Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells

The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.     

Differential expression of murine CGI-105 gene in 3T3-L1 cells by adrenocorticotropic hormones

The effects of adrenocorticotropic hormones on murine CGI-105 gene expression were investigated in 3T3-L1 cells. Expression was markedly increased in differentiated cells and it was up-regulated 2-fold in cells induced to differentiate with dexamethasone.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.     

The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.     

27-Hydroxycholesterol suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells

Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.     

Uncoupling of 3T3-L1 gene expression from lipid accumulation during adipogenesis

Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.     

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.     

Bezafibrate regulates the expression and enzyme activity of 11beta-hydroxysteroid dehydrogenase type 1 in murine adipose tissue and 3T3-L1 adipocytes

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR(alpha, -gamma, and -delta) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11beta-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11beta-HSD1 preferentially in adipose tissue of db/db mice (-47%, P<0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P<0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (-34%, P<0.01) and enzyme activity (-32%, P<0.01) of 11beta-HSD1, whereas the treatment substantially augmented the expression (+71%, P<0.01) and secretion (+27%, P<0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11beta-HSD1 by siRNA confirmed that 11beta-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11beta-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11beta-HSD1 and adiponectin in murine adipocytes, both of which may contribute to metabolically-beneficial effects by bezafibrate.     

Wortmannin and LY294002 inhibit myo-inositol accumulation by cultured bovine aorta endothelial cells and murine 3T3-L1 adipocytes

We have previously reported that myo-inositol uptake and metabolism is reduced in human fibroblasts derived from patients with ataxia telangiectasia (AT). Treating normal fibroblasts with 10-100 microM wortmannin duplicates some of the phenotypic properties of AT fibroblasts including the decrease in myo-inositol accumulation. In the present study we examined whether treatment of other types of mammalian cells with wortmannin or LY294002 altered myo-inositol uptake. Cultured bovine aorta endothelial cells or 3T3-L1 adipocytes were incubated with either wortmannin or LY294002, and afterwards, myo-inositol uptake and SMIT mRNA levels were determined. Incubating cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 caused a time- and concentration-dependent decrease in myo-inositol accumulation that was independent of changes in SMIT mRNA levels. The effect of wortmannin and LY294002 on myo-inositol accumulation was not due to an increase in myo-inositol secretion. The effect of LY294002 on myo-inositol accumulation was reversible. Furthermore, the LY294002-induced decrease in myo-inositol accumulation was specific since the uptake of serine or choline by cultured bovine aorta endothelial cells and 3T3-L1 adipocytes treated with LY294002 was not significantly decreased. Co-incubation of cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 and hyperosmotic medium caused a significant decrease in the induction of myo-inositol accumulation by hyperosmolarity without significantly affecting the hyperosmotic-induced increase in SMIT mRNA levels. These data suggest that myo-inositol accumulation is regulated post-translationally by wortmannin and LY294002.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Regulation of lipoprotein lipase mRNA content in 3T3-L1 cells by tumour necrosis factor.

U-937 cells differentiated by exposure to dibutyryl cyclic AMP respond to complement fragment C5a with a marked increase in cytoskeletal F-actin, which can be detected by fluorescence-activated cell sorting (f.a.c.s.) analysis of their rhodamine phalloidin-stained cytoskeletons. The C5a-induced increase in F-actin content can be prevented by prior exposure of the cells to cytochalasin B and pertussis toxin. It is insensitive to removal of extra cellular Ca2+, to cholera toxin or to neomycin. Phorbol myristate acetate (PMA), an activator of protein kinase C, does not induce actin polymerization in the differentiated cells. Both C5a and PMA stimulate superoxide production. The action of C5a on superoxide formation is also inhibited by neomycin, a phospholipase inhibitor. These results suggest that the cytoskeletal response to C5a requires activation of a G protein, but probably does not involve phospholipase C and protein kinase C, and is not highly dependent on the availability of Ca2+. Phospholipase C and kinase C may, however, be components of the pathway leading from C5a binding to superoxide production.     

Regulation of apelin mRNA expression by insulin and glucocorticoids in mouse 3T3-L1 adipocytes

The novel 36-amino acid peptide, apelin, is the endogenous ligand for the orphan receptor APJ. Apelin may play important roles in the regulation of the cardiovascular system and the hypothalamic-pituitary axis. It is a potent hypotensive agent and one of the most potent stimulators of cardiac contractility. In this study, we investigated the roles of apelin derived from adipocytes in the regulation of cardiovascular homeostasis. We found that both apelin and APJ mRNAs were expressed in isolated mouse adipocytes and that apelin mRNA levels increased during the differentiation of 3T3-L1 cells to adipocytes. We also found that the administration of insulin (1 nM-100 nM) increased, while that of dexamethasone (0.1 nM-100 nM) decreased the apelin mRNA levels in 3T3-L1 adipocytes in a dose-dependent manner, suggesting that insulin and glucocorticoids regulate apelin gene expression in adipocytes. We speculate that high glucocorticoid levels suppress apelin production and stimulate angiotensin II production in adipocyte, decreasing the counter-regulatory activity of apelin against the pressor action of angiotensin II, which might partly be involved in the mechanism underlying the development of obesity-related hypertension.