We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.

Growth hormone (GH),

A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMPstimulated increase in PEPCK gene transcription.

Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiationand hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution.Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery. -Liao, J., R. Sportsman, J. Harris, and A. Stahl. Real-time quantification of fatty acid uptake using a novel fluorescence assay. J. Lipid Res. 2005. 46: 597-602.Supplementary key words long-chain fatty acids • fatty acid uptake assay • quencher dyes Uptake of long-chain fatty acids (LCFAs) plays an important role in the absorption of dietary lipids as well as the delivery of metabolic energy to a variety of tissues. Besides their function as substrates for ␤ -oxidation, fatty acids also contribute to membrane synthesis, protein modifications, immune responses, and activation of protein kinases and nuclear hormone receptors (1, 2). Recent findings have also directly implicated increased intracellular levels of LCFAs in the obesity-associated insulin desensitization of skeletal muscle and liver (1, 3).LCFA uptake in adipocytes is mainly facilitated by membrane proteins, particularly members of the fatty acid transport protein (FATPs/SLC27) family. Mammalian genomes have been shown to contain six FATP genes (4). The identification of this fatty acid transporter family and other fatty acid uptake-enhancing proteins such as CD36 has allowed a better understanding of the mechanisms and regulation of LCFA transport on a cellular level, yielding insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity. In addition, these cell surface proteins represent new targets for the inhibition of LCFA uptake.To gain a better understanding of the molecular dynamics of fatty acid uptake and the development of small molecular inhibitors of this process, robust and physiologically relevant measurements of LCFA uptake kinetics are required. Here, we report the development of a fluorescence assay bas...

No abstract

For insight into the mechanisms of gene regulation by growth hormone (GH), the regulation of transcription factors associated with the serum response element (SRE) located upstream of c-fos was examined. The SRE can mediate induction of reporter expression in response to GH. For insight into the mechanism by which GH regulates transcription factors, regulation of SREassociated proteins by GH was examined. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increased binding of two complexes; binding returned to control values within 30 min. The two GH-stimulated complexes were supershifted by antibodies against the serum response factor (SRF), indicating that they contained SRF or an antigenically related protein. One of the GH-stimulated complexes was supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH was lost when the SRF binding site in the SRE was mutated, and mutation of either the SRF or TCF binding site altered the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibited the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression was mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulated the serine phosphorylation of Elk-1. The increase was evident within 10 min and subsided after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1 contributing to GH-promoted transcriptional activation via the SRE.

A bidirectional communication exists between the nervous system and the immune system. Evidence has accumulated suggesting that cytokines-immune peptides influence sympathetic neuronal survival and that cytokines can promote the secretion of catecholamines. Using an isolated perfused rat liver (IPRL) preparation, we have shown that the liver is an important source of circulating cytokines in response to lipopolysaccharide (LPS) and that corticosterone dose dependently influenced LPS-induced production of tumor necrosis factor (TNF) and interleukin-6 (IL-6). In this study, we investigated the direct effect of epinephrine (another stress hormone) on the production of TNF and IL-6 in liver. We demonstrated that epinephrine (1 microM/ml) alone did not induce TNF bioactivity but significantly increased IL-6 bioactivity from IPRL effluent. When the IPRL was infused with LPS, epinephrine significantly decreased TNF bioactivity. Epinephrine in LPS-treated livers also significantly increased IL-6 bioactivity. Both responses were totally inhibited by the beta-blocker propranolol (10 microM/ml). Anisomycin, a protein synthesis inhibitor, infused into the IPRL completely blocked the rise in TNF and IL-6 concentrations in the effluent leaving the IPRL, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. We then attempted to determine whether epinephrine exerts similar effects in vitro. Using isolated Kupffer cells and hepatocytes, we found that epinephrine alone had no effect on TNF and IL-6 production in Kupffer cells and hepatocytes but significantly decreased LPS-induced TNF bioactivity and increased LPS-induced IL-6 bioactivity in Kupffer cells. Our data support the hypothesis that epinephrine can promote IL-6 secretion from IPRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Using an isolated perfused rat liver (IPRL) preparation, we assessed whether corticosterone may contribute to the rise in tumor necrosis factor (TNF) and interleukin-6 (IL-6) in rats after injection with lipopolysaccharide (LPS) or exposure to psychological stress. Intravenous infusion of LPS into the IPRL led to dose-dependent increases in TNF and IL-6 concentrations in the effluent. Anisomycin, a protein synthesis inhibitor, completely blocked the rise in TNF and IL-6 concentration in the IPRL effluent, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. Intravenous infusion of corticosterone at nonstressed (35 ng/ml) and stressed levels (350 ng/ml) increased TNF and/or IL-6 release. However, when LPS was combined with corticosterone, the lower dose of corticosterone facilitated the release of cytokines, whereas the higher dose of corticosterone suppressed the release of cytokines. We then showed that isolated Kupffer cells were capable of significant TNF and IL-6 production and that corticosterone decreased LPS-induced cytokine production in these cells. Our data support the hypothesis that the liver is an important source of circulating cytokines in response to LPS. In addition, although in vitro data generally support the hypothesis that corticosterone suppresses the production of cytokines, our in situ data support the hypothesis that physiological levels of corticosterone cause an increase in TNF and IL-6.