Reduced expression of the Indy (‘I am Not Dead, Yet’) gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane associated citrate transporter expressed highly in the liver, protects mice from high-fat diet and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We aimed to study a possible role of mIndy in human hepatic fat metabolism. In obese, insulin resistant patients with NAFLD, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In non-human primates, a two year high fat, high sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription via the IL-6-receptor (IL-6R) and activation of the transcription factor Stat3 and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-Stat3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and non-human primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 via mINDY. Targeting human mINDY may have therapeutic potential in obese patients with NAFLD.
Reduced expression of the INDY (I'm not dead yet) tricarboxylate carrier increased the life span in different species by mechanisms akin to caloric restriction. Mammalian INDY homolog (mIndy, SLC13A5) gene expression seems to be regulated by hormonal and/or nutritional factors. The underlying mechanisms are still unknown. The current study revealed that mIndy expression and [14C]-citrate uptake was induced by physiological concentrations of glucagon via a cAMP-dependent and cAMP-responsive element–binding protein (CREB)–dependent mechanism in primary rat hepatocytes. The promoter sequence of mIndy located upstream of the most frequent transcription start site was determined by 5′-rapid amplification of cDNA ends. In silico analysis identified a CREB-binding site within this promoter fragment of mIndy. Functional relevance for the CREB-binding site was demonstrated with reporter gene constructs that were induced by CREB activation when under the control of a fragment of a wild-type promoter, whereas promoter activity was lost after site-directed mutagenesis of the CREB-binding site. Moreover, CREB binding to this promoter element was confirmed by chromatin immunoprecipitation in rat liver. In vivo studies revealed that mIndy was induced in livers of fasted as well as in high-fat-diet–streptozotocin diabetic rats, in which CREB is constitutively activated. mIndy induction was completely prevented when CREB was depleted in these rats by antisense oligonucleotides. Together, these data suggest that mIndy is a CREB-dependent glucagon target gene that is induced in fasting and in type 2 diabetes. Increased mIndy expression might contribute to the metabolic consequences of diabetes in the liver.
Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E 2 (PGE 2 ) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE 2 and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE 2 while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE 2 induced suppressors of cytokine signaling (SOCS3). PGE 2 but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE 2 -dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE 2 pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. Conclusion: PGE 2 might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulindependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE 2 may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3. (HEPATOLOGY 2009;50: 781-790.)
Abstract-Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (Ϫlog EC50, mol/L:9.0Ϯ0.1). A second application of UII after 30 minutes elicited a reduced contraction (36Ϯ4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ( 125 I-UII), Ϸ70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t h ]: 5.6Ϯ0.2 minutes), but recycled quantitatively within 60 minutes (t h 31.9Ϯ2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. Key Words: urotensin II Ⅲ vascular tone Ⅲ urotensin receptor Ⅲ recycling Ⅲ internalization U rotensin II (UII) is among the most potent mammalian vasoconstrictors identified so far. 1 UII was characterized as being 1 to 2 orders of magnitude more potent than endothelin-1 (ET-1) 2 and acts on the urotensin (UT) receptor, formerly known as GPR14. 3 Stimulation of the UT receptor results in a phospholipase C-mediated increase in cytosolic Ca 2ϩ and the activation of rho-kinase. 4,5 Both signaling pathways promote an increase in MLCK phosphorylation, resulting in a strong contraction of vascular smooth muscle cells. 6 In addition, stimulation of the UT receptor also results in an activation of tyrosine kinases, the mitogen-activated protein kinase (MAPK) p38, and extracellular signal regulated kinases (ERK)1/2. 6,7 UII-mediated vasoactive effects are variable and depend on the species and the disease state investigated. 8,9 In humans, UII peptide and human UT (hUT) receptor mRNAs are found in the heart (atrial and ventricular myocytes, fibroblasts) 10 and kidney (epithelia of tubules and ducts, renal capillary and glomerular endothelial cells). 11 Low levels of UII binding sites have been identified in the coronary arteries, kidney, left ventricle, and skeletal muscle. 12 A dose-dependent reduction of forearm blood flow in healthy subjects and vasoconstricto...
Prostaglandin E 2 receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G q , EP2 and EP4 to G s , and EP3 to G i . The EP4-R, in contrast to the EP3␤-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G i -coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E 2 , or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAGepitope-tagged rat EP3␤-R (rEP3␤-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3␤-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E 2 stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time-and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAGrEP3␤-R. Agonist-induced phosphorylation of the FLAGhEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.Prostaglandin E 2 receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domains (Negishi, 1994). There are four subtypes of E-prostaglandin receptors (EP-Rs) that differ in their affinity to synthetic ligands and their G protein coupling specificity. EP1-Rs are linked to G q and increase inositol trisphosphate (InsP 3 ) and, hence, cytosolic Ca 2ϩ concentration. EP2-Rs and EP4-Rs are coupled to G s and increase intracellular cAMP. EP3-Rs are coupled to G i and decrease hormone-stimulated cyclic AMP (cAMP) formation ( Fig. 1) (Coleman, 1994). These receptors display an overall sequence homology of about 50%; the putative transmembrane domains are the most conserved (Coleman, 1994).
Prostaglandin E 2 receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EPiR are coupled to G q , EP 2 and EP 4 R to G s and EP 3 R to G;. Different C-terminal splice variants of the bovine EP 3 R are coupled to different G-proteins. A mouse EP 3 R whose C-terminal domain had been partially truncated no longer showed agonist-induced Gj -protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP 3 hEP 4 R, containing the N-terminal main portion of the Gj-coupled rat EP 3 ßR including the 7th transmembrane domain and the intracellular C-terminal domain of the G s -coupled human EP 4 R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP 3 hEP4R cDNA expressed a plasma membrane PGE 2 binding site with a slightly lower K d value for PGE 2 but an identical binding profile for receptorspecific ligands as cells transfected with the native rat EP 3 ßR. In HepG 2 cells stably transfected with the chimeric rEP 3 hEP 4 R cDNA PGE 2 did not increase cAMP formation characteristic of G s coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of Gj coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP 3ß R. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.
For the five principal prostanoids PGD P , PGE P , PGF PK , prostacyclin and thromboxane A P eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF PK receptor, which is conserved among all PGF PK receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by sitedirected mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF PK with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF PK . Wild type receptor and H81Q showed a shallow pH optimum for PGF2K K binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF PK with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF PK receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.z 1999 Federation of European Biochemical Societies.
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