Phillip H. Pekala

Glucose transporter gene expression: Regulation of transcription and mRNA stability

The facilitated diffusion of D-glucose across the plasma membrane is carried out by a set of stereospecific transport proteins known as the glucose transporters. These integral membrane proteins are members of a gene family where tissue-specific expression of one or more members will determine in part the net rate of glucose entry into the cell. The regulation of glucose transporter gene expression is a critical feature of cellular homeostasis, as defects in specific transporter expression can lead to profound alterations in cellular physiology. In this review, we provide a brief descriptive background on the family of glucose transporters and examine in depth the regulation of the two transporters expressed in adipose tissue, GLUTI, a basal growth-related transporter and GLUT4, the insulin-responsive glucose transporter.

Regulation of lipoprotein lipase synthesis by recombinant tumor necrosis factor--the primary regulatory role of the hormone in 3T3-L1 adipocytes.

Tumor necrosis factor (TNF), a protein homologous to cachectin, has been implicated in mediating cachexia. This effect at least in part has been suggested to occur through the influence of the hormone on adipose tissue metabolism. Using fully differentiated 3T3-L1 adipocytes as a model system, we have been investigating the effects of recombinant TNF (rTNF) on key features of adipocyte metabolism. Exposure of fully differentiated 3T3-L1 adipocytes to recombinant tumor necrosis factor resulted in a dose and time-dependent suppression of the activity of lipoprotein lipase. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. No effect of rTNF on the half-life of the enzyme was observed. General protein synthesis, as judged by [35S]methionine incorporation into acid-insoluble protein, was minimally affected by exposure of the cells to rTNF; this was further confirmed by sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. As opposed to our previously reported results with crude preparations of TNF, no effect on either the ability of the adipocytes to synthesize and store or mobilize triacylglycerol was observed. Our results are consistent with the hypothesis that other hormones present in crude preparations of TNF acting either alone or synergistically with TNF play a major role in the further metabolic derangements associated with adipose tissue during cachexia.

Regulation of GLUT4 gene expression by arachidonic acid. Evidence for multiple pathways, one of which requires oxidation to prostaglandin E2.

We have previously described the ability of arachidonic acid (AA) to regulate GLUT4 gene expression (Tebbey, P. W., McGowan, K. M., Stephens, J. M., Buttke, T. M., and Pekala, P. H.(1994) J. Biol. Chem. 269, 639-644). Chronic exposure (48 h) of fully differentiated 3T3-L1 cells to AA resulted in an 90% suppression of GLUT4 mRNA accumulation. This decrease was demonstrated to be due to a 50% decrease in GLUT4 gene transcription as well as a destabilization of the GLUT4 message (t decreased from 8.0 to 4.6 h). In the current study we have identified, at least in part, the mechanism by which AA exerts its effects on GLUT4 expression. Compatible with a cyclooxygenase mediated event, the AA-induced suppression of GLUT4 mRNA was abolished by pretreating the cells with the inhibitor, indomethacin. Consistent with this observation, exposure of the cells to 10 μM PGE mimicked the effect of AA, in contrast to products of the lipoxygenase pathway which were unable to suppress GLUT4 mRNA content. Quantification of the conversion of AA to PGE demonstrated a 50-fold increase in PGE released into the media within 7 h of AA addition. Cyclic AMP levels were also increased 50-fold with AA treatment consistent with PGE activation of adenylate cyclase. Various long chain fatty acids, including the nonmetabolizable analog of AA, eicosatetraenoic acid (ETYA), also decreased GLUT4 mRNA levels. The effect of ETYA, a potent inhibitor of both lipo- and cyclooxygenases and a potent activator of peroxisome proliferator activated receptors (PPARs), suggested the presence of a second pathway where nonmetabolized fatty acid functioned to suppress GLUT4 mRNA levels. Further support for a PPAR-mediated mechanism was obtained by exposure of the cells to the classic PPAR activator, clofibrate, which resulted in a 75% decrease in GLUT4 mRNA content. Nuclear extracts prepared from the adipocytes contained a protein complex that bound to the PPAR responsive element (PPRE) found in the promoter of the fatty acyl-CoA oxidase gene. When the adipocytes were treated with either AA or ETYA, binding to the PPRE was disrupted, consistent with an ability of these fatty acids to control gene expression by altering the occupation of a PPRE. However, a perfect PPRE has yet to be identified in the GLUT4 promoter, but this does not rule the possibility of a PPAR playing an indirect role in the AA-induced GLUT4 mRNA suppression.

Regulation of lipoprotein lipase synthesis and 3T3-L1 adipocyte metabolism by recombinant interleukin 1

When fully differentiated 3T3-L1 adipocytes were exposed to purified, recombinant murine interleukin 1 (rIL-1), a dose-dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to an effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate [35S]methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporation into acid precipitable protein; however, after a 17 h exposure of the 3T3-L1 cells to recombinant interleukin 1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several-fold. When the effect of Il-1 on the major metabolic pathways of the adipocyte was investigated, lipolysis as measured by glycerol release from the cells was markedly enhanced after a 17 h incubation with the hormone, while no effect was observed on de novo fatty acid synthesis. These effects on the metabolism of the adipocytes occur at concentration on a basis of molecules per cell, similar (only a 3-fold difference) to those required for stimulation of [3H]thymidine incorporation into mouse thymocyte DNA, suggesting that IL-1 may be a physiologically significant effector of adipocyte metabolism.

Tumor Necrosis Factor-α Regulation of Glucose Transporter (GLUT1) mRNA Turnover CONTRIBUTION OF THE 3′-UNTRANSLATED REGION OF THE GLUT1 MESSAGE

In the current study we report on the contribution of the GLUT1 3′-untranslated region (UTR) to the stability of the GLUT1 mRNA. To facilitate these investigations, a hybrid construct was prepared by insertion of the GLUT1 3′-UTR into a normally stable reporter gene coding for preproinsulin. The GLUT1 3′-UTR conferred lability to the otherwise long lived construct and transferred an ability to be stabilized in response to treatment with the cytokine, tumor necrosis factor-α (TNF). The destabilizing element has been mapped to a region located between bases 2242 and 2347 of the GLUT1 3′-UTR; this same region also mediates the stabilization response to TNF. In vitro RNA-protein binding assays using protein extracts from control and TNF-treated cells demonstrated that two proteins, one of 37 kDa and the other of 40 kDa, recognized sequence elements within the stability-determining region and were up-regulated in response to TNF treatment. The RNA-binding activity of these proteins coincides with the stabilization of the GLUT1 message, suggesting that they may be involved in regulation of the turnover of this message.

TUMOR NECROSIS FACTOR-ALPHA INITIATED SIGNAL TRANSDUCTION IN 3T3-L1 ADIPOCYTES

Examination of the ability of tumor necrosis factor‐α (TNF) to activate both the p44/42 and p38 MAP kinase cascades in fully differentiated 3T3‐L1 adipocytes indicated a rapid MEK1/2‐dependent activation of p44/42 MAP kinase. Use of the MEK1/2 inhibitor PD98059 indicated that this pathway at least in part was responsible for nuclear localization of the transcription factor NF‐κB. The stress/cytokine‐activated p38 MAP kinase was observed to be constitutively active, and its phosphorylation (activation) status was not altered with TNF treatment. However, TNF treatment did result in activation of the transcription factor, ATF‐2, a primary downstream target of p38 MAP kinase. Use of the p38 MAP kinase inhibitors SB202190 and SB202580 did not interfere with the ability of TNF to activate ATF‐2, suggesting that either the γ isoform of p38 MAP kinase or a p38‐independent pathway was utilized by TNF to increase the phosphorylated fraction of ATF‐2. In previous studies we had demonstrated the ability of TNF to suppress the transcription of the GLUT4 gene. Prevention of activation of either the p44/42 MAP kinase pathway (PD98059) or the p38 MAP kinase pathway (SB202190 and SB202580) indicated that these pathways did not control GLUT4 transcription. J. Cell. Physiol. 179:58–66, 1999.

Response of bone marrow stromal cells to adipogenic antagonists.

Adipocytes constitute a major part of the bone marrow stroma in vivo and may play an active role in lymphohematopoiesis. Earlier studies had shown that the bone marrow stromal cell clone BMS2 was capable of adipocyte differentiation in vitro, in addition to its well-defined ability to support B lymphopoiesis. We now demonstrate that the process of adipogenesis in this functional bone marrow stromal cell clone can be inhibited by the cytokines interleukin-1 alpha, tumor necrosis factor, and transforming growth factor beta. Exposure of preadipocyte BMS2 cells to these agents blocked the induction of adipocyte differentiation as assessed by morphologic criteria and analysis of the neutral lipid content. Both interleukin-1 alpha and tumor necrosis factor elicited a rapid transient elevation in the steady-state mRNA levels of c-fos, c-jun, and JE. When added to differentiated adipocytes, the three cytokines continued to act as adipogenic antagonists. This was indicated by concentration- and time-dependent decreases in the activity of an adipocyte-specific enzyme, lipoprotein lipase. These changes in enzyme activity correlated directly with a decrease in steady-state levels of lipoprotein lipase mRNA. Another RNA marker of adipocyte differentiation (adipsin) was less influenced by the adipogenic antagonists. This may reflect the longer half-life of this mRNA transcript compared with those of lipoprotein lipase. Our results dramatically demonstrate that the differentiation state of bone marrow stromal cells can be modulated by exogenous factors in vitro. It is also the first report that transformation growth factor beta regulates the activity of lipoprotein lipase. These data suggest potential physiologic actions for these cytokines in vivo within the overall context of lymphohematopoiesis.

Monokine regulation of glucose transporter mRNA in L6 myotubes

Endotoxin-induced macrophage secretory proteins (monokines) have been shown to stimulate hexose uptake in L6 myotubes (1). In those studies a doubling of the Vmax for hexose uptake was observed which correlated with elevated numbers of glucose transporters (GT) in both plasma and microsomal membranes. To determine if these changes in transporter populations were due to increased GT mRNA, we performed Northern blot analysis using L6 cell RNA and a cDNA to the HepG2 glucose transporter. The L6 myotubes contained a single 2.8 kb species of GT mRNA that increased 2.5-fold after an 8h exposure to the monokine preparation. beta-Actin mRNA levels were unaltered by the treatment, indicating specificity of monokine action. Glucose transporter mRNA content appeared to reach a maximum 8 h after exposure to the monokine. Over the next 16 h the levels of this mRNA gradually decreased, approaching control levels. Data obtained from nuclear transcription run-on assays suggest that increased levels of CT mRNA are due to an increased rate of gene transcription. A second transporter, the insulin-sensitive glucose transporter, was also observed to be expressed in the L6 cells. Monokine treatment resulted in a 60% suppression of the mRNA coding for this protein.

Interleukin 11 signaling in 3T3-L1 adipocytes

Interleukin 11 (IL‐11) is an anti‐inflammatory cytokine with receptors located on most cell types and tissues throughout the body. Its anti‐inflammatory properties are mediated through suppression of cytokine synthesis, in large part by prevention of NF‐κB activation. As adipose tissue synthesizes and secretes cytokines involved in establishing insulin resistance and due to the ability of IL‐11 to suppress cytokine synthesis, we initiated an investigation to determine the signal transduction pathways initiated by IL‐11 in adipose tissue. Using the 3T3‐L1 adipocyte cell culture model we demonstrate the rapid activation of the p44/42MAP kinase, PI3‐kinase, and STATs 1 and 3. Activation of MAP kinase is demonstrated to lead to the downstream activation of p90 RSK (ribosomal S6 kinase) as well as ATF‐1 and CREB. PI3‐kinase appears to activate the downstream target of p70 S6 kinase resulting in phosphorylation of ribosomal protein S6. STAT phosphorylation appears to be initiated through PI3‐kinase and to a lesser degree through p44/42 MAP kinase. These studies demonstrate the activation of three major signaling pathways and support a role for IL‐11 in the regulation of both transcription and protein synthesis in fully differentiated adipocytes.

Poly ADP-ribosylation of protein.

Publisher Summary This chapter discusses poly ADP-ribosylation of protein. ADP-ribosylation is a postsynthetic modification of protein that involves the covalent attachment of the ADP-ribose moiety of NAD to specific amino-acid residues or to another ADP-ribose moiety to form a poly(ADP-ribose) molecule. There are two classes of enzymes responsible for this modification: (1) the mono(ADP-ribosyl) transferases and (2) poly(ADP-ribosyl) synthetases. Some bacterial toxins alter the activity of critical metabolic pathways by catalyzing the ADP-ribosylation of a key regulatory enzyme. Diphtheria toxin and Pseudomonas aeruginosa exotoxin A catalyze the mono ADP-ribosylation of eukaryotic elongation factor II, resulting in the inhibition of protein synthesis; choleragen and E. coli heat-labile enterotoxin catalyze the ADP-ribosylation of a regulatory protein of adenylate cyclase, resulting in the activation of the enzyme. Poly(ADP-ribose) synthetase is localized primarily in the nucleus of eukaryotic cells.