Suppression of 11beta-hydroxysteroid dehydrogenase type 1 with RNA interference substantially attenuates 3T3-L1 adipogenesis.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which regulates the local level of glucocorticoids, has been suggested to be involved in the development of obesity. A definitive functional role for 11beta-HSD1 in adipogenesis, however, remains to be established. We developed 3T3-L1 cell lines stably transfected with a small hairpin RNA (shRNA) targeting 11beta-HSD1. A shRNA containing two nucleotide substitutions was used as a control. Silencing of 11beta-HSD1 substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes, which was induced by a mixture containing either corticosterone or dexamethasone. Silencing of 11beta-HSD1 increased the concentration of 11-dehydrocorticosterone in the culture supernatant but did not significantly affect the levels of corticosterone or dexamethasone. Translocation of glucocorticoid receptors to the nucleus in response to glucocorticoids was significantly attenuated by silencing 11beta-HSD1. The number of cells entering the S phase of the cell cycle following the induction of adipogenesis was significantly reduced by silencing 11beta-HSD1. 11beta-HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation, however, did not affect the progression of adipogenesis. These results indicate that 11beta-HSD1 plays a significant functional role in the initiation of 3T3-L1 adipogenesis and provide new mechanistic insights into the role of 11beta-HSD1 in the development of obesity and related diseases.     

11β-hydroxysteroid Dehydrogenase 1 in Adipocytes: Expression is Differentiation-dependent and Hormonally Regulated

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the reversible metabolism of physiological glucocorticoids (cortisol, corticosterone) to inactive metabolites (cortisone, 11-dehydrocorticosterone), thus regulating glucocorticoid access to receptors. 11β-HSD-1 expression is regulated during development and by hormones in a tissue specific manner. The enzyme is highly expressed in liver, where it may influence glucocorticoid action on fuel metabolism, processes also important in adipose tissue. Here we show that 11β-HSD-1 is expressed in white adipose tissue, in both the adipocyte and stromal/vascular compartments, and in the adipocyte cell lines 3T3-F442A and 3T3-L1. In these cells, 11β-HSD-1 expression is induced upon differentiation into adipocytes and is characteristic of a ‘late differentiation’ gene, with maximal expression 6-8 days after confluence is reached. In intact 3T3-F442A adipocytes the enzyme direction is predominantly 11β-reduction, activating inert glucocorticoids. The expression of 11β-HSD-1 mRNA is altered in fully differentiated 3T3-F442A adipocytes treated with insulin, dexamethasone or a combination of the hormones, in an identical manner to glycerol-3-phosphate dehydrogenase (GPDH) mRNA (encoding a key enzyme in triglyceride synthesis and a well-characterised marker of adipocyte differentiation). The demonstration of 11β-HSD-1 expression in adipocytes and its predominant reductase activity in intact 3T3-F442A adipocytes suggests that 11β-HSD-1 may play an important role in potentiating glucocorticoid action in these cells. 3T3-F442A and 3T3-L1 represent useful model systems in which to examine the factors which regulate 11β-HSD-1 gene expression and the role of 11β-HSD-1 in modulating glucocorticoid action in adipose tissue.     

Fructose promotes the differentiation of 3T3-L1 adipocytes and accelerates lipid metabolism

Excessive fructose consumption and elevated glucocorticoids contribute to metabolic syndrome. We show that fructose as the only carbohydrate source is sufficient for the differentiation of 3T3-L1 fibroblasts into adipocytes. Differentiation of cells in fructose containing medium resulted in increased 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) expression and activity. Experiments with transfected HEK-293 cells suggested more efficient NADPH generation by fructose compared with glucose in the endoplasmic reticulum (ER). Adipocytes differentiated in the presence of fructose showed increased FABP4 expression, C/EBPα to C/EBPβ ratio and lipolysis. Thus, excessive fructose may cause adverse metabolic effects by enhancing 11β-HSD1 activity and increasing lipolysis in adipocytes.     

Dexamethasone induces high-amplitude rhythms in preadipocytes,But hinders circadian expression in differentiated adipocytes

Glucocorticoids induce circadian gene expression in cultured cells and change the phase of circadian gene expression in vivo. In addition, glucocorticoids induce differentiation of preadipocyte to adipocytes. We set out to test the effect of dexamethasone, a glucocorticoid receptor agonist, on circadian rhythms in 3T3-L1 differentiated adipocytes. Our results show that differentiated adipocytes exhibit robust circadian rhythms without dexamethasone. Dexamethasone induces phase changes and increases the amplitude of circadian gene expression in nondifferentiated 3T3-L1 preadipocytes. However, dexamethasone had an opposite effect on differentiated adipocytes, leading to low-amplitude circadian expression. In conclusion, although glucocorticoids reset circadian rhythms, once rhythms are reset, glucocorticoid administration hinders circadian expression.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA levels for PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2-deoxy glucose uptake both in basal and insulin-stimulated state of adipocytes, which may contribute, at least partly, to its insulin-sensitizing ability.     

Quantitation and cellular localization of 11beta-HSD1 expression in murine thymus

11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1), an NADPH-dependent reductase, functions in intact cells to convert inactive 11-keto metabolites of glucocorticoids into biologically active glucocorticoids. The enzyme is thus capable of amplifying glucocorticoid action in tissues in which it is expressed. In the experiments presented here, we show that 11beta-HSD1 is expressed in the murine thymus and that expression increases from late fetal development to maximal levels in the adult thymus. Quantitative real time-PCR, immunoblots, and assays of enzymatic activity reveal adult thymic expression of 11beta-HSD1 mRNA and protein at levels approximately 6-7% of those observed in liver. Immunofluorescence experiments show that the enzyme is expressed in the medullary thymocytes and thymocytes present at the corticomedullary junction. These experiments extend our recognition of 11beta-HSD1 expression in cells of the immune system and lend support to the notion that glucocorticoid signaling and amplification of those signals by regeneration of active glucocorticoids from inactive 11-keto metabolites might impact intrathymic T cell development and the establishment of the immune repertoire.     

The functional consequences of 11beta-hydroxysteroid dehydrogenase expression in adipose tissue.

Clinical observations have highlighted the link between glucocorticoids and obesity. While exogenous glucocorticoids in excess predispose to the development of central obesity, we have focused on cortisol metabolism within human adipose tissue. 11beta-hydroxysteroid dehydrogenase (11beta-HSD) inter-converts the active glucocorticoid, cortisol, and inactive cortisone. 11beta-HSD1, the only isoform expressed in adipose tissue, acts predominantly as an oxoreductase to generate cortisol. Expression is higher in omental compared to subcutaneous preadipocytes and activity and expression are potently regulated by growth factors and cytokines. Mice over-expressing 11beta-HSD1 specifically within adipocytes develop central obesity. However, the situation is less clear in humans. Globally, there appears to be inhibition of the enzyme, but expression in human obesity is still not fully characterized; its functional role in adipocyte biology remains to be elucidated. In vitro, 11beta-HSD1 appears to function in promoting adipocyte differentiation and limiting preadipocyte proliferation, but the impact of these effects in vivo upon the regulation of fat mass remains to be defined. Clinical studies utilizing selective 11beta-HSD1 inhibitors may help to answer this question.     

Regulation of the expression of pp160, a putative insulin receptor signal protein, by insulin, dexamethasone, and 1-methyl-3-isobutylxanthine in 3T3-L1 adipocytes.

pp160, a cytosolic protein with Mr of approximately 160,000, is phosphorylated on tyrosine in response to insulin and is considered to be involved in signaling from the insulin receptor. The expression of pp160 during the differentiation of 3T3-L1 fibroblasts to adipocytes and in adipocytes has been investigated using quantitative immunoblotting with antibodies against a peptide from pp160. Between day 6 and day 8 of differentiation induced by insulin, dexamethasone (Dex), and 1-methyl-3-isobutylxanthine (Mix), pp160 expression increased 10-20-fold over the amount present in confluent fibroblasts. Omission of either insulin or Dex resulted in reduced expression of pp160 and in incomplete adipogenesis. Chronic treatment of fully differentiated adipocytes for 24 h with either insulin, Dex, or Mix alone in the presence of serum resulted in a decrease in the expression of pp160 by 70-85%. Chronic exposure to insulin caused a significant increase in the apparent size of pp160 to 172 kDa. Alkaline phosphatase treatment lowered the Mr of pp160 from both insulin-treated and basal cells to 150,000. These results demonstrate that pp160 is expressed in 3T3-L1 adipocytes during the time when insulin receptors are expressed in large numbers and that the maintenance of pp160 concentrations in adipocytes can be regulated by insulin, Mix, and Dex. The decreased expression of pp160 caused by these factors may be related to postreceptor insulin resistance.     

Protocol for effective differentiation of 3T3-L1 cells to adipocytes

In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 μM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.     

Stearoyl-CoA desaturase 2 is required for peroxisome proliferator-activated receptor gamma expression and adipogenesis in cultured 3T3-L1 cells

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells.     

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.