Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells.

AIMS/HYPOTHESIS: Recent experiments in liver and adipocyte cell lines indicate that palmitate can induce endoplasmic reticulum (ER) stress. Since it has been shown that ER stress can interfere with insulin signalling, our hypothesis was that the deleterious action of palmitate on the insulin signalling pathway in muscle cells could also involve ER stress. METHODS: We used C2C12 and human myotubes that were treated either with palmitate or tunicamycin. Total lysates and RNA were prepared for western blotting or quantitative RT-PCR respectively. Glycogen synthesis was assessed by [¹4C]glucose incorporation. RESULTS: Incubation of myotubes with palmitate or tunicamycin inhibited insulin-stimulated protein kinase B (PKB)/ v-akt murine thymoma viral oncogene homologue 1 (Akt). In parallel, an increase in ER stress markers was observed. Pre-incubation with chemical chaperones that reduce ER stress only prevented tunicamycin but not palmitate-induced insulin resistance. We hypothesised that ER stress activation levels induced by palmitate may not be high enough to induce insulin resistance, in contrast with tunicamycin-induced ER stress. Indeed, tunicamycin induced a robust activation of the inositol-requiring enzyme 1 (IRE-1)/c-JUN NH2-terminal kinase (JNK) pathway, leading to serine phosphorylation of insulin receptor substrate 1 (IRS-1) and a decrease in IRS-1 tyrosine phosphorylation. In contrast, palmitate only induced a very weak activation of the IRE1/JNK pathway, with no IRS1 serine phosphorylation. CONCLUSIONS/INTERPRETATION: These data show that insulin resistance induced by palmitate is not related to ER stress in muscle cells.

Tumor necrosis factor-alpha-induced adipose-related protein (TIARP), a cell-surface protein that is highly induced by tumor necrosis factor-alpha and adipose conversion.

Tumor necrosis factor-alpha (TNF alpha) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNFalpha on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF alpha-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF alpha exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF alpha effects on the fat cell as a channel or a transporter.

Adiponutrin, a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage.

We have used a mRNA differential display technique to identify new genes involved in the reprogramming of gene expression during the adipose conversion of mouse 3T3 preadipocyte cell lines. We report here on the identification and cloning of a novel adipose-specific cDNA encoding a predicted membrane protein of 413 amino acids. The level of the corresponding 3.2-kilobase mRNA is tremendously increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes. A single, very abundant 3.2-kilobase transcript is also found in inguinal and epididymal white adipose tissues and in interscapular brown adipose tissue but not in any other tissues examined. Its expression in adipose tissue is under tight nutritional regulation. The level of this novel 3.2-kilobase transcript becomes virtually nondetectable during fasting but is dramatically increased when fasted mice are refed a high carbohydrate diet. Based on its adipose specificity and dietary regulation, the novel gene product has been designated adiponutrin. The expression of adiponutrin mRNA is also 50-fold elevated in genetically obese fa/fa rats, indicating a link between adiponutrin and obesity. Western blot and confocal imagery analyses with epitope-tagged protein transiently expressed in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly localizes to membranes and is absent from the cytosol. Sequence analysis reveals homologies with several other members of related eukaryotic proteins, including a human paralog, which has been recently described in vesicular transport mechanisms. This leads us to suggest that adiponutrin could be involved in vesicular targeting and protein transport restricted to the adipocyte function.

Id3 prevents differentiation of preadipose cells.

We have studied the expression of the Id1, Id2, and Id3 genes during adipose differentiation of 3T3-F442A cells. All three Id mRNAs are present in preadipose cells, but the mRNA for Id3 is the most abundant. All three Id mRNAs sharply decline in the course of adipose differentiation, and their virtual disappearance precedes differentiation. The decrease in Id2 and Id3 is associated with adipose differentiation rather than with growth arrest since it is not observed in 3T3-C2 cells, a fibroblast line with a very low susceptibility to adipose conversion. The decline in Id2 and Id3 mRNAs is associated with a reduced transcription rate of the two genes. Id1 mRNA is reduced in amount during adipose conversion of 3T3-F442A cells, but the decrease is also observed in resting 3T3-C2 cells and is associated with very little decrease in transcription of the gene. Addition of fresh serum reactivates Id3 gene expression in quiescent 3T3-C2 cells but not in adipose 3T3-F442A cells. Stably transformed preadipose cells expressing an Id3 cDNA under the control of a viral promoter are virtually unable to differentiate. We postulate that the Id3 protein is a negative regulator of fat cell formation and presumably acts by preventing an as yet unidentified basic helix-loop-helix protein from activating the program of differentiation.

Forskolin induces the reorganization of extracellular matrix fibronectin and cytoarchitecture in 3T3-F442A adipocytes: its effect on fibronectin gene expression.

We studied the effect of forskolin on fibronectin and actin gene expression in 3T3-F442A adipogenic cell line. The structural organizations of extracellular matrix fibronectin and actin cytoskeleton were investigated in parallel. Immunofluorescence experiments showed that preadipocytes treated for 48 h with 10 microM forskolin exhibited an intensified network of both actin and fibronectin when compared to control. A similar picture was obtained with adipocytes given long-term exposure to forskolin. As determined by Western analysis, fibronectin protein levels were increased by 50-75% over control, both in preadipocytes and adipocytes. A parallel increase of fibronectin mRNA content was observed in forskolin-treated cells. In contrast, forskolin treatment of preadipocytes and adipocytes did not elicit any change in the steady-state level of either actin mRNA or protein. Nuclear run-on experiments showed that forskolin increased the fibronectin gene transcription rate but left that of the actin gene in adipocytes unchanged. These findings suggest the reorganization of the actin network in forskolin-treated adipocytes to be a consequence of fibronectin-enhanced biosynthesis and reorganization.

Beta-adrenergic-cyclic AMP signalling pathway modulates cell function at the transcriptional level in 3T3-F442A adipocytes.

We studied the role of cAMP in the regulation of the expression of the adipsin gene and of some other adipose-specific genes including lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), and adipocyte P2 (aP2) in 3T3-F442A adipocytes. Northern blot analysis of isoproterenol (10(-6) M)-, forskolin (10(-5) M)- or 8-bromo-cAMP (10(-3) M)-treated adipocytes showed that the steady-state levels of adipsin mRNA were strongly reduced in a time-dependent and reversible manner. The concentration of isoproterenol giving a half-maximal effect in the down-regulation of the adipsin message was approximately 5 x 10(-8) M. Similarly, cell treatment by forskolin elicited a down-regulation of LPL and G3PDH mRNA levels but did not alter aP2 mRNA level. As determined by nuclear run-on assays, the rate of transcription of adipsin, LPL and G3PDH in isoproterenol-treated adipocytes was respectively 3, 3, and 2 times lower than in control adipocytes. These results indicate (1) that cAMP plays a dominant antilipogenic role in the fat cell through the transcriptional down-regulation of the expression of two major genes involved in triglyceride biosynthesis; (2) that cAMP does not reverse the adipocyte character; (3) hence, that cAMP suppresses adipsin expression at the transcriptional level, providing additional support for the role of adipsin protein in adipocyte metabolism.

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipsin gene expression in 3T3-F442A adipocytes is posttranscriptionally down-regulated by retinoic acid.

Retinoic acid (RA) has been shown to inhibit the differentiation of 3T3 adipogenic cell lines. In this report, the steady-state levels of several adipose-specific mRNAs were studied in mature adipocytes treated with RA. Northern blot analysis showed that, following a 24-h exposure of 3T3-F442A adipocytic cultures to RA (10 microM), there was a 4-5-fold decrease of adipsin mRNA level. In contrast, actin, adipocyte P2, lipoprotein lipase, and glycerophosphate dehydrogenase mRNA levels were unchanged during the same interval. The rate of adipsin and actin gene transcription, assessed by nuclear run-on assays, remained unchanged in adipocytes exposed to RA. The half-life (t1/2) of adipsin mRNA, determined by pulse-chase with [3H] uridine, was greatly shortened in RA-treated adipocytes (t1/2 approximately 7.3 h) as compared with untreated cells (t1/2 approximately 37.6 h). Conversely, actin mRNA stability was not altered by the drug. These results suggest that RA can specifically down-regulate adipsin expression in adipocytes at a posttranscriptional level without inducing the reversal of adipocyte differentiation.

Differential regulation of beta 1- and beta 2-adrenergic receptor protein and mRNA levels by glucocorticoids during 3T3-F442A adipose differentiation.

The regulation by dexamethasone of beta 1- and beta 2-adrenergic receptor expression during the adipose differentiation of 3T3-F442A cells was investigated at the receptor protein and mRNA level. Preadipocytes were poorly responsive to beta-adrenergic receptor (beta-AR) agonists and expressed few beta-ARs (approximately 3,000 sites/cell) solely of beta 1 subtype. Differentiation increased adrenergic sensitivity and total beta-AR number (approximately 16,000 sites/cell) with a beta 1/beta 2 ratio of approximately 90/10. Long term exposure of either differentiating cells or mature adipocytes to dexamethasone induced down-regulation of (-)-isoproterenol-sensitive adenylate cyclase activity which paralleled a 2- to 3.5-fold decrease in beta-ARs, while the beta 1/beta 2 ratio switched to approximately 20/80. The ratios of beta 1/beta 2 binding sites were always consistent with the rank order of potency of beta-adrenergic agonists in stimulating the adenylate cyclase system. The action of steroid agonists and antagonist suggested a glucocorticoid receptor-mediated mechanism. The beta 1-AR mRNA (3.2 kilobases) was stimulated 3-4.7 times in differentiated cells, as compared with preadipose cells; this beta 1-AR transcript was repressed in dexamethasone-treated cells. The beta 2-AR mRNA species (2.3 kilobases), absent in preadipocytes, was expressed at low levels in untreated adipocytes, but reached 11-fold this level in dexamethasone-exposed cells. The switch in receptor subtype protein and mRNA levels elicited by dexamethasone demonstrates the differential genetic control by glucocorticoids of beta-AR subtype expression in 3T3-F442A cells. We suggest that this regulation of beta-AR gene expression requires interactions of glucocorticoid receptors with specific DNA targets and with one (or several) transcription factor(s) that are cell- and differentiation state-dependent.

Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes: insulin and dexamethasone control.

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day 1, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.

The antiglucocorticoid RU38486 is a potent accelerator of adipose conversion of 3T3-F442A cells.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Differential effects of retinoic acid upon early and late events in adipose conversion of 3T3 preadipocytes.

When confluent 3T3-F442A cell cultures (Day 0) were grown for 3 days in fetal calf serum-supplemented medium containing isobutyl methyl xanthine and dexamethasone (induction phase) and then shifted to serum-free hormone-defined medium (expression phase), they spontaneously exhibited a sharp rise in lipoprotein lipase activity (LPL); at the peak (Day 7) the LPL activity was about 25 times higher than in control cultures and was further enhanced by insulin. Although this expression of LPL activity was spontaneous, the emergence of glycerophosphate dehydrogenase (G3PDH) activity was completely dependent upon insulin as well as upon the expression of the differentiated phenotype. In committed cells, insulin elicited sustained DNA synthesis associated with limited cell proliferation. The addition of retinoic acid during the phase of expression inhibited insulin-dependent terminal differentiation (i.e., the emergence of G3PDH activity and acquisition of the differentiated phenotype). In addition, retinoic acid counteracted the stimulating effect of insulin upon LPL activity, but affected neither the mitotic process nor the spontaneous emergence of LPL activity. When added during the phase of induction, it prevented the overall process of adipogenic differentiation. Thus, the use of retinoic acid can indicate independent control of the mitogenic and lipogenic effects of insulin following commitment to adipogenic differentiation.

Control of the adipogenic differentiation of 3T3-F442A cells by retinoic acid, dexamethasone, and insulin: a topographic analysis.

Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.

Dihydrocytochalasin B promotes adipose conversion of 3T3 cells.

Differentiation of preadipose 3T3-F442A cells into adipose cells is accelerated by the addition of dihydrocytochalasin B. The effect of the drug on 3T3-C2 cells is more marked: these cells are practically unable to differentiate in the absence of H2CB but a long-term exposure to the drug enables the cells to accumulate lipid droplets in medium supplemented with fetal calf serum and insulin. During their differentiation under these conditions the 3T3-C2 cells develop markers typical of adipose cells: glycerophosphate dehydrogenase, ATP-citrate lyase, fatty acid synthetase and glycerophosphate acyltransferase.