From stem cells to pancreatic ß-cells: strategies, applications, and potential treatments for diabetes.

Loss and functional failure of pancreatic ß-cells results in disruption of glucose homeostasis and progression of diabetes. Although whole pancreas or pancreatic islet transplantation serves as a promising approach for ß-cell replenishment and diabetes therapy, the severe scarcity of donor islets makes it unattainable for most diabetic patients. Stem cells, particularly induced pluripotent stem cells (iPSCs), are promising for the treatment of diabetes owing to their self-renewal capacity and ability to differentiate into functional ß-cells. In this review, we first introduce the development of functional ß-cells and their heterogeneity and then turn to highlight recent advances in the generation of ß-cells from stem cells and their potential applications in disease modeling, drug discovery and clinical therapy. Finally, we have discussed the current challenges in developing stem cell-based therapeutic strategies for improving the treatment of diabetes. Although some significant technical hurdles remain, stem cells offer great hope for patients with diabetes and will certainly transform future clinical practice.

Pancreatic ß-cell failure, clinical implications, and therapeutic strategies in type 2 diabetes.

ABSTRACT: Pancreatic ß-cell failure due to a reduction in function and mass has been defined as a primary contributor to the progression of type 2 diabetes (T2D). Reserving insulin-producing ß-cells and hence restoring insulin production are gaining attention in translational diabetes research, and ß-cell replenishment has been the main focus for diabetes treatment. Significant findings in ß-cell proliferation, transdifferentiation, pluripotent stem cell differentiation, and associated small molecules have served as promising strategies to regenerate ß-cells. In this review, we summarize current knowledge on the mechanisms implicated in ß-cell dynamic processes under physiological and diabetic conditions, in which genetic factors, age-related alterations, metabolic stresses, and compromised identity are critical factors contributing to ß-cell failure in T2D. The article also focuses on recent advances in therapeutic strategies for diabetes treatment by promoting ß-cell proliferation, inducing non-ß-cell transdifferentiation, and reprograming stem cell differentiation. Although a significant challenge remains for each of these strategies, the recognition of the mechanisms responsible for ß-cell development and mature endocrine cell plasticity and remarkable advances in the generation of exogenous ß-cells from stem cells and single-cell studies pave the way for developing potential approaches to cure diabetes.

JunB condensation attenuates vascular endothelial damage under hyperglycemic condition.

Endothelial damage is the initial and crucial factor in the occurrence and development of vascular complications in diabetic patients, contributing to morbidity and mortality. Although hyperglycemia has been identified as a damaging effector, the detailed mechanisms remain elusive. In this study, identified by ATAC-seq and RNA-seq, JunB reverses the inhibition of proliferation and the promotion of apoptosis in human umbilical vein endothelial cells treated with high glucose, mainly through the cell cycle and p53 signaling pathways. Furthermore, JunB undergoes phase separation in the nucleus and in vitro, mediated by its intrinsic disordered region and DNA-binding domain. Nuclear localization and condensation behaviors are required for JunB-mediated proliferation and apoptosis. Thus, our study uncovers the roles of JunB and its coacervation in repairing vascular endothelial damage caused by high glucose, elucidating the involvement of phase separation in diabetes and diabetic endothelial dysfunction.

Liraglutide ameliorates hepatic steatosis via retinoic acid receptor-related orphan receptor α-mediated autophagy pathway.

Liraglutide, an analog of human glucagon-like peptide-1 (GLP-1), has been found to improve hepatic steatosis in clinical practice. However, the underlying mechanism remains to be fully defined. Increasing evidence suggests that retinoic acid receptor-related orphan receptor α (RORα) is involved in hepatic lipid accumulation. In the current study, we investigated whether the ameliorating impact of liraglutide on lipid-induced hepatic steatosis is dependent on RORα activity and examined the underlying mechanisms. Cre-loxP-mediated, liver-specific Rorα knockout (Rora LKO) mice, and littermate controls with a Roraloxp/loxp genotype were established. The effects of liraglutide on lipid accumulation were evaluated in mice challenged with a high-fat diet (HFD) for 12 weeks. Moreover, mouse AML12 hepatocytes expressing small interfering RNA (siRNA) of Rora were exposed to palmitic acid to explore the pharmacological mechanism of liraglutide. The results showed that liraglutide treatment significantly alleviated HFD-induced liver steatosis, marked by reduced liver weight and triglyceride accumulation, improved glucose tolerance and serum levels of lipid profiles and aminotransferase. Consistently, liraglutide also ameliorated lipid deposits in a steatotic hepatocyte model in vitro. In addition, liraglutide treatment reversed the HFD-induced downregulation of Rora expression and autophagic activity in mouse liver tissues. However, the beneficial effect of liraglutide on hepatic steatosis was not observed in Rora LKO mice. Mechanistically, the ablation of Rorα in hepatocytes diminished liraglutide-induced autophagosome formation and the fusion of autophagosomes and lysosomes, resulting in weakened autophagic flux activation. Thus, our findings suggest that RORα is essential for the beneficial impact of liraglutide on lipid deposition in hepatocytes and regulates autophagic activity in the underlying mechanism.

Nuclear factor-Y mediates pancreatic ß-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress.

BACKGROUND: Pancreatic ß-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in ß-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of ß-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in ß-cell compensatory response to insulin resistance in obesity is unclear. METHODS: In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in ß-cells ( ob , Nf-ya ßKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to ß-cell compensation upon metabolic stress. RESULTS: Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of ß-cell Nf-ya in obese mice worsened glucose intolerance and resulted in ß-cell dysfunction, which was attributable to augmented ß-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya ßKO mice were sensitive to palmitate-induced ß-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 ß-cell line could rescue palmitate-induced ß-cell apoptosis, dysfunction, and mitochondrial impairment. CONCLUSION: Pancreatic NF-Y might be an essential regulator of ß-cell compensation under metabolic stress.

Novel association of SNP rs2297828 in PRDM16 gene with predisposition to type 2 diabetes.

OBJECTIVES: Transcriptional regulator PRD1-BF-1-RIZ1 homology (PR) domain containing protein-16 (PRDM16) has a fundamental function in maintaining energy homeostasis and regulating glucose and lipid metabolism, which are responsible for the development of type 2 diabetes (T2D). However, the impact of genetic variation of PRDM16 gene on T2D risk remains to be investigated. Thus, we evaluated the possible association between genetic variants within PRDM16 region and T2D development in Chinese individuals. METHODS: A total of 427 T2D patients and 408 healthy controls were enrolled. Ten single-nucleotide variants across PRDM16 gene were screened with the SNaPshot assay. The effect of genotypes and alleles of different variant on the T2D risk was examined under diverse genetic models. The impact of genetic variant on promoter activity was determined using an in vitro luciferase reporter gene assay. RESULTS: Genotypic frequency of rs2297828 in the PRDM16 promoter region was significantly different between patients with T2D and controls (P = 0.004). The minor allele A of rs2297828 was potentially associated with a higher T2D susceptibility in a dominant model (AG + AA vs GG: OR = 1.54, 95 % CI: 1.12-1.12; P = 0.007), and the subjects with either an AA homozygote or an AG heterozygote displayed increased fasting blood levels of glucose and lipids. Reporter gene assays demonstrated that rs2297828 can influence the activity of the PRDM16 promoter. CONCLUSIONS: We firstly observed that PRDM16 variation might influence T2D occurrence, and rs2297828 might be a functional variant that can influence the expression of PRDM16.

The suppressive functions of Rora in B lineage cell proliferation and BCR/ABL1-induced B-ALL pathogenesis.

RORA plays an important role in regulating circadian rhythms, inflammation, metabolism and cellular development. Herein, we explore the roles of Rora in B cell proliferation and differentiation, as well as in Ph+ B-ALL. By using Roraloxp/loxp Mx-1-Cre mice, Rora was deleted in hematopoietic cells post Pipc induction. Rora deficiency mice were associated with an obvious accumulation of B cells in the peripheral blood, bone marrow, and spleen. On the other hand, activation of Rora with Cholesterol sulfate (CS) was associated with decreased B cell numbers. RNA-seq analysis revealed that the transcription level of Lmo1 was decreased in Rora deficient B cells. Moreover, the expression of RORA was shown to be decreased in Ph+ B-ALL cells compared to peripheral blood derived B cells from healthy donors. The overexpression of Rora in BaF3 cells with BCR/ABL1 was also associated with impeded the cell growth and an increased apoptotic rate compared to cells transduced with BCR/ABL1 alone. The co-expression of BCR/ABL1 and Rora induced B-ALL mouse model was associated with the significant inhibition of BCR/ABL1-transformed cell growth and prolonged the survival of the diseased mice. These results suggest a novel role for Rora in B cell development and Ph+ leukemogenesis.

Cholesterol Sulfate Exerts Protective Effect on Pancreatic ß-Cells by Regulating ß-Cell Mass and Insulin Secretion.

Rational: Cholesterol sulfate (CS) is the most abundant known sterol sulfate in human plasma, and it plays a significant role in the control of metabolism and inflammatory response, which contribute to the pathogenesis of insulin resistance, ß-cell dysfunction and the resultant development of diabetes. However, the role of CS in ß-cells and its effect on the development of diabetes remain unknown. Here, we determined the physiological function of CS in pancreatic ß-cell homeostasis. Materials and Methods: Blood CS levels in streptozotocin (STZ)- or high-fat diet-induced diabetic mice and patients with type 1 or 2 diabetes were determined by LC-MS/MS. The impact of CS on ß-cell mass and insulin secretion was investigated in vitro in isolated mouse islets and the ß-cell line INS-1 and in vivo in STZ-induced diabetic mice. The molecular mechanism of CS was explored by viability assay, EdU incorporation analysis, flow cytometry, intracellular Ca2+ influx analysis, mitochondrial membrane potential and cellular ROS assays, and metabolism assay kits. Results: Plasma CS levels in mice and humans were significantly elevated under diabetic conditions. CS attenuated diabetes in a low-dose STZ-induced mouse model. Mechanistically, CS promoted ß-cell proliferation and protected ß-cells against apoptosis under stressful conditions, which in turn preserved ß-cell mass. In addition, CS supported glucose transporter-2 (GLUT2) expression and mitochondrial integrity, which then resulted in a less reactive oxygen species (ROS) generation and an increase in ATP production, thereby enabling insulin secretion machinery in the islets to function adequately. Conclusion: This study revealed a novel dual role of CS in integrating ß-cell survival and cell function, suggesting that CS might offer a physiologic approach to preserve ß-cells and protect against the development of diabetes mellitus.

Nuclear Factor-Y in Mouse Pancreatic ß-Cells Plays a Crucial Role in Glucose Homeostasis by Regulating ß-Cell Mass and Insulin Secretion.

Pancreatic ß-cell mass and insulin secretion are determined by the dynamic change of transcription factor expression levels in response to altered metabolic demand. Nuclear factor-Y (NF-Y) is an evolutionarily conserved transcription factor playing critical roles in multiple cellular processes. However, the physiological role of NF-Y in pancreatic ß-cells is poorly understood. The current study was undertaken in a conditional knockout of Nf-ya specifically in pancreatic ß-cells (Nf-ya ßKO) to define the essential physiological role of NF-Y in ß-cells. Nf-ya ßKO mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced ß-cell proliferation resulting in decreased ß-cell mass was observed in these mice, which was associated with disturbed actin cytoskeleton. NF-Y-deficient ß-cells also exhibited impaired insulin secretion with a reduced Ca2+ influx in response to glucose, which was associated with an inefficient glucose uptake into ß-cells due to a decreased expression of GLUT2 and a reduction in ATP production resulting from the disruption of mitochondrial integrity. This study is the first to show that NF-Y is critical for pancreatic islet homeostasis and function through regulation in ß-cell proliferation, glucose uptake into ß-cells, and mitochondrial energy metabolism. Modulating NF-Y expression in ß-cells may therefore offer an attractive approach for therapeutic intervention.

Mitochondria-Associated Endoplasmic Reticulum Membranes in the Pathogenesis of Type 2 Diabetes Mellitus.

The endoplasmic reticulum (ER) and mitochondria are essential intracellular organelles that actively communicate via temporally and spatially formed contacts called mitochondria-associated membranes (MAMs). These mitochondria-ER contacts are not only necessary for the physiological function of the organelles and their coordination with each other, but they also control the intracellular lipid exchange, calcium signaling, cell survival, and homeostasis in cellular metabolism. Growing evidence strongly supports the role of the mitochondria-ER connection in the insulin resistance of peripheral tissues, pancreatic ß cell dysfunction, and the consequent development of type 2 diabetes mellitus (T2DM). In this review, we summarize current advances in the understanding of the mitochondria-ER connection and specifically focus on addressing a new perspective of the alterations in mitochondria-ER communication in insulin signaling and ß cell maintenance.

Effects of genetic variations in Acads gene on the risk of chronic obstructive pulmonary disease.

Short-chain acyl-CoA dehydrogenase (SCAD), encoded by the Acads gene, functions in the mitochondrial ß-oxidation of saturated short-chain fatty acids. SCAD deficiency results in mitochondrial dysfunction, which is one underlying biological mechanism of chronic obstructive pulmonary disease (COPD) pathogenesis. In this case-control study, we aimed to examine the effects of Acads gene polymorphisms on the susceptibility to COPD. A total of 16 tagging single-nucleotide polymorphisms (SNPs) in Acads gene region was identified and genotyped in 646 unrelated ethnic Chinese Han individuals including 279 patients with COPD and 367 healthy controls, their allelic and genotypic associations with COPD were determined by different genetic models. Furthermore, we estimated the linkage disequilibrium and haplotypes from these tested variants and determined the effects of haplotypes on the risk of COPD. The allelic and genotypic frequencies of SNPs rs2239686 and rs487915 in Acads gene were significantly different between COPD patients and controls, no statistically significant results were observed for other SNPs. Minor alleles A of rs2239686 and T of rs487915 were associated with a decreased pulmonary function and an increased COPD risk in a dominant manner. Functional analysis indicated that the risk allele A of rs2239686 could increase Acads expressions and the intracellular reactive oxygen species content. Haplotype analysis revealed that the haplotypes CTCCT in block 2 (rs3794216-rs3794215-rs34491494-rs558314-rs7312316) as well as GC in block 3 (rs2239686-rs487915) were protective against COPD, while haplotypes CTCGC in block 2 and AT in block 3 exhibited significant associations with the increased susceptibility to COPD. Our results suggest that Acads gene could potentially be a risk factor of COPD and thus its genetic variants might be as genetic biomarkers to predict the COPD susceptibility.

Deficiency in the short-chain acyl-CoA dehydrogenase protects mice against diet-induced obesity and insulin resistance.

Acyl-CoA dehydrogenases (CADs) participate in mitochondrial fatty acid oxidation; abnormal fatty acid oxidation is associated with obesity and related metabolic disorders. We decipher the impact of short-chain CAD (SCAD) on adiposity and insulin resistance. BALB/cBy strain mice derived from BALB/c strain are deficient in SCAD activity because of a spontaneous deletion in the acyl-CoA dehydrogenases (Acads) gene. Adiposity, lipogenesis, and insulin sensitivity were compared in BALB/c and BALB/cBy mice subjected to high-fat diets (HFDs). A whole hepatic transcriptome profiling experiment with microarrays was performed to evaluate the mechanisms by which SCAD deficiency protects against insulin resistance. Acads-deficient mice were significantly resistant to HFD-induced obesity and insulin resistance as compared with control mice. Reduced obesity results from decreased triglyceride content due to activation of AMPK in liver that would reduce hepatic content of malonyl-CoA, resulting in decreased hepatic de novo lipogenesis. Improved insulin sensitivity was associated with reduced diacylglycerol content commensurate with reduced PKC-ε activity and increased protein kinase B (AKT) activation in liver and skeletal muscle. Additionally, Acads-deficient mice displayed significantly higher expression of the endoplasmic chaperone 78-kDa glucose-regulated protein, which was further associated with the AKT activation in the primary hepatocytes. Modulation of SCAD expression may therefore be a novel therapeutic approach to manage and prevent obesity and related metabolic diseases, such as diabetes.-Chen, Y., Chen, J., Zhang, C., Yang, S., Zhang, X., Liu, Y., Su, Z. Deficiency in the short-chain acyl-CoA dehydrogenase protects mice against diet-induced obesity and insulin resistance.

Effects of Genetic Variants of Nuclear Receptor Y on the Risk of Type 2 Diabetes Mellitus.

Nuclear factor-Y (NF-Y) consists of three evolutionary conserved subunits including NF-YA, NF-YB, and NF-YC; it is a critical transcriptional regulator of lipid and glucose metabolism and adipokine biosynthesis that are associated with type 2 diabetes mellitus (T2DM) occurrence, while the impacts of genetic variants in the NF-Y gene on the risk of T2DM remain to be investigated. In the present study, we screened five single-nucleotide polymorphisms (SNPs) with the SNaPshot method in 427 patients with T2DM and 408 healthy individuals. Subsequently, we analyzed the relationships between genotypes and haplotypes constructed from these SNPs with T2DM under diverse genetic models. Furthermore, we investigated the allele effects on the quantitative metabolic traits. Of the five tagSNPs, we found that three SNPs (rs2268188, rs6918969, and rs28869187) exhibited nominal significant differences in allelic or genotypic frequency between patients with T2DM and healthy individuals. The minor alleles G, C, and C at rs2268188, rs6918969, and rs28869187, respectively, conferred a higher T2DM risk under a dominant genetic model, and the carriers of these risk alleles (either homozygotes of the minor allele or heterozygotes) had statistically higher levels of fasting plasma glucose, cholesterol, and triglycerides. Haplotype analysis showed that SNPs rs2268188, rs6918969, rs28869187, and rs35105472 formed a haplotype block, and haplotype TTAC was protective against T2DM (OR = 0.76, 95% CI = 0.33-0.82, P = 0.004), while haplotype GCCG was associated with an elevated susceptibility to T2DM (OR = 2.33, 95% CI = 1.43-3.57, P = 0.001). This study is the first ever observation to our knowledge that indicates the genetic variants of NF-YA might influence a Chinese Han individual's occurrence of T2DM.

Mutation spectrum in GNAQ and GNA11 in Chinese uveal melanoma.

Uveal melanoma is the most common intraocular cancer in the adult eye. R183 and Q209 were found to be mutational hotspots in exon 4 and exon 5 of GNAQ and GNA11 in Caucasians. However, only a few studies have reported somatic mutations in GNAQ or GNA11 in uveal melanoma in Chinese. We extracted somatic DNA from paraffin-embedded biopsies of 63 Chinese uveal melanoma samples and sequenced the entire coding regions of exons 4 and 5 in GNAQ and GNA11. The results showed that 33% of Chinese uveal melanoma samples carried Q209 mutations while none had R183 mutation in GNAQ or GNA11. In addition, seven novel missense somatic mutations in GNAQ (Y192C, F194L, P170S, D236N, L232F, V230A, and M227I) and four novel missense somatic mutations in GNA11 (R166C, I200T, S225F, and V206M) were found in our study. The high mutation frequency of Q209 and the novel missense mutations detected in this study suggest that GNAQ and GNA11 are common targets for somatic mutations in Chinese uveal melanoma.

Regulation of hepatic gluconeogenesis by nuclear factor Y transcription factor in mice.

Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular mechanisms in the regulation of hepatic gluconeogenesis remain to be fully defined. In this study, using murine hepatocytes and a liver-specific knockout mouse model, we explored the physiological role of nuclear factor Y (NF-Y) in regulating hepatic glucose metabolism and the underlying mechanism. We found that NF-Y targets the gluconeogenesis pathway in the liver. Hepatic NF-Y expression was effectively induced by cAMP, glucagon, and fasting in vivo Lentivirus-mediated NF-Y overexpression in Hepa1-6 hepatocytes markedly raised the gluconeogenic gene expression and cellular glucose production compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated gluconeogenic gene expression and glucose production. We also provide evidence indicating that CRE-loxP-mediated, liver-specific NF-YA knockout compromises hepatic glucose production. Mechanistically, luciferase reporter gene assays and ChIP analysis indicated that NF-Y activates transcription of the gluconeogenic genes Pck1 and G6pc, by encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase), respectively, via directly binding to the CCAAT regulatory sequence motif in their promoters. Of note, NF-Y enhanced gluconeogenesis by interacting with cAMP-responsive element-binding protein (CREB). Overall, our results reveal a previously unrecognized physiological function of NF-Y in controlling glucose metabolism by up-regulating the gluconeogenic genes Pck1 and G6pc Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.

Unraveling the Regulation of Hepatic Gluconeogenesis.

Hepatic gluconeogenesis, de novo glucose synthesis from available precursors, plays a crucial role in maintaining glucose homeostasis to meet energy demands during prolonged starvation in animals. The abnormally increased rate of hepatic gluconeogenesis contributes to hyperglycemia in diabetes. Gluconeogenesis is regulated on multiple levels, such as hormonal secretion, gene transcription, and posttranslational modification. We review here the molecular mechanisms underlying the transcriptional regulation of gluconeogenesis in response to nutritional and hormonal changes. The nutrient state determines the hormone release, which instigates the signaling cascades in the liver to modulate the activities of various transcriptional factors through various post-translational modifications like phosphorylation, methylation, and acetylation. AMP-activated protein kinase (AMPK) can mediate the activities of some transcription factors, however its role in the regulation of gluconeogenesis remains uncertain. Metformin, a primary hypoglycemic agent of type 2 diabetes, ameliorates hyperglycemia predominantly through suppression of hepatic gluconeogenesis. Several molecular mechanisms have been proposed to be metformin's mode of action.

Retinoic acid receptor-related orphan receptor α stimulates adipose tissue inflammation by modulating endoplasmic reticulum stress.

Adipose tissue inflammation has been linked to metabolic diseases such as obesity and type 2 diabetes. However, the molecules that mediate inflammation in adipose tissue have not been addressed. Although retinoic acid receptor-related orphan receptor α (RORα) is known to be involved in the regulation of inflammatory response in some tissues, its role is largely unknown in adipose tissue. Conversely, it is known that endoplasmic reticulum (ER) stress and unfolding protein response (UPR) signaling affect the inflammatory response in obese adipose tissue, but whether RORα regulates these processes remains unknown. In this study, we investigate the link between RORα and adipose tissue inflammation. We showed that the inflammatory response in macrophages or 3T3-L1 adipocytes stimulated by lipopolysaccharide, as well as adipose tissue in obese mice, markedly increased the expression of RORα. Adenovirus-mediated overexpression of RORα or treatment with the RORα-specific agonist SR1078 enhanced the expression of inflammatory cytokines and increased the number of infiltrated macrophages into adipose tissue. Furthermore, SR1078 up-regulated the mRNA expression of ER stress response genes and enhanced phosphorylations of two of the three mediators of major UPR signaling pathways, PERK and IRE1α. Finally, we found that alleviation of ER stress using a chemical chaperone followed by the suppression of RORα induced inflammation in adipose tissue. Our data suggest that RORα-induced ER stress response potentially contributes to the adipose tissue inflammation that can be mitigated by treatment with chemical chaperones. The relationships established here between RORα expression, inflammation, and UPR signaling may have implications for therapeutic targeting of obesity-related metabolic diseases.

[The Mechanisms of Trimetazidine Alleviating the Oxidative Stress in Adipose-derived Mesenchymal Stem Cells].

OBJECTIVES: To investigate the effects of trimetazidine (TMZ) on the oxidative stress injury in adipose-derived mesenchymal stem cells (ADSCs). METHODS: ADSCs derived from adipose tissue of SD rats were characterized by flow cytometry and multiline age differentiation. ADSCs apoptosis was induced by H2O2 in vitro , Dirrerent concentration of TMZ (250 µmol/L, 500 µmol/L) was used to protect ADSCs from apoptosis. The morphological features of apoptotic ADSCs were analyzed by Hoechst 33342, mitochondrial potential and structure was analyzed by JC-1 staining and electron microscope, respectively. The apoptotic proteins were detected by Western blot. The effect of TMZ on antioxidant capacity of ADSCs was evaluated by detecting reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA). RESULTS: The isolated ADSCs expressed high levels of CD29 and CD90, low levels of CD34 and CD45 and no expression of CD31. ADSCs could be induced to adipocyte and osteoplastic cells. After being treated by H2O2, ADSCs displayed apoptosis characteristics with increased number of apoptotic cells, decreased mitochondrial transmembrane potential and damaged mitochondria. The expressions of apoptotic proteins, including Bax, Bad, and Caspase3, were dramatically increased compared to the controls; however, the anti-apoptotic protein Bcl2 was decreased. At the meantime, the contents of ROS and MDA were elevated, but the concentrations of SOD and GSH were reduced. The treatment of TMZ could partly reverse above negative impacts to ADSCs. CONCLUSION: TMZ could improve the survival rate of ADSCs by enhancing anti-oxidant defense systems to remove excessive ROS and regulating the expression of protective protein.

Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

One-Step Self-Assembling Nanomicelles for Pirarubicin Delivery To Overcome Multidrug Resistance in Breast Cancer.

Tumor cells can acquire multidrug resistance (MDR) as a result of drug efflux mediated by P-glycoprotein (P-gp). Here we report a targeted delivery system to carry pirarubicin (THP) to MDR breast cancer both in vitro and in vivo. PEG-derivatized vitamin E (PAMV6) amphiphiles loaded with THP were self-assembled in a single step. The PAMV6 micelles showed unimodal size distribution and high drug loading efficiency. Cytotoxicity of PAMV6/THP was higher than that of free THP on MCF-7/ADR cells but comparable to that of THP on MCF-7 cells. PAMV6/THP was able to reverse MDR more than free THP in MCF-7/ADR cells, likely reflecting the remarkably higher intracellular THP concentration in micelle-treated cells and PAMV6-mediated inhibition of P-gp activity. PAMV6/THP micelles were internalized into MCF-7/ADR cells via macropinocytosis and caveolin-mediated endocytosis, further avoiding P-gp-mediated efflux. Mechanistic studies revealed that blank PAMV6 micelles inhibited P-gp activity but did not affect P-gp expression, by significantly reducing mitochondrial membrane potential and slightly decreasing intracellular ATP levels. In a nude mouse xenograft model, PAMV6/THP led to much greater THP accumulation in tumors and much slower tumor growth than free THP. At the same time, PAMV6/THP was associated with significantly less severe bone marrow suppression and organ toxicity than free THP. Our results indicate that this PAMV6-based micelle system holds promise for combating MDR in cancer therapy.