脂联素球状结构域对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响

目的:通过探讨脂联素球状结构域(gAd)对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响,进而探讨gAd促进脂肪细胞摄取的葡萄糖是否经磷酸戊糖途径代谢。方法:用gAd干预分化成熟的3T3-L1脂肪细胞,干预结束后测定细胞残液的葡萄糖浓度,并以实时荧光定量PCR(RT-PCR)法检测各组细胞磷酸戊糖途径关键酶葡萄糖-6-磷酸酶(G6PD)转录水平的表达情况,进行统计学分析。结果:各实验组细胞残液中葡萄糖浓度均显著低于对照组(均P     

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency: nucleotide inhibition of G6PD and the pentose phosphate shunt

We evaluated the erythrocytes of two patients with hereditary pyrimidine 5'-nucleotidase deficiency. Significant findings included an increased reduced glutathione content, increased incubated Heinz body formation, a positive ascorbate cyanide test, and decreased intraerythrocytic pH. The pentose phosphate shunt activity of the patients' red cells as measured by the release of 14CO2 from 14C-1- glucose was decreased compared to high reticulocyte controls. Glucose-6- phosphate dehydrogenase (G6PD) activity in hemolysates from control erythrocytes was inhibited 43% by 5.5 mM cytidine 5'-triphosphate (CTP) and 50% by 5.5 mM in uridine 5'-triphosphate (UTP) at pH 7.1. CTP was a competitive inhibitor for G6P (Ki = 1.7 mM) and a noncompetitive inhibitor for NADP+ (Ki = 7.8 mM). Glutathione peroxidase, glutathione reductase, and 6-phosphogluconate dehydrogenase were not affected by these compounds. Pentose phosphate shunt activity in control red cell hemolysate at pH 7.1 was inhibited to a similar degree by 5.5 mM CTP or UTP. Since the intracellular concentrations of G6P and NADP+ are below their KmS for G6PD, these data suggest that high concentrations of pyrimidine 5'-nucleotides depress pentose phosphate shunt activity in pyrimidin 5'-nucleotidase deficiency. Thus, this impairment of the pentose phosphate pathway appears to contribute to the pathogenesis of hemolysis in pyrimidine 5'-nucleotidase deficiency hemolytic anemia.     

Constant expression of hexose-6-phosphate dehydrogenase during differentiation of human adipose-derived mesenchymal stem cells

The reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11beta-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.     

Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency

S ummary . Pentose phosphate shunt activity was studied by the release of ¹⁴CO₂ from ¹⁴C-1-glucose and ¹⁴C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7·1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate ( K _i= 1·05 mM) and a noncompetitive inhibitor with NADP ( K _i= 3·3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their K _ms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.     

Effect of dehydroepiandrosterone on cell growth and mitochondrial function in TM-3 cells

Dehydroepiandrosterone (DHEA), a major steroid hormone, decreases with age, and this reduction has been shown to be associated with physical health. In the present study, the effect of DHEA on cell growth and mitochondrial function was investigated using TM-3 cells, a Leydig cell line. The growth of TM-3 cells exposed to 100 μM DHEA for 24h was inhibited due to cell cycle arrest, primarily in the S and G2/M phases, and this effect was caused by decreased activity of glucose-6-phosphate dehydrogenase (G6PD) and reduced expression of cyclinA and cyclinB mRNA. A novel finding was that DHEA improved TM-3 cell viability in a markedly time-dependent manner. Although no differences were observed in the configuration or number of TM-3 cell mitochondria following DHEA treatment, mitochondrial membrane permeability and the activity of succinate dehydrogenase (SDH) increased subsequent to 24h treatment of cells with 100 μM DHEA. Overall, the data demonstrate that DHEA inhibited TM-3 cell growth by decreasing G6PD activity and the expression of cyclin mRNAs, whereas it improved TM-3 cell viability by increasing mitochondrial membrane permeability and the activity of SDH. This could be one of mechanisms of DHEA exerts its biological function.     

Insulin down-regulates resistin mRNA through the synthesis of protein(s) that could accelerate the degradation of resistin mRNA in 3T3-L1 adipocytes

AIMS/HYPOTHESIS: Resistin is a peptide secreted by adipocytes and recognized as a hormone that could link obesity to insulin resistance. This study was designed to examine the effect and mechanism(s) of insulin on resistin expression in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were stimulated with insulin and resistin mRNA expression was examined by Northern blot analysis. In some experiments, the insulin signal was blocked by several chemical inhibitors or overexpression of a dominant negative form (Deltap85) of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). RESULTS: Insulin treatment caused a reduction of resistin mRNA in time-dependent and dose-dependent manners in 3T3-L1 adipocytes. Pre-treatment with PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, or SB203580, an inhibitor of p38 mitogen-activated protein-kinase (p38 MAP-kinase) pathway, did not influence insulin-induced reduction of resistin mRNA. Inhibition of PI 3-kinase by LY294002 or Deltap85 also failed to block insulin-induced reduction of resistin mRNA. Cycloheximide, a protein synthesis inhibitor, completely blocked insulin-induced reduction of resistin mRNA. Actinomycin D, a RNA synthesis inhibitor, also blocked insulin-induced reduction of resistin mRNA, and the decreasing rate of resistin mRNA in cells treated with insulin alone was faster than that with actinomycin D. CONCLUSION/INTERPRETATION: Insulin downregulates resistin mRNA via PI 3-kinase, ERK or p38 MAP-kinase independent pathways in 3T3-L1 adipocytes. The downregulation mechanism of resistin mRNA by insulin would be an indirect event through the synthesis of novel protein(s) that could accelerate the degradation of resistin mRNA.     

Insulin-stimulated methylaminoisobutyric acid uptake in 3T3-L1 fibroblasts and fat cells

3T3-L1 fibroblasts and fat cells have been extensively used to study the development of insulin-stimulated glucose and lipid metabolism during adipocyte differentiation in vitro. In this paper we explore the ability of insulin to stimulate amino acid uptake in 3T3-L1 cells using the nonmetabolizable amino acid analog methylaminoisobutyric acid (MAIB). In differentiated 3T3-L1 fat cells, a 12-h preincubation with insulin was required for maximal stimulation of MAIB uptake. In contrast, in the undifferentiated fibroblasts, insulin stimulation peaked between 6 and 8 h and then declined significantly. Maximal stimulation of MAIB uptake in the differentiated fat cell exceeded that in the fibroblast phenotype. This increased ability of insulin to stimulate MAIB uptake in fat cells appeared within the first day after removal of the differentiation medium. 3T3-L1 fat cells were 30 times more sensitive to the effects of insulin on MAIB transport than the undifferentiated fibroblasts. These findings are consistent with previous data on insulin-stimulated deoxyglucose uptake, in that the increased sensitivity to insulin with differentiation is more than can be accounted for by the increase in receptor number. The activity of porcine proinsulin indicates that this stimulation reflects the known characteristics of the insulin receptor. The stimulation of MAIB uptake by insulin in 3T3-L1 fat cells was blocked by inhibitors of protein synthesis (cycloheximide and puromycin) and mRNA synthesis (actinomycin D). Colchicine, an inhibitor of microtubule function, showed little inhibition of insulin-stimulated MAIB uptake. Insulin stimulation of MAIB uptake was greater when 3T3-L1 cells were preincubated with insulin in the absence of essential amino acids. Basal transport in 3T3-L1 cells was not influenced by the presence or absence of amino acids. Thus, amino acid deprivation appears specifically to enhance the ability of insulin to stimulate amino acid transport in cultured adipocytes.     

Potent inhibition of thyroid cancer cells by the MEK inhibitor PD0325901 and its potentiation by suppression of the PI3K and NF-kappaB pathways

BACKGROUND: We recently demonstrated inhibition of thyroid cancer cells by the MEK inhibitor CI-1040. The objective of this study was to use a potent new-generation MEK inhibitor PD0325901 to further investigate the therapeutic potential of specifically targeting MEK in the MAP kinase pathway for thyroid cancer. METHODS: We examined the effects of PD0325901 on a variety of cellular and molecular activities of thyroid cancer cell lines with distinct genotypes. RESULTS: PD0325901 remarkably inhibited MAP kinase pathway signaling in the thyroid cancer cells tested. It potently inhibited cell proliferation (IC(50) = 0.059-0.783 microM) and arrested cell cycle at the G0/G1 phase of cells harboring BRAF or RAS mutations but not cells harboring wild-type alleles or the RET/PTC1 rearrangement. Synergistic inhibitory effects were observed when PD0325901 was combined with phosphatidylinositol 3-kinase (PI3K) or NF-kappaB pathway inhibitors in most cells, including the RET/PTC1-harboring cells. PD0325901 could inhibit invasion and anchorage-independent growth of thyroid cancer cells independently of the type of genetic alterations. This compound did not seem to have significant proapoptotic effects, however. CONCLUSIONS: The MEK inhibitor PD0325901 has a wide range of potent inhibitory effects on thyroid cancer cells, some of which seemed to be genotype-selective, consistent with the results previously observed with an early-generation MEK inhibitor, CI-1040. The data provide further evidence that targeted inhibition of MEK may be therapeutically effective for thyroid cancer, particularly if the PI3K and NF-kappaB pathways are concurrently inhibited.     

Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.

Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts.     

Resistin promotes 3T3-L1 preadipocyte differentiation

OBJECTIVE: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. DESIGN: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. METHODS: The rat resistin gene was inserted into the pDual GC and pEFGP-N2 expression vectors for examination of the effects of resistin overexpression in 3T3-L1 cells before and after differentiation was stimulated with 3-isobutyl-1-methyxanthine (MIX), insulin and dexamethasone (DEX). Smaller conserved fragments were inserted into short interference RNA (siRNA) expression vectors, for examination of the effect of targeted resistin inhibition on differentiation of resistin-overexpressing 3T3-L1 cells. RESULTS: Prior to stimulation, the resistin-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells. Following stimulation, differentiation in the resistin-transfected 3T3-L1 cells was dramatically promoted, especially in the early stages. Stimulation of differentiation was also observed in non-transfected 3T3-L1 cells grown in resistin protein-containing conditioned medium. The expression of adipocyte differentiation-associated markers such as CCAAT enhancer binding protein (C/EBPalpha), retinoid X receptor (RXRalpha) and lipoprotein lipase (LPL) was upregulated in resistin-overexpressing cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. In addition, expression of two of the three tested siRNAs inhibited the adipoconversion process, providing further evidence that resistin promotes the differentiation of preadipocytes to adipocytes. CONCLUSION: Resistin can promote preadipocyte differentiation. Based on this, we propose that resistin may be an important candidate mediator of obesity-induced insulin resistance.     

Endothelin-1 regulates adiponectin gene expression and secretion in 3T3-L1 adipocytes via distinct signaling pathways

Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase's ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1.     

Trypanosoma cruzi strains, Tulahuen 2 and Y, besides the difference in resistance to oxidative stress, display differential glucose-6-phosphate and 6-phosphogluconate dehydrogenases activities

The drugs currently available for Chagas'disease treatment are unsatisfactory due to limited efficacy and toxic side effects, making the search for more specific pharmacological agents a priority. The components of the Trypanosoma cruzi trypanothione-dependent antioxidant system have been pointed out as potential chemotherapeutic targets for the development of more specific drugs. To work properly, this system must have a current supply of NADPH, provided by glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). Here, we compare two T. cruzi strains, Tulahuen 2 and Y, regarding growth rate, cytosolic tryparedoxin peroxidase (TcCPX) concentration and pentose phosphate pathway dehydrogenases activities. Tulahuen 2 cells show higher values as compared to the Y strain when the following parameters are compared: TcCPX concentration, resistance to H2O2, growth index and G6PD activity. Different patterns of G6PD and 6PGD activities were observed among strains along the growth curve and when cells were challenged with H2O2. These data reinforce the heterogeneity within T. cruzi populations and also the importance of G6PD in protecting the parasite against reactive oxygen species.     

ACTH and alpha-MSH inhibit leptin expression and secretion in 3T3-L1 adipocytes: model for a central-peripheral melanocortin-leptin pathway

Leptin is the 167 amino-acid protein product of the Lep (obese) gene that is released predominantly from adipose tissue and circulates at levels related to the amount of fat. Leptin expression is hormonally regulated: insulin and glucocorticoids are stimulators, while inhibitors include beta-adrenergic agonists and testosterone. Recently, adenylate cyclase-coupled melanocortin receptors have been identified in murine adipose tissue, the 3T3-L1 adipocyte cell line, and in human fat tissue. These studies prompted us to evaluate the effects of pro-opiomelanocortin (POMC)-derived peptides on leptin production and expression in 3T3-L1 adipocytes in culture. 3T3-L1 pre-adipocytes differentiated by the insulin/indomethacin (I/I) method produced leptin at levels that were two times higher than those obtained in cells differentiated by the more traditional insulin/dexamethasone/isobutylmethylxanthine (I/D/M) method. By RT-PCR studies, 3T3-L1 cells expressed both the melanocortin 2 receptors (MC2-R) and melanocortin 5 receptors (MC5-R) isoforms of the melanocortin receptor at an early stage of differentiation. When I/I differentiated 3T3-L1 adipocytes were incubated with different concentrations of dibutyryl cAMP (db-cAMP) or POMC-derived peptides (ACTH and alpha-MSH), ACTH and alpha-MSH stimulated cAMP production after 30 min (2-fold increase) associated with a dose-dependent inhibition of leptin secretion (ACTHz.Gt;alpha-MSH; IC(50)=3.2+/-0.4 SE and 36+/-5 nM, respectively), maximal after 3 h of incubation (30% inhibition). In addition, 100 nM ACTH and alpha-MSH induced a 60% reduction in leptin expression by RT-PCR. Incubation of cells with 0.5 mM db-cAMP led to a more prominent inhibition of leptin expression and secretion (up to 80% at 1 and 24 h, respectively). The ACTH and alpha-MSH inhibitory effects on leptin secretion were mediated by activation of the MC2-R and MC5-R and were reversed by the MC-R antagonists ACTH(11-24) and ACTH(7-38). In summary, we have shown that POMC-peptides are potent inhibitors of leptin expression and production in 3T3-L1 adipocytes. The finding of ACTH/alpha-MSH receptor-induced inhibition of leptin production and expression in adipocytes support the possibility that there is a control mechanism for modulation of adipose tissue function via a melanocortin-leptin axis.     

Increased prevalence of proliferative retinopathy in patients with type 1 diabetes who are deficient in glucose-6-phosphate dehydrogenase

Aims/hypothesis  

Impaired activity of the pentose phosphate pathway of glucose metabolism caused by hereditary deficiency of its key regulatory enzyme glucose-6-phosphate dehydrogenase (G6PD) has consequences that may worsen or attenuate the course of diabetic complications. Decreased availability of NADPH can predispose to oxidative stress and endothelial dysfunction, but can also limit the activity of the polyol pathway and cholesterol synthesis. Reduced availability of pentose phosphates for nucleic acid synthesis could impair cell proliferation. We sought to learn in which direction G6PD deficiency affects diabetic retinopathy.