Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1-bearing astrocytes

Impaired glutamate uptake function of astrocytes associated with accumulation of extracellular glutamate is a well-documented feature of amyotrophic lateral sclerosis (ALS). Enhancing the uptake function of astrocytic glutamate transport 1 (GLT1) may be a potential treatment for this disease. Human adipose-derived stem cells (hADSCs) are capable of secreting a large number of cytokines which exhibit diverse pharmacological effects. Therefore, we investigate the influence of the soluble factors released by hADSCs on the GLT1 in primary astrocytes cultured from SOD1^G93A mice, a widely studied mutant human SOD1 transgenic model of ALS. Our data indicate that soluble factors from hADSCs significantly upregulate the expression of GLT1 in SOD1^G93A-bearing astrocytes, which result in enhanced glutamate uptake function. The upregulation of GLT1 is accompanied by the inhibition of caspase-3 activation in mutant astrocytes. In addition, we find that hADSCs cocultured with SOD1^G93A-bearing astrocytes produce more VEGF, HGF and IGF-1, which are reported to have neuroprotective effects. Our results suggest that hADSCs may be a potential candidate in cellular therapy for ALS.     

Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro

Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1⁺ and subsequent PDX1⁺/NKX6.1⁺ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1⁺/NKX6.1⁺ progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.     

Amelioration of streptozotocin-induced diabetes in mice with cells derived from human marrow stromal cells

Background

Pluri-potent bone marrow stromal cells (MSCs) provide an attractive opportunity to generate unlimited glucose-responsive insulin-producing cells for the treatment of diabetes. We explored the potential for human MSCs (hMSCs) to be differentiated into glucose-responsive cells through a non-viral genetic reprogramming approach.

Methods and Findings

Two hMSC lines were transfected with three genes: PDX-1, NeuroD1 and Ngn3 without subsequent selection, followed by differentiation induction in vitro and transplantation into diabetic mice. Human MSCs expressed mRNAs of the archetypal stem cell markers: Sox2, Oct4, Nanog and CD34, and the endocrine cell markers: PDX-1, NeuroD1, Ngn3, and Nkx6.1. Following gene transfection and differentiation induction, hMSCs expressed insulin in vitro, but were not glucose regulated. After transplantation, hMSCs differentiated further and ∼12.5% of the grafted cells expressed insulin. The graft bearing kidneys contained mRNA of insulin and other key genes required for the functions of beta cells. Mice transplanted with manipulated hMSCs showed reduced blood glucose levels (from 18.9+/−0.75 to 7.63+/−1.63 mM). 13 of the 16 mice became normoglycaemic (6.9+/−0.64 mM), despite the failure to detect the expression of SUR1, a K⁺-ATP channel component required for regulation of insulin secretion.

Conclusions

Our data confirm that hMSCs can be induced to express insulin sufficient to reduce blood glucose in a diabetic mouse model. Our triple gene approach has created cells that seem less glucose responsive in vitro but which become more efficient after transplantation. The maturation process requires further study, particularly the in vivo factors influencing the differentiation, in order to scale up for clinical purposes.     

Obesity Resistance and Enhanced Insulin Sensitivity in Ahnak-/- Mice Fed a High Fat Diet Are Related to Impaired Adipogenesis and Increased Energy Expenditure

ObjectiveRecent evidence has suggested that AHNAK expression is altered in obesity, although its role in adipose tissue development remains unclear. The objective of this study was to determine the molecular mechanism by which Ahnak influences adipogenesis and glucose homeostasis.DesignWe investigated the in vitro role of AHNAK in adipogenesis using adipose-derived mesenchymal stem cells (ADSCs) and C3H10T1/2 cells. AHNAK-KO male mice were fed a high-fat diet (HFD; 60% calories from fat) and examined for glucose and insulin tolerances, for body fat compositions, and by hyperinsulinemic-euglycemic clamping. Energy expenditures were assessed using metabolic cages and by measuring the expression levels of genes involved in thermogenesis in white or brown adipose tissues.ResultsAdipogenesis in ADSCs was impaired in AHNAK-KO mice. The loss of AHNAK led to decreased BMP4/SMAD1 signaling, resulting in the downregulation of key regulators of adipocyte differentiation (P<0.05). AHNAK directly interacted with SMAD1 on the Pparγ2 promoter. Concomitantly, HFD-fed AHNAK-KO mice displayed reduced hepatosteatosis and improved metabolic profiles, including improved glucose tolerance (P<0.001), enhanced insulin sensitivity (P<0.001), and increased energy expenditure (P<0.05), without undergoing alterations in food intake and physical activity.ConclusionAHNAK plays a crucial role in body fat accumulation by regulating adipose tissue development via interaction with the SMAD1 protein and can be involved in metabolic homeostasis.     

Glut4 is upregulated despite decreased insulin signaling during prolonged fasting in northern elephant seal pups

Postprandial cellular glucose uptake is dependent on an insulin-signaling cascade in muscle and adipose tissue, resulting in the translocation of the insulin-dependent glucose transporter 4 (Glut4) into the plasma membrane. Additionally, extended food deprivation is characterized by suppressed insulin signaling and decreased Glut4 expression. Northern elephant seals are adapted to prolonged fasts characterized by high levels of plasma glucose. To address the hypothesis that the fasting-induced decrease in insulin is associated with reduced insulin signaling in prolonged fasted seals, we compared the adipose protein levels of the cellular insulin-signaling pathway, Glut4 and plasma glucose, insulin, cortisol, and adiponectin concentrations between Early (n = 9; 2-3 wks postweaning) and Late (n = 8; 6-8 wks postweaning) fasted seals. Plasma adiponectin (230 ± 13 vs. 177 ± 11 ng/ml), insulin (2.7 ± 0.4 vs. 1.0 ± 0.1 μU/ml), and glucose (9.8 ± 0.5 vs. 8.0 ± 0.3 mM) decreased, while cortisol (124 ± 6 vs. 257 ± 30 nM) doubled with fasting. Glut4 increased (31%) with fasting despite the significant decreases in the cellular content of phosphatidylinositol 3-kinase as well as phosphorylated insulin receptor, insulin receptor substrate-1, and Akt2. Increased Glut4 may have contributed to the decrease in plasma glucose, but the decrease in insulin and insulin signaling suggests that Glut4 is not insulin-dependent in adipose tissue during prolonged fasting in elephant seals. The reduction of plasma glucose independent of insulin may make these animals an ideal model for the study of insulin resistance.     

Puerarin ameliorates hyperglycemia in HFD diabetic mice by promoting β-cell neogenesis via GLP-1R signaling activation

BackgroundDiabetes is characterized by β-cell loss and dysfunction. A strategy for diabetes treatment is to promote new β-cell formation. Puerarin is an isoflavone from the root of Pueraria lobata (Willd.) Ohwi. Our previous study demonstrated puerarin could ameliorate hyperglycemia in diabetic mice. However, related mechanisms and potential roles of puerarin in β-cell neogenesis have not been elucidated.PurposeThe present study aims to investigate whether anti-diabetic effect of puerarin is dependent on promoting β-cell neogenesis via GLP-1R signaling activation.MethodsA high-fat diet (HFD) induced diabetic mouse model was applied to investigate effects of puerarin in vivo, exendin-4 (GLP-1R agonist) and metformin were used as positive controls. Moreover, related mechanisms and GLP-1R downstream signal transduction were explored in isolated cultured mouse pancreatic ductal cells.ResultsPuerarin improved glucose homeostasis in HFD diabetic mice significantly. Markers of new β-cell formation (insulin, PDX1 and Ngn3) were observed in pancreatic ducts of HFD mice treated by puerarin. Of note, efficacy of puerarin in vivo was suppressed by GLP-1R antagonist exendin9-39, but enhanced by exendin-4 respectively. In cultured mouse pancreatic ductal cells, puerarin induced expressions of insulin and PDX1, upregulated GLP-1R expression and activated β-catenin and STAT3 subsequently. Expressions of insulin and PDX1 in ductal cells could be blocked by exendin9-39, or β-catenin inhibitor ICG001, or JAK2 inhibitor AG490.ConclusionThese data clarified puerarin ameliorated hyperglycemia of HFD mice via a novel mechanism involved promoting β-cell neogenesis. Our finding highlights the potential value of puerarin developing as an anti-diabetic agent.     

Effect of T3 hormone on neural differentiation of human adipose derived stem cells

Human adult stem cells, which are capable of self‐renewal and differentiation into other cell types, can be isolated from various tissues. There are no ethical and rejection problems as in the case of embryonic stem cells, so they are a promising source for cell therapy. The human body contains a great amount of adipose tissue that contains high numbers of mesenchymal stem cells. Human adipose‐derived stem cells (hADSCs) could be easily induced to form neuron‐like cells, and because of its availability and abundance, we can use it for clinical cell therapy. On the other hand, T₃ hormone as a known neurotropic factor has important impressions on the nervous system. The aim of this study was to explore the effects of T₃ treatment on neural differentiation of hADSCs. ADSCs were harvested from human adipose tissue, after neurosphere formation, and during final differentiation, treatment with T₃ was performed. Immunocytochemistry, real‐time RT‐PCR, Western blotting techniques were used for detection of nestin, MAP2, and GFAP markers in order to confirm the effects of T₃ on neural differentiation of hADSCs. Our results showed an increase in the number of glial cells but reduction in neuronal cells number fallowing T₃ treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Insulin Stimulated-Glucose Transporter Glut 4 Is Expressed in the Retina

The vertebrate retina is a very metabolically active tissue whose energy demands are normally met through the uptake of glucose and oxygen. Glucose metabolism in this tissue relies upon adequate glucose delivery from the systemic circulation. Therefore, glucose transport depends on the expression of glucose transporters. Here, we show retinal expression of the Glut 4 glucose transporter in frog and rat retinas. Immunohistochemistry and in situ hybridization studies showed Glut 4 expression in the three nuclear layers of the retina: the photoreceptor, inner nuclear and ganglionar cell layers. In the rat retina immunoprecipitation and Western blot analysis revealed a protein with an apparent molecular mass of 45 kDa. ¹⁴C-glucose accumulation by isolated rat retinas was significantly enhanced by physiological concentrations of insulin, an effect blocked by inhibitors of phosphatidyl-inositol 3-kinase (PI3K), a key enzyme in the insulin-signaling pathway in other tissues. Also, we observed an increase in ³H-cytochalasin binding sites in the presence of insulin, suggesting an increase in transporter recruitment at the cell surface. Besides, insulin induced phosphorylation of Akt, an effect also blocked by PI3K inhibition. Expression of Glut 4 was not modified in retinas of a type 1 diabetic rat model. To our knowledge, our results provide the first evidence of Glut4 expression in the retina, suggesting it as an insulin- responsive tissue.     

Phosphorylated S6K1 (Thr389) is a molecular adipose tissue marker of altered glucose tolerance

Molecular tissue markers of altered glucose metabolism will be useful as potential targets for antidiabetic drugs. S6K1 is a downstream signal of insulin action. We aimed to evaluate ^pThr389S6K1 and total S6K1 levels in human and rat fat depots as candidate markers of altered glucose metabolism. ^pThr389S6K1 and total S6K1 levels were measured using enzyme linked immune sorbent assay (ELISA) in 49 adipose tissue samples from subjects with morbid obesity and in 18 peri-renal white adipose tissue samples from rats. The effects of high glucose and rosiglitazone have been explored in human preadipocytes. ^pThr389S6K1/_totalS6K1 in subcutaneous adipose tissue was significantly increased subjects with Type 2 diabetes (0.78±0.26 vs. 0.55±0.14, P=.02) and associated with fasting glucose (r=0.46, P=.04) and glycated hemoglobin (r=0.63, P=.02) in SAT. Similar associations with fasting glucose (r=0.43, P=.03) and IRS1 (r=-0.41, P=.04) gene expression were found in visceral adipose tissue. In addition, rat experiments confirmed the higher ^pThr389S6K1/totalS6K1 levels in adipose tissue in association with obesity-associated metabolic disturbances. ^pThr389S6K1/totalS6K1 was validated using western blot in rat adipose tissue. Both ELISA and western blot data significantly correlated (r=0.85, P=.005). In human preadipocytes, high glucose medium led to increased ^pThr389S6K1/total S6K1 levels in comparison with normal glucose medium, which was significantly decreased under rosiglitazone administration. In conclusion, in human and rat adipose tissue, phosphorylated S6K1 is a marker for increased glucose levels.     

Insulin-regulated Glut4 Translocation: MEMBRANE PROTEIN TRAFFICKING WITH SIX DISTINCTIVE STEPS*

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (k_en = 0.2 min⁻¹ versus 0.6 min⁻¹). Differentiation decreases Glut4 k_en 40% (k_en = 0.12 min⁻¹). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (k_ex = 0.025–0.038 min⁻¹), not through the fast Tf receptor pathway (k_ex = 0.2 min⁻¹). The k_ex measured in adipocytes after insulin stimulation is similar (k_ex = 0.027 min⁻¹). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (k_sort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (k_seq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (k_fuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.     

脂肪干细胞的诱导分化及其来源的研究

目的:通过组织块培养法得到脂肪干细胞(adipose-derived stem cells,ADSCs),探讨其诱导分化潜能,并初步研究ADSCs的来源。方法:用脂肪组织块培养法培养原代人ADSCs。第三代ADSCs进行成脂和成骨诱导分化,分别用油红O和茜素红S染色进行鉴定。脂肪组织块培养七天后取脂肪组织进行Hematoxylin-eosin Staining(HE)染色观察ADSCs组织分布。结果:用脂肪组织块培养法成功培养出原代人ADSCs。ADSCs传代到第8代,依然保持着良好的增殖能力和细胞形态。ADSCs能成功诱导成脂肪细胞和骨细胞。通过对培养七天后的脂肪组织块进行HE染色,发现ADSCs主要分布在脂肪组织的间质血管和结缔组织周围。结论:用脂肪组织块培养出来的ADSCs具有成脂和成骨分化的潜能。ADSCs主要定位于间质血管和结缔组织周围。     

Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR

BackgroundInsulin-like growth factor 2 (IGF2), an essential component of the stem cell niche, has been reported to modulate the proliferation and differentiation of stem cells. Previously, a continuous expression of IGF2 in tissues was reported to maintain the self-renewal ability of several types of stem cells. Therefore, in this study, we investigated the expression of IGF2 in adipose tissues and explored the effects of IGF2 on adipose-derived stromal cells (ADSCs) in vitro.MethodsThe expression pattern of IGF2 in rat adipose tissues was determined by gene expression and protein analyses. The effect of IGF2 on proliferation, stemness-related marker expression and adipogenic and osteogenic differentiation was systematically investigated. Furthermore, antagonists of IGF2-specific receptors—namely, BMS-754807 and picropodophyllin—were added to explore the underlying signal transduction mechanisms.ResultsIGF2 levels displayed a tendency to decrease with age in rat adipose tissues. After the addition of IGF2, isolated ADSCs displayed higher proliferation and expression of the stemness-related markers NANOG, OCT4 and SOX2 and greater differentiation potential to adipocytes and osteoblasts. Additionally, both type 1 insulin-like growth factor receptor (IGF-1R) and insulin receptor (IR) participated in the IGF2-mediated promotion of stemness in ADSCs.ConclusionsOur findings indicate that IGF2 could enhance the stemness of rat ADSCs via IGF-1R and IR and may highlight an effective method for the expansion of ADSCs for clinical application.     

Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Glucose Regulates Steady-state Levels of PDX1 via the Reciprocal Actions of GSK3 and AKT Kinases

The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured β cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.     

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2)

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2^−/−). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in β-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-β-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2^−/− islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2^−/− mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2^−/− mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9–39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca²⁺ entry through L-type voltage-dependent Ca²⁺ channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.     

原儿茶酸促进人脂肪干细胞体外增殖的研究

为了寻找能够促进干细胞增殖的药物,观察了中药益智仁（Alpinia oxyphylln）中提取的原儿茶酸对人脂肪干细胞体外增殖的影响,并对其作用机制进行了初步的探讨．人脂肪干细胞能在体外分化为神经元样细胞,并对凋亡的PC-12细胞起到保护作用．原儿茶酸能够促进人脂肪干细胞的增殖,且呈现明显的剂量依赖性和时间依赖性．流式细胞术检测细胞DNA含量的结果显示,原儿茶酸处理组细胞S期所占比例明显增加,其中,1．5mmol／L原儿茶酸处理组细胞S期所占比例与对照组相比增加2倍以上．同时,该组细胞G2／M期所占比例明显增加,G0／G1期所占比例明显下降．蛋白质免疫印迹结果显示,1．5mmol／L原儿茶酸处理组细胞周期素D1（cyclinD1）的表达明显升高．cyclin D1-siRNA转染显著抑制了原儿茶酸对人脂肪干细胞体外增殖的促进作用．流式细胞术检测细胞表面标志物,成骨诱导和脂肪诱导的结果显示,原儿茶酸处理后,人脂肪干细胞仍保持间充质干细胞多分化潜能的特性．上述结果提示,原儿茶酸有可能在人脂肪干细胞介导的干细胞移植治疗中发挥作用．