1, and Tos3, which are three upstream protein kinases of Snf1 in Saccharomyces cerevisiae, in response to high-glucose and heat shock stresses. Results suggested that changing the gene dosage of ELM1/SAK1/TOS3 had different effects under high-glucose and heat shock stresses. ELM1 and SAK1 overexpressions could enhance the tolerance of S. cerevisiae to high-glucose and heat shock stresses, respectively. Nevertheless, the overexpression of TOS3 decreased the tolerance to high-glucose stress, and a native level of Tos3 was important for the normal adaptation to heat shock condition. The overexpression of ELM1 increased the accumulation of trehalose and ergosterol and altered the composition of fatty acids with altered gene expressions involved in the metabolism of three metabolites. Enhanced resistance to heat shock stress in SAK1 overexpression might be related to the enhanced accumulation of trehalose and ergosterol and upregulated transcription of genes related to the metabolism of trehalose and ergosterol. Furthermore, Elm1 might regulate the metabolism of trehalose, ergosterol, and fatty acids in a Snf1-independent form under high-glucose stress. A Snf1-independent pathway might be involved in the regulation of trehalose metabolism by Sak1 under heat shock condition. However, Sak1 and Snf1 may have an indirect relationship in the regulation of ergosterol synthesis. KEY POINTS: • Altering the gene dosage of ELM1/SAK1/TOS3 had different effects on stress responses • Elm1 regulated high-glucose response in a Snf1-independent manner • Sak1 and Snf1 had an indirect relationship in the regulation of heat shock response.

Differential MicroRNA Expression Pattern in Endothelial Progenitor Cells During Diabetic Retinopathy.

Endothelial progenitor cells (EPCs) are involved in the pathogenesis of microvascular dysfunction in diabetic retinopathy (DR). MicroRNAs (miRNAs) serve as crucial regulators in many biological process and human diseases. Herein, to investigate the expression profile and possible role of miRNAs in EPCs, small RNA sequencing was conducted to identify EPC dysfunction-related miRNAs in DR. A total of 72 miRNAs were differentially expressed in EPCs following high glucose stress. Based on Gene Ontology (GO) analysis, the target genes of differentially expressed miRNAs were targeted to “protein binding,” “cell differentiation,” and “cytoskeleton.” Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that cGMP-PKG signaling pathway was tightly associated with miRNA-mediated EPC function. Furthermore, miR-375–3p was verified to be up-regulated in the clinical samples of DR patients. Inhibition of miR-375–3p protected against hyperglycemic stress- or hypoxic stress-induced EPC injury, which increased the viability, proliferation, migration, and tube formation ability of EPCs and retarded the development of apoptosis. Collectively, this study provides a novel insight into the pathogenesis of EPC dysfunction in DR. miR-375–3p is a potential target for the diagnosis or treatment of DR.

Chronic metabolic and induced stress impacts mesenchymal stromal cell differentiation and modulation of dental origin in-vitro

The impact of induced (smoking) and metabolic stress (diabetes) on dental stem cells with respect to pre-impact consideration on differentiation and bone formation were investigated. The progenitor stem cells isolated from dental pulp, follicle and gingival tissues were phenotyped and subjected to nicotine and high glucose stress mimicking the smoking and diabetic condition in-vitro. The results showed that the cellular viability post treatment with 100 µM nicotine and 10uM glucose was about 86% to 89% respectively in all the three cell types while about 73% in combined nicotine and glucose treatment. No variation in the expression of pro-inflammatory TNF-α, IL-1β and IL-12 in all the three cell types were noticed. The observed viability in nicotine treated cells were due to elevated IL-6, IL-10 while in glucose was due to brain derived neurotropic factor (BDNF). Higher expression of IL-4, IL-6, IL-10, TGF-β and heme oxygenase −1 (HO-1) were found high in both stressors treated cells. Differentiation and mineralization markers Alkaline phosphatase (ALP), Collagenase I (COL1), Osteocalcin, Runt related transcription factor 2 (RUNX2), Osteopontin and Bone sialoprotein were expressed in the dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) at varying levels post nicotine or glucose treatment while not significantly observed in dental follicular stem cells (DFSCs). Therefore, it is evident that the stem cells of varied dental origin responded to the stress are more or less uniform with physiological delay in differentiation into osteoblast. It is evident from the study that, the metabolic or induced stress subverts the process of regenerative healing by mesenchymal stromal cells with their anatomical niche.

Monomethyl branched-chain fatty acids are critical for Caenorhabitis elegans survival in elevated glucose conditions

The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose.

Metabolic Imbalance Effect on Retinal Müller Glial Cells Reprogramming Capacity: Involvement of Histone Deacetylase SIRT6.

Retinal Müller glial cells (MGs) are among the first to demonstrate metabolic changes during retinal disease and are a potential source of regenerative cells. In response to a harmful stimulus, they can dedifferentiate acquiring neural stem cells properties, proliferate and migrate to the damaged retinal layer and differentiate into lost neurons. However, it is not yet known how this reprogramming process is regulated in mammals. Since glucose and oxygen are important regulatory elements that may help directing stem cell fate, we aimed to study the effect of glucose variations and oxidative stress in Müller cells reprogramming capacity and analyze the participation the histone deacetylase SIRT6, as an epigenetic modulator of this process. We found that the combination of high glucose and oxidative stress induced a decrease in the levels of the marker glutamine synthetase, and an increase in the migration capacity of the cells suggesting that these experimental conditions could induce some degree of dedifferentiation and favor the migration ability. High glucose induced an increase in the levels of the pluripotent factor SOX9 and a decrease in SIRT6 levels accompanied by the increase in the acetylation levels of H3K9. Inhibiting SIRT6 expression by siRNA rendered an increase in SOX9 levels. We also determined SOX9 levels in retinas from mice with a conditional deletion of SIRT6 in the CNS. To further understand the mechanisms that regulate MGs response under metabolic impaired conditions, we evaluated the gene expression profile and performed Gene Ontology enrichment analysis of Müller cells from a murine model of Diabetes. We found several differentially expressed genes and observed that the transcriptomic change involved the enrichment of genes associated with glucose metabolism, cell migration, development and pluripotency. We found that many functional categories affected in cells of diabetic animals were directly related to SIRT6 function. Transcription factors enrichment analysis allowed us to predict several factors, including SOX9, that may be involved in the modulation of the differential expression program observed in diabetic MGs. Our results underline the heterogeneity of Müller cells response and the challenge that the study of metabolic impairment in vivo represents.

Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK−/− C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK−/− cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

Effect of Metformin on Lactate Metabolism in Normal Hepatocytes under High Glucose Stress in Vitro

Objective: To study the effect of metformin on lactate metabolism in hepatocytes in vitro under high glucose stress. Method: LO2 hepatocytes was cultured in vitro, hepatocytes were randomly divided into blank control group, 25 mmol/L glucose solution, 27 mmol/L glucose solution, 29 mmol/L glucose solution, 31 mmol/L glucose solution, 33 mmol/L glucose solution, 35 mmol/L glucose solution treatment group, after determining the optimal concentration as 31 mmol/L, use 30 mmol/L metformin solution, and then divided into blank control group, normal hepatocytes + the optimal concentration of glucose solution, normal hepatocytes + metformin solution , normal hepatocytes+. The optimal concentration of glucose solution normal hepatocytes + metformin solution, calculate the number of hepatocytes on cell count plate respectively in the 12 h, 24 h, 48 h, and use the lactic acid kit to determine the lactic acid value of the cell culture medium of normal liver cells + optimal concentration glucose solution and normal liver cells + optimal concentration glucose solution + metformin solution at 12 h, 24 h, and 48 h, respectively. Results: There was no significant change in the lactic acid concentration but significant increase in the number of surviving hepatocytes in the high-glycemic control group compared with that in the high-glycemic control group without metformin. Conclusions: Metformin has no significant effect on lactic acid metabolism of hepatocytes under high glucose stress in vitro, and has a protective effect on hepatocytes under high glucose stress. Based on this, it is preliminarily believed that metformin is not the direct factor leading to diabetic lactic acidosis.

CHIP-overexpressing Wharton's jelly-derived mesenchymal stem cells attenuate hyperglycemia-induced oxidative stress-mediated kidney injuries in diabetic rats

Accumulating studies have demonstrated the protective roles of mesenchymal stem cells against several disorders. However, one of their crucial limitations is reduced viability under stress conditions, including the hyperglycemia induced by diabetes. The molecular mechanisms involved in diabetes-induced kidney injuries are not fully elucidated. In this study, we found that high glucose (HG) reduced human proximal tubular epithelial cell viability. Further, hyperglycemia induced oxidative stress-mediated apoptosis and fibrosis in HK-2 cells via activation of the mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinase JNK and p38 kinase. Carboxyl terminus of HSP70 interacting protein (CHIP) overactivation considerably rescued cell viability under HG stress. Moreover, Western blot analysis, flow cytometry, and MitoSOX staining revealed that hyperglycemia-induced mitochondrial oxidative stress production and apoptosis were attenuated in CHIP-overexpressing Wharton's jelly-derived mesenchymal stem cells (WJMSCs). Co-culture with CHIP-expressing WJMSCs maintained HK-2 cell viability, and inhibited apoptosis and fibrosis by attenuating HG-induced ROS-mediated MAPK activation. CHIP-overexpressing WJMSCs also rescued the decreased kidney weight and hyperglycemia-induced kidney damage observed in streptozotocin-induced diabetic rats. Cumulatively, the current research findings demonstrate that CHIP suppresses hyperglycemia-induced oxidative stress and confers resistance to MAPK-induced apoptosis and fibrosis, and suggests that CHIP protects WJMSCs and the high quality WJMSCs have therapeutic effects against diabetes-induced kidney injuries.

Mitofusin-2 Enhances Mitochondrial Contact With the Endoplasmic Reticulum and Promotes Diabetic Cardiomyopathy.

Diabetic cardiomyopathy has been associated with mitochondrial damage. Mitochondria–endoplasmic reticulum (ER) contact is an important determinant of mitochondrial function and ER homeostasis. We therefore investigated whether hyperglycemia can damage the mitochondria by increasing their contact with the ER in cardiomyocytes. We found that hyperglycemia induced mitochondria–ER contact in cardiomyocytes, as evidenced by the increased MMM1, MDM34, and BAP31 expressions. Interestingly, the silencing of Mfn2 reduced the cooperation between the mitochondria and the ER in cardiomyocytes. Mfn2 silencing improved cardiomyocyte viability and function under hyperglycemic conditions. Additionally, the silencing of Mfn2 markedly attenuated the release of calcium from the ER to the mitochondria, thereby preserving mitochondrial metabolism in cardiomyocytes under hyperglycemic conditions. Mfn2 silencing reduced mitochondrial reactive oxygen species production, which reduced mitochondria-dependent apoptosis in hyperglycemia-treated cardiomyocytes. Finally, Mfn2 silencing attenuated ER stress in cardiomyocytes subjected to high-glucose stress. These results demonstrate that Mfn2 promotes mitochondria–ER contact in hyperglycemia-treated cardiomyocytes. The silencing of Mfn2 sustained mitochondrial function, suppressed mitochondrial calcium overload, prevented mitochondrial apoptosis, and reduced ER stress, thereby enhancing cardiomyocyte survival under hyperglycemic conditions.

E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein (CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages.

Recent studies indicate that umbilical cord stem cells are cytoprotective against several disorders. One critical limitation in using stem cells is reduction in their viability under stressful conditions, such as diabetes. However, the molecular intricacies responsible for diabetic conditions are not fully elucidated. In this study, we found that high glucose (HG) conditions induced loss of chaperone homeostasis, stabilized PTEN, triggered the downstream signaling cascade, and induced apoptosis and oxidative stress in Wharton's jelly derived mesenchymal stem cells (WJMSCs). Increased Carboxyl terminus of Hsc70 interacting protein (CHIP) expression promoted phosphatase and tensin homolog (PTEN) degradation via the ubiquitin‐proteasome system and shortened its half‐life during HG stress. Docking studies confirmed the interaction of CHIP with PTEN and FOXO3a with the Bim promoter region. Further, it was found that the chaperone system is involved in CHIP‐mediated PTEN proteasomal degradation. CHIP depletion stabilizes PTEN whereas PTEN inhibition showed an inverse effect. CHIP overactivation suppressed the binding of FOXO3a with bim. Coculturing CHIP overexpressed WJMSCs suppressed HG‐induced apoptosis and oxidative stress in embryo derived cardiac cell lines. CHIP overexpressing and PTEN silenced WJMSCs ameliorated diabetic effects in streptozotocin (STZ) induced diabetic rats and further improved their body weight and heart weight, and rescued from hyperglycemia‐induced cardiac injury. Considering these, the current study suggests that CHIP confers resistance to apoptosis and acts as a potentiation factor in WJMSCs to provide protection from degenerative effects of diabetes.

Cassia auriculata leaf extract ameliorates diabetic nephropathy by attenuating autophagic necroptosis via RIP-1/RIP-3-p-p38MAPK signaling.

Diabetic nephropathy (DN) is the most common manifestation of high glucose induced diabetes mellitus. In this study, we report the effects of Cassia auriculata ethanol leaf extract (CALE) on DN-associated cell toxicity and complications. The effects of CALE were screened in vitro using RGE cells. Cell viability was assessed using MTT and flow cytometry. Male Sprague-Dawley rats were divided into control, DN and treatment groups (n=8). The DN and treatment groups received 60mg/kg/bw of streptozotocin in citrate buffer, while the treatment group was administered 150mg/kg/bw of CALE for 10weeks. Biochemical analysis was conducted using spectrophotometry. Kidney tissues were analyzed using hematoxylin and eosin staining and transmission electron microscopy. CD365-KIM-1 expression was assessed using flow cytometry and signalling proteins were detected using western blotting. Treatment with 30-mM glucose reduced the viability of RGE cells in a time-dependent manner and increased the population of dead RGE cells. Cotreatment with CALE reduced cell death and glucose induced protein expression of LC3-II, RIP-1 and RIP-3 in a dose-dependent manner. In addition, CALE improved the biochemical complications, renal dysfunction and pathophysiology of rats with DN and partially or fully restored the expression of key DN-associated signalling proteins, such as KIM-1 LC3-II, RIP-1, RIP-3 and p-p38MAPK in kidney cells. CALE showed protective effects, and improved DN-associated complications in RGE cells under high glucose stress conditions, potentially by inhibiting autophagic-necroptosis signals. Additionally, CALE improved the biochemical and pathological features of kidney injury while reducing autophagic-necroptosis in rat renal cells via the LC3-II-RIP-p38MAPK pathway. PRACTICAL APPLICATIONS: Results from the current investigation will add information to the literature on glucose induced renal toxicity and the protective effects of CALE over the complications of diabetic nephropathy (DN). The mechanistic investigations of the study will add light on the autophagic/necroptosis signals in DN and open new routes of investigations to study the efficacy of CALE in diabetes-related complications.

Regulation of Erythritol Metabolism, a Biomarker of Cardiometabolic Disease

Abstract Objectives Serum erythritol is a predictive biomarker of chronic disease development and complications. In cohort studies, elevated serum erythritol at baseline was predictive of central adiposity gain, T2DM, and cardiovascular disease development up to 20 years later. Erythritol is a polyol that has been well characterized as a non-nutritive sweetener. Erythritol was recently discovered to be synthesized endogenously in humans from glucose through the pentose phosphate pathway (PPP). The regulation of erythritol synthesis in mammals, however, is not characterized. The purpose of this study was to assess stimuli that modulate intracellular erythritol levels and the distribution of erythritol in key metabolic tissues. Methods First, we fed 8-week-old C57BL/6J mice a defined diet (AIN93G) for two weeks, after which plasma and tissue erythritol were measured by GC-MS. To further evaluate the cellular regulation of erythritol synthesis, we used A549 cells for their high PPP activity. Using siRNA, we knocked down SORD (a potential erythritol-synthesizing enzyme) and glucose-6-phosphate dehydrogenase (G6PD) under normal (5 mM) and high (25 mM) glucose conditions and measured intracellular erythritol. We also exposed A549’s to hydrogen peroxide to assess if erythritol synthesis is elevated by oxidative stress, an additional stimulator of the PPP. Results Plasma erythritol ranged from 0.44 uM to 0.73 uM in young, healthy mice. Erythritol differed significantly based on tissue type (p < 0.0001). The liver and kidney had the highest relative erythritol, followed by quadriceps, whereas white adipose tissue contained the least erythritol. SORD knockdown significantly reduced erythritol synthesis under high glucose, but not normal glucose conditions (p < 0.01). Knockdown of G6PD did not impact erythritol synthesis at either glucose concentration. Finally, treatment with hydrogen peroxide significantly increased intracellular erythritol (p < 0.0001). Conclusions In conclusion, erythritol is present in mouse plasma, SORD-expressing tissues, and human cells. Erythritol was elevated by high glucose and oxidative stress–two key factors in the pathogenesis of cardiometabolic diseases. Together, these data suggest that erythritol synthesis occurs parallel to elevated PPP flux in response to metabolic stressors. Funding Sources N/A

High glucose induced c-Met activation promotes aggressive phenotype and regulates expression of glucose metabolism genes in HCC cells

Hepatocellular carcinoma (HCC) is strongly associated with metabolic dysregulations/deregulations and hyperglycemia is a common metabolic disturbance in metabolic diseases. Hyperglycemia is defined to promote epithelial to mesenchymal transition (EMT) of cancer cells in various cancers but its molecular contribution to HCC progression and aggressiveness is relatively unclear. In this study, we analyzed the molecular mechanisms behind the hyperglycemia-induced EMT in HCC cell lines. Here, we report that high glucose promotes EMT through activating c-Met receptor tyrosine kinase via promoting its ligand-independent homodimerization. c-Met activation is critical for high glucose induced acquisition of mesenchymal phenotype, survival under high glucose stress and reprogramming of cellular metabolism by modulating glucose metabolism gene expression to promote aggressiveness in HCC cells. The crucial role of c-Met in high glucose induced EMT and aggressiveness may be the potential link between metabolic syndrome-related hepatocarcinogenesis and/or HCC progression. Considering c-Met inhibition in hyperglycemic patients would be an important complementary strategy for therapy that favors sensitization of HCC cells to therapeutics.

Isoquercitrin protects HUVECs against high glucose‑induced apoptosis through regulating p53 proteasomal degradation.

High glucose (HG)-induced endothelial apoptosis serves an important role in the vascular dysfunction associated with diabetes mellitus (DM). It has been reported that isoquercitrin (IQC), a flavonoid glucoside, possesses an anti-DM effect, but the mechanism requires further investigation. The present study investigated the effect of IQC against HG-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and explored its molecular mechanism. HUVECs were treated with 5 or 30 mM glucose for 48 h. Endothelial cell viability was monitored using the Cell Counting Kit-8 assay. Mitochondrial membrane potential was detected by JC-1 staining. Apoptosis was observed by TUNEL staining and flow cytometry. Western blotting was used for the analysis of apoptosis-associated proteins Bax, Bcl-2, cleaved (C)-caspase3, total-caspase3, p53 and phosphorylated p53. Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of Bax, Bcl-2 and p53. Immunofluorescence staining was utilized to detect the expression levels and distribution of p53 and ubiquitin specific peptidase 10 (USP10) in HUVECs. The results revealed that IQC significantly attenuated HG-induced endothelial apoptosis, as shown by decreased apoptotic cells observed by TUNEL, JC-1 staining and flow cytometry. Moreover, under HG stress, IQC treatment markedly inhibited the increased expression levels of the pro-apoptotic proteins p53, Bax and C-caspase3, and increased the expression levels of the anti-apoptotic protein Bcl-2 in HUVECs. However, the anti-apoptotic effect of IQC against HG was partially blunted by increasing p53 protein levels in vitro. IQC influenced the mRNA expression levels of Bax and Bcl-2 in response to HG, but it did not affect the transcription of p53. Notably, IQC inhibited the HG-induced phosphorylation of p53 at Ser15 and the nuclear transport of USP10, destabilizing p53 and increasing the proteasomal degradation of the p53 protein. The current findings revealed that IQC exerted a protective effect against the HG-induced apoptosis of endothelial cells by regulating the proteasomal degradation of the p53 protein, suggesting that IQC may be used as a novel therapeutic compound to ameliorate DM-induced vascular complications.

Activation of Insulin-Like Growth Factor-2 Ameliorates Retinal Cell Damage and Exerts Protection in in vitro Model of Diabetic Retinopathy

Background: The major event in the development of diabetes-related blindness and vision impairment is the onset of retinal cell damage. Overall awareness of insulin-like growth factor-2 (IGF2) mechanisms emphasizes its protective behavior in retinal cells that help to provide new information about the development of treatment for retinal complications. Objectives: This study analyzes the effect of in vitro changes associated with the cell survival and rescue mechanism in IGF2 inhibition and activation using chromeceptin and IGF2 peptides in ARPE-19 cells cultured in high glucose conditions. Method: Cell death was induced using high glucose (15 mmol/L), IGF2 inhibition was done using chromeceptin (1 µM) (Sigma Aldrich, Saint Louis, MO, USA), and IGF2 activation was done using IGF2 peptide (10 ng/mL). The cells were analyzed for changes in cell proliferation, apoptosis markers, antioxidant molecules, and alteration of cytokines. Results: The study demonstrated that cells lacking IGF2 exhibited a significant increase in reactive oxygen levels with apoptosis patterns. Also, gene expression analysis by qRT-PCR demonstrated a significant increase in Yes-associated protein 1, CDK2, TNF-α, and BIRC5 genes in cells under high glucose stress and IGF inhibition compared to control. Further, the cytokine analysis also revealed that cells devoid of IGF2 activated an increase in cytokines such as IL-8, CX43, ICAM-1, IL-17, CCL3, and MCP-1 and decreased paraoxonase compared to normal control cells. On the other hand, ARPE-19 cells grown in high glucose shows that IGF2 increases the survival genes with reduced levels of inflammatory cytokines. Conclusion: The finding of the investigation, therefore, shows that the use of IGF2 activators may prevent the progression of ocular dysfunction in the control of diabetes-related complications.

Regulation and Distribution of Endogenous Erythritol, a Biomarker of Cardiometabolic Disease

Serum erythritol is a novel predictive biomarker of chronic disease development and complications. In cohort studies, elevated serum erythritol at baseline was predictive of central adiposity gain, T2DM, and cardiovascular disease development. Higher erythritol has also been associated with complications of these chronic diseases such as diabetic retinopathy and nephropathy. Erythritol is a 4-carbon polyol that has been well characterized as a non-nutritive sweetener. More recently, erythritol was discovered to be synthesized endogenously in humans from glucose through the pentose phosphate pathway (PPP). The PPP generates NADPH for anabolic reactions and to combat oxidative stress. We identified two human enzymes that catalyze the conversion of erythrose-4-phosphate to erythritol: sorbitol dehydrogenase (SORD) and alcohol dehydrogenase 1 (ADH1). SORD knockdown significantly decreases erythritol synthesis in vitro. The regulation of erythritol synthesis in mammals is not characterized. The purpose of this study was to assess stimuli that modulate intracellular erythritol levels and the distribution of erythritol in key metabolic tissues. We hypothesize that erythritol synthesis will be induced by stimuli that increase PPP activity, and that erythritol will be present in tissues with high SORD expression. First, we fed 8-week-old C57BL/6J mice a defined diet (AIN93G) for two weeks. After two weeks, plasma and tissue erythritol including liver, kidney, white adipose, and skeletal muscle, were measured by GC-MS. To further evaluate the cellular regulation of erythritol synthesis, we used A549 cells due to their high PPP activity. Using siRNA, we knocked down SORD and glucose-6-phosphate dehydrogenase (G6PD) under normal (5mM) and high (25mM) glucose conditions, then measured intracellular erythritol. We also exposed A549’s to hydrogen peroxide to assess if erythritol synthesis is elevated by oxidative stress. Plasma erythritol ranged from 0.44 uM to 0.73 uM in young, healthy mice. Erythritol was significantly different based on tissue type (p < 0.0001, one-way ANOVA). The liver and kidney had the highest relative erythritol, followed by quadriceps, whereas white adipose tissue contained the least erythritol. Interestingly, SORD knockdown significantly reduced erythritol synthesis under high glucose, but not normal glucose conditions (p < 0.01, Sidak's multiple comparisons test). Knockdown of G6PD did not impact erythritol synthesis at either glucose concentration. Finally, treatment with hydrogen peroxide significantly increased intracellular erythritol (p < 0.0001, student's t-test). In mice, erythritol was highest in tissues known to have high SORD expression. Unexpectedly, erythritol was lowest in adipose, which is known to have high PPP activity. This finding is supported by the reduction of intracellular erythritol induced by SORD, but not G6PD knockdown. Erythritol synthesis was also elevated by two factors known to stimulate the PPP: high glucose and oxidative stress. Together, these data suggest that erythritol synthesis is linked to PPP flux, but is regulated downstream of the oxidative phase of the PPP.