Lauren M. Cagen

Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements.

The enhanced synthesis of fatty acids in the liver and adipose tissue in response to insulin is critically dependent on the transcription factor SREBP-1c (sterol-regulatory-element-binding protein 1c). Insulin increases the expression of the SREBP-1c gene in intact liver and in hepatocytes cultured in vitro. To learn the mechanism of this stimulation, we analysed the activation of the rat SREBP-1c promoter and its truncated or mutated congeners driving a luciferase reporter gene in transiently transfected rat hepatocytes. The rat SREBP-1c promoter contains binding sites for LXR (liver X receptor), Sp1, NF-Y (nuclear factor-Y) and SREBP itself. We have found that each of these sites is required for the full stimulatory response of the SREBP-1c promoter to insulin. Mutation of either the putative LXREs (LXR response elements) or the SRE (sterol response element) in the proximal SREBP-1c promoter reduced the stimulatory effect of insulin by about 50%. Insulin and the LXR agonist TO901317 increased the association of SREBP-1 with the SREBP-1c promoter. Ectopic expression of LXRalpha or SREBP-1c increased activity of the SREBP-1c promoter, and this effect is further enhanced by insulin. The Sp1 and NF-Y sites adjacent to the SRE are also required for full activation of the SREBP-1c promoter by insulin. We propose that the combined actions of the SRE, LXREs, Sp1 and NF-Y elements constitute an insulin-responsive cis-acting unit of the SREBP-1c gene in the liver.

Insulin enhances post-translational processing of nascent SREBP-1c by promoting its phosphorylation and association with COPII vesicles

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding SREBP-1c (sterol regulatory element-binding protein-1c). Nascent SREBP-1c is synthesized and embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi in coatomer protein II (COPII) vesicles where two sequential cleavages generate the transcriptionally active NH2-terminal fragment, nSREBP-1c. There is limited indirect evidence to suggest that insulin may also regulate the posttranslational processing of the nascent SREBP-1c protein. Therefore, we designed experiments to directly assess the action of insulin on the post-translational processing of epitope-tagged full-length SREBP-1c and SREBP-2 proteins expressed in cultured hepatocytes. We demonstrate that insulin treatment led to enhanced post-translational processing of SREBP-1c, which was associated with phosphorylation of ER-bound nascent SREBP-1c protein that increased affinity of the SREBP-1c cleavage-activating protein (SCAP)-SREBP-1c complex for the Sec23/24 proteins of the COPII vesicles. Furthermore, chemical and molecular inhibitors of the phosphoinositide 3-kinase pathway and its downstream kinase protein kinase B (PKB)/Akt prevented both insulin-mediated phosphorylation of nascent SREBP-1c protein and its posttranslational processing. Insulin had no effect on the proteolysis of nascent SREBP-2 under identical conditions. We also show that in vitro incubation of an active PKB/Akt enzyme with recombinant full-length SREBP-1c led to its phosphorylation. Thus, insulin selectively stimulates the processing of SREBP-1c in rat hepatocytes by enhancing the association between the SCAP-SREBP-1c complex and COPII proteins and subsequent ER to Golgi transport and proteolytic cleavage. This effect of insulin is tightly linked to phosphoinositide 3-kinase and PKB/Akt-dependent serine phosphorylation of the precursor SREBP-1c protein.

Expression of the Rat Sterol Regulatory Element-binding Protein-1c Gene in Response to Insulin Is Mediated by Increased Transactivating Capacity of Specificity Protein 1 (Sp1)

The induction of genes involved in lipid biosynthesis by insulin is mediated in part by the sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c is directly regulated by insulin by transcriptional and post-transcriptional mechanisms. Previously, we have demonstrated that the insulin-responsive cis-acting unit of the rat SREBP-1c promoter is composed of several elements that include a sterol regulatory element, two liver X receptor elements, and a number of conserved GC boxes. Here we systematically dissected the role of these GC boxes and report that five bona fide Sp1-binding elements of the SREBP-1c promoter determine its basal and insulin-induced activation. Luciferase expression driven by the rat SREBP-1c promoter was accelerated by ectopic expression of Sp1, and insulin further enhanced the transactivation potential of Sp1. Introduction of a small interfering RNA against Sp1 reduced both basal and insulin-induced activation of the SREBP-1c promoter. We also found that Sp1 interacted with both SREBP-1c and LXRα proteins and that insulin promoted these interactions. Chromatin immunoprecipitation studies revealed that insulin facilitated the recruitment of the steroid receptor coactivator-1 to the SREBP-1c promoter. These studies identify a novel mechanism by which maximal activation of the rat SREBP-1c gene expression by insulin is mediated by Sp1 and its enhanced ability to interact with other transcriptional regulatory proteins.

Stability of prostaglandin I2 in human blood

Prostaglandin I2 (PGI2) is hydrolyzed more slowly in human plasma than in buffer at the same pH; this stabilization appears to be due to binding of PGI2 to albumin. The stability of the antiaggregatory potency of PGI2 is similar in human blood and in plasma buffered to pH 7.55. No measurable conversion of [3H]-PGI2 to radioactive or nonradioactive metabolites occurred during incubation with blood.

Hepatic Gene Expression in Morbidly Obese Women: Implications for Disease Susceptibility

The objective of this study was to determine the molecular bases of disordered hepatic function and disease susceptibility in obesity. We compared global gene expression in liver biopsies from morbidly obese (MO) women undergoing gastric bypass (GBP) surgery with that of women undergoing ventral hernia repair who had experienced massive weight loss (MWL) following prior GBP. Metabolic and hormonal profiles were examined in MO vs. MWL groups. Additionally, we analyzed individual profiles of hepatic gene expression in liver biopsy specimens obtained from MO and MWL subjects. All patients underwent preoperative metabolic profiling. RNAs were extracted from wedge biopsies of livers from MO and MWL subjects, and analysis of mRNA expression was carried out using Affymetrix HG‐U133A microarray gene chips. Genes exhibiting greater than twofold differential expression between MO and MWL subjects were organized according to gene ontology and hierarchical clustering, and expression of key genes exhibiting differential regulation was quantified by real‐time–polymerase chain reaction (RT‐PCR). We discovered 154 genes to be differentially expressed in livers of MWL and MO subjects. A total of 28 candidate disease susceptibility genes were identified that encoded proteins regulating lipid and energy homeostasis (PLIN, ENO3, ELOVL2, APOF, LEPR, IGFBP1, DDIT4), signal transduction (MAP2K6, SOCS‐2), postinflammatory tissue repair (HLA‐DQB1, SPP1, P4HA1, LUM), bile acid transport (SULT2A, ABCB11), and metabolism of xenobiotics (GSTT2, CYP1A1). Using gene expression profiling, we have identified novel candidate disease susceptibility genes whose expression is altered in livers of MO subjects. The significance of altered expression of these genes to obesity‐related disease is discussed.

Insulin increases expression of apobec-1, the catalytic subunit of the apolipoprotein B mRNA editing complex in rat hepatocytes

We have previously shown that chronic insulin treatment of rat hepatocytes increases the fraction of edited apolipoprotein B (apoB) mRNA from approximately 50% to as much as 90%. We have now examined the effect of insulin on apobec-1 mRNA abundance and demonstrate that increased editing of apoB mRNA following insulin treatment is accompanied by elevated apobec-1 mRNA levels in primary rat hepatocytes. Time-course measurements of the effects of insulin on apoB mRNA editing and apobec-1 mRNA abundance showed that both were elevated almost maximally within 48 hours and sustained for at least 5 days of insulin treatment.

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Abstract Saline washed red blood cells of the toadfish convert [1- 14 C] arachidonic acid to products that cochromatograph with prostaglandin E 2 and prostaglandin F 2α . This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E 2 was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1- 14 C]-arachidonic acid and [5,6,8,9,11,12,14,15,- 3 H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F 2α was produced by reduction of prostaglandin E 2 . The capacity of toadfish red blood cells to reduce prostaglandin E 2 to prostaglandin F 2α was confirmed by incubation of the cells with [1- 14 C] prostaglandin E 2 .

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Abstract Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1- 14 C]-arachidonic acid and [5,6,8,9,11,12,14,15- 3 H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E 2 ; (b) prostaglandin F 2 α was formed by reduction of prostaglandin H 2 and not by reduction of prostaglandin E 2 ; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.

Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat

The effect of gonadectomy and treatment with sex-steroids on renal prostaglandin 9-ketoreductase activity in 10-11 week old male and female rats was determined. Rats were gonadectomized or subjected to sham operation at 3 weeks of age. During week 7, rats were injected s.c. twice over a 6-day interval with vehicle (peanut oil, 0.5 ml X kg-1) or with depot forms of testosterone (5 mg X kg-1), estradiol (0.02 mg X kg-1), progesterone (5 mg X kg-1), or estradiol and progesterone combined. Renal prostaglandin 9-ketoreductase activity was about 50% higher in female rats than in males. Gonadectomy decreased 9-ketoreductase activity in females, but not in males, and eliminated the gender difference in enzyme activity. Treatment with estradiol elevated 9-ketoreductase activity in males and females, while treatment with testosterone or progesterone was without effect. Progesterone did, however, antagonize the elevation in 9-ketoreductase activity produced by estradiol.

Attenuation by acetaminophen of arachidonic acid-induced coronary vasolidation and output of prostaglandins in the isolated rat heart

We studied the effect of acetaminophen on the vascular actions of arachidonic acid and on the output of prostaglandins in the isolated rat heart. Arachidonic acid (33 nmol), administered as a bolus into the heart through the aortic cannula produced vasoconstriction followed by a long lasting vasodilation. Arachidonic acid also markedly increased the output of PGE2 and 6-keto-PGF1 alpha from the heart. In the presence of acetaminophen (0.1-1.0 mM) both the output of prostaglandins and the duration of the coronary vasodilation were attenuated. We suggest that the major component of arachidonic acid-induced coronary vasodilation is mediated through prostaglandin synthesis and is blocked by acetaminophen.