A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Effects of thiazolidinediones on differentiation, proliferation, and apoptosis

Thiazolidinediones induce adipocyte differentiation and thereby limit proliferative potential; hence, early investigations focused on their ability to modulate cellular proliferation and apoptosis. Several lines of evidence indicate significant thiazolidinedione-mediated antitumor activity. An emerging view is that some antitumor effects are totally or partially peroxisome proliferator-activated receptor-gamma (PPARgamma) dependent, whereas others are PPARgamma independent. The aim of this review is to examine the current evidence about the molecular mechanisms by which thiazolidinediones augment cellular differentiation, inhibit cellular proliferation, and induce apoptosis. We first address the role of thiazolidinediones and/or PPARgamma on Wnt/beta-catenin signaling pathway as it affects cellular differentiation and then discuss other pathways that are also involved in differentiation as well as proliferation and apoptosis.     

Subcutaneous Abdominal Adipose Tissue Subcompartments: Potential Role in Rosiglitazone Effects

Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial‐SAT (sSAT) and deep‐SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin‐sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot‐specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin‐sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata‐specific differences in differentiation including peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport‐4 (GLUT4), glucocorticoid metabolism with 11β‐hydroxysteroid dehydrogenase type‐1 (11βHSD1), and alterations in the adipokine leptin. While depot‐specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR‐γ, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT‐specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11βHSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.     

Alterations of peroxisome proliferator-activated receptor delta activity affect fatty acid-controlled adipose differentiation

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARgamma or PPARdelta promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARdelta in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARdelta mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARdelta enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARgamma and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARdelta mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARdelta plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARdelta expression or activity could affect adaptive responses of white adipose tissue to nutritional changes.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

Molecular and biochemical events during differentiation of the HD3 chicken erythroblastic cell line

The chicken erythroblast cell line HD3 is transformed by a temperature-sensitive mutant of avian erythroleukemia virus. Upon shift to the non-permissive temperature in the presence of inducers (hemin and butyric acid), HD3 cells differentiate to an erythrocyte phenotype and provide a model system for analyzing events associated with this process. Expression of some cell surface proteins undergoes drastic changes as cells mature to the erythrocyte stage with a selective loss of membrane proteins that appears to be species-specific. Specific changes also occur in the expression and activities of cytosolic enzymes reflecting alterations of metabolism. HD3 differentiation is characterized by increased transferrin receptor (TFR) expression and increased hemoglobin (Hb) synthesis, a marker for the erythrocyte. In parallel, there is a decrease in glucose transport and an increase in nucleoside transport signifying a switch from glycolytic hexose metabolism to metabolism of pentose from nucleoside. Likewise the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD) declines while glucose-6-phosphate dehydrogenase (G6PDH) activity remains constant. Commitment to the erythrocyte lineage alters expression of specific genes: TFR mRNA level increases while expression decreases for GLUT1 and GLUT3 glucose transporter mRNAs and GAD mRNA. However, the relationship between GAD activity and GAD mRNA was complex indicating modulation of GAD mRNA and protein half-lives. Serine/threonine and tyrosine phosphorylation and cAMP levels were shown to regulate the level of these messages. In this review, we describe how HD3 differentiation involves changes in plasma membrane composition, metabolism and gene expression that are orchestrated at different levels of control by multiple signaling modalities.     

Microarray analysis of brain RNA in mice with methylenetetrahydrofolate reductase deficiency and hyperhomocysteinemia

Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common genetic cause of hyperhomocysteinemia, which is associated with increased risk for cardiovascular disease, stroke and possibly other neurological disorders. Microarray analysis of brain RNA from day 14 Mthfr(-/-) mice revealed several genes with altered expression. Expression changes in inositol 1,4,5-triphosphate receptor, type 1 (Itpr1), proteolipid protein (Plp), neurogenic differentiation factor 1 (Neurod1), S100 calcium binding protein A8 (S100a8), and methylenetetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) were confirmed by RT-PCR. We propose that neuronal damage by hyperhomocysteinemia may involve disruption of intracellular calcium.     

A selective peroxisome proliferator-activated receptor-gamma (PPARgamma) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists such as the thiazolidinediones are insulin sensitizers used in the treatment of type 2 diabetes. These compounds induce adipogenesis in cell culture models and increase weight gain in rodents and humans. We have identified a novel PPARgamma ligand, LG100641, that does not activate PPARgamma but selectively and competitively blocks thiazolidinedione-induced PPARgamma activation and adipocyte conversion. It also antagonizes target gene activation as well as repression in agonist-treated 3T3-L1 adipocytes. This novel PPARgamma antagonist does not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR), the heterodimeric partner for PPARgamma, or by a differentiation cocktail containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Surprisingly, LG100641, like the PPARgamma agonist rosiglitazone, increases glucose uptake in 3T3-L1 adipocytes. Such selective PPARgamma antagonists may help determine whether insulin sensitization by thiazolidinediones is mediated solely through PPARgamma activation, and whether there are PPARgamma-ligand-independent pathways for adipocyte differentiation. If selective PPARgamma modulators block adipogenesis in vivo, they may prevent obesity, lower insulin resistance, and delay the onset of type 2 diabetes.     

The metabolism of proline,a stress substrate,modulates carcinogenic pathways

The resurgence of interest in tumor metabolism has led investigators to emphasize the metabolism of proline as a “stress substrate” and to suggest this pathway as a potential anti-tumor target. Proline oxidase, a.k.a. proline dehydrogenase (POX/PRODH), catalyzes the first step in proline degradation and uses proline to generate ATP for survival or reactive oxygen species for programmed cell death. POX/PRODH is induced by p53 under genotoxic stress and initiates apoptosis by both mitochondrial and death receptor pathways. Furthermore, POX/PRODH is induced by PPARγ and its pharmacologic ligands, the thiazolidinediones. The anti-tumor effects of PPARγ may be critically dependent on POX/PRODH. In addition, it is upregulated by nutrient stress through the mTOR pathway to maintain ATP levels. We propose that proline is made available as a stress substrate by the degradation of collagen in the microenvironmental extracellular matrix by matrix metalloproteinases. In a manner analogous to autophagy, this proline-dependent process for bioenergetics from collagen in extracellular matrix can be designated “ecophagy”.     

Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.     

Asiatic Acid Inhibits Adipogenic Differentiation of Bone Marrow Stromal Cells

Bone marrow mesenchymal stromal cells (BMSCs) are the common precursors for both osteoblasts and adipocytes. With aging, BMSC osteoblast differentiation decreases whereas BMSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. In the present study, we investigated the effect of asiatic acid (AA) on adipocytic differentiation of BMSCs. AA inhibited the adipogenic induction of lipid accumulation, activity of glycerol-3-phosphate dehydrogenase, and expression of marker genes in adipogenesis: peroxisome proliferation-activated receptor (PPAR)γ, adipocyte fatty acid-binding protein (ap) 2, and adipsin. Further, we found that AA did not alter clonal expansion rate and expression of C/EBPβ, upstream key regulator of PPARγ, and binding activity of C/EBPβ to PPARγ promoter was not affected by AA as well. These findings suggest that AA may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes, and inhibition of PPARγ by AA is through C/EBPβ-independent mechanisms. Thus, AA could be a potential candidate for a novel drug against osteoporosis.     

Trypsinization of chick glial cells before seeding: Effects on energy metabolism enzymes and glutamine synthetase

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.     

Expression of γ-chain cytokine receptors on CD8+ T cells in HIV infection with a focus on IL-7Rα (CD127)

When interleukin-2 (IL-2) receptor γ-chain (γ(C))-sharing cytokine receptors on T cells bind their specific ligands (IL-2, -4, -7, -9, -15 or -21), they initiate a variety of cell signals that promote survival, differentiation or antiviral or antitumor cytolytic functions. Although expression of the γ(C) is constitutive across T-cell subsets, the varying expression of other receptor complex components can regulate cytokine signalling and function. Impaired γ(C) cytokine activity in HIV infection, and the role of γ(C) cytokines in CD8(+) T-cell function and homeostasis, implicates these molecules among potential contributors to the observed decline of cytolytic activity (CTL) in HIV disease. In particular, this review will be highlighting information about the IL-7 receptor (IL-7R) complex, which is composed of the γ(C) and the IL-7Rα (CD127) chains. There has been an abundance of HIV-related CD127 research and its important role in CD8(+) T-cell survival and function. The expression of CD127 undergoes dramatic changes throughout the course of T-cell responses in HIV infection. The expression of CD127 is significantly decreased in progressive HIV disease, whereas effective antiretroviral therapy results in its recovery. Observations of impaired IL-7 activity in HIV(+) individuals have suggested that CD127 has an important role in HIV immunopathogenesis. In addition, a soluble form of CD127 (sCD127) is upregulated in the plasma of HIV(+) individuals. Hence, CD127 is being increasingly considered as a marker of disease prognosis, and related information may provide insight into understanding the expression and role of other γ(C) receptors in HIV disease and contribute to the development of novel cytokine-based therapeutics.