Varvara Nikolaidou-Neokosmidou

Vassilis I Zannis

Dimitris Kardassis
email: kardasis@imbb.forth.gr

Inhibition of Hepatocyte Nuclear Factor 4 transcriptional activity by the Nuclear Factor kappa B pathway

Keywords: HNF-4, NF-κB, transcriptional inhibition, APOC3, TNFα, coactivators Activation Function, APOA1: apolipoprotein A-I, APOA4: apolipoprotein A-IV, APOC3: apolipoprotein C-III, CBP: CREB binding protein

Hepatocyte Nuclear Factor 4 (HNF-4) is a key regulator of liver specific gene expression in mammals. We have shown previously that the activity of the human apolipoprotein C-III (APOC3) promoter is positively regulated by the antiinflammatory cytokine Transforming Growth Factor β (TGFβ) and its effectors Smad3 and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine Tumor Necrosis Factor α (TNFα) antagonizes TGFβ for the regulation of APOC3 gene expression in hepatocytes. TNFα was a strong inhibitor of the activity of apolipoprotein promoters that harbor HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFα-induced signaling pathways it was shown that inhibition of the APOC3 promoter by TNFα involved Nuclear Factor kappa B (NF-κB). Latent Membrane Protein 1 of Epstein Barr Virus which is an established, potent activator of NF-κB as well as wild type forms of various NF-κB signaling mediators also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFα had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells but inhibited the binding of HNF-4 to the proximal APOC3 HRE. Using the GAL4 system, we showed that both Activation Functions of HNF-4 (AF-1 and AF-2) are inhibited by TNFα and that this inhibition was abolished by overexpression of different HNF-4 coactivators including PGC-1, CBP and SRC3. In summary, our findings indicate that TNFα or other factors that trigger an NF-κB response in hepatic cells inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and

INTRODUCTION

Hepatocyte Nuclear Factor 4 (HNF-4) belongs to the hormone nuclear receptor gene superfamily of transcription factors on the basis of its domain architecture which is characterized by the presence of an N-terminal Activation Function 1 (AF-1), a DNA binding domain (DBD) of the zinc-finger type and a multifunctional ligand binding domain (LBD) which harbors Activation Function 2 (AF-2) and is connected with the DBD by a hinge region [START_REF] Mangelsdorf | The nuclear receptor superfamily: the second decade[END_REF][START_REF] Sladek | Nuclear Receptors and Genetic Disease[END_REF][START_REF] Giguere | Orphan Nuclear Receptors: From Gene to Function[END_REF][START_REF] Hadzopoulou-Cladaras | Functional Domains of the Nuclear Receptor Hepatocyte Nuclear Factor 4[END_REF]. Although HNF-4 belongs to a subclass of nuclear receptors termed "orphan", it was recently shown that fatty acids may constitute endogenous ligands of HNF-4 [START_REF] Duda | Structural Basis for HNF-4α Activation by Ligand and Coactivator Binding[END_REF][START_REF] Dhe-Paganon | Crystal Structure of the HNF-4α Ligand Binding Domain in Complex with Endogenous Fatty Acid Ligand[END_REF][START_REF] Wisely | Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids[END_REF] HNF-4 also harbors an inhibitory C-terminal domain which modulates the binding of coactivators to the adjacent AF-2 [START_REF] Hadzopoulou-Cladaras | Functional Domains of the Nuclear Receptor Hepatocyte Nuclear Factor 4[END_REF][START_REF] Ruse | Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain[END_REF]. The AF-1 and AF-2 domains of HNF-4 have been shown to interact with different members of the p160 family of coactivators including SRC-1 and GRIP1/TIF2 [START_REF] Sladek | Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1[END_REF][START_REF] Wang | SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4[END_REF][START_REF] Iordanidou | Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha[END_REF], as well as with the histone acetyltransferase CREBbinding protein (CBP) [START_REF] Dell | CREB-binding Protein Is a Transcriptional Coactivator for Hepatocyte Nuclear Factor-4 and Enhances Apolipoprotein Gene Expression[END_REF]. CBP acetylates HNF-4 and this modification has been shown to regulate several functions of HNF-4 including DNA binding, nuclear accumulation and interaction with coactivators [START_REF] Soutoglou | Acetylation regulates transcription factor activity at multiple levels[END_REF]. In addition, HNF-4 interacts with the peroxisome proliferator activated receptor gamma coactivator 1 (PGC-1), a protein that plays important roles in hepatic gluconeogenesis [START_REF] Iordanidou | Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha[END_REF][START_REF] Yoon | Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1[END_REF]. HNF-4 is predominantly expressed in the liver, intestine, pancreas and kidney and regulates the expression of genes and gene networks with important physiological roles in the above organs [START_REF] Sladek | Nuclear Receptors and Genetic Disease[END_REF]. Conditional inactivation of HNF-4 in the liver of mice caused weight loss, increased rate of mortality and lipid abnormalities due to impaired expression of HNF-4-regulated genes involved in lipid and bile acid metabolism and transport [START_REF] Hayhurst | Hepatocyte Nuclear Factor 4α (Nuclear Receptor 2A1) Is Essential for Maintenance of Hepatic Gene Expression and Lipid Homeostasis[END_REF]. Among the genes that were transcriptionally silenced by the inactivation of HNF-4 in the liver was the apolipoprotein C-III gene [START_REF] Hayhurst | Hepatocyte Nuclear Factor 4α (Nuclear Receptor 2A1) Is Essential for Maintenance of Hepatic Gene Expression and Lipid Homeostasis[END_REF]. Apolipoprotein C-III (APOC3) has been implicated in the modulation of binding of apoE-containing lipoproteins to cell receptors and their subsequent catabolism [START_REF] Herbert | The Metabolic Basis of Inherited Diseases[END_REF][START_REF] Brown | Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein[END_REF][START_REF] Shelburne | Effect of apoproteins on hepatic uptake of triglyceride emulsions in the rat[END_REF][START_REF] Windler | Regulation of the hepatic uptake of triglyceride-rich lipoproteins in the rat. Opposing effects of homologous apolipoprotein E and individual C apoproteins[END_REF][START_REF] Krauss | Further observations on the activation and inhibition of lipoprotein lipase by apolipoproteins[END_REF]. The importance of APOC3 in triglyceride homeostasis is supported by studies in transgenic mice in which overexpression of the APOC3 gene was found to be associated with severe hypertriglyceridemia due to the defective clearance of triglyceride-rich lipoprotein remnants [21,22].

The human APOC3 gene is closely linked to the human apoA1 and apoA4 genes on the long arm of chromosome 11 [23]. In vitro mutagenesis established that three hormone response elements (HREs), located in the proximal promoter and enhancer as well as three Sp1 binding sites located in the APOC3 enhancer are important for the APOC3 gene expression in hepatic cells [24][25][26][START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF]. Two of the above HREs (elements B and I) bind HNF-4 and other orphan and liganddependent nuclear receptors [25][26][START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF].

Previous studies have demonstrated that the APOC3 gene is downregulated during the acute phase-response due to the action of proinflammatory cytokines such as tumor necrosis factor-α (TNFα) and interleukin-1 (IL-1) [START_REF] Lacorte | Activation of CAAT enhancer-binding protein delta (C/EBPdelta) by interleukin-1 negatively influences apolipoprotein C-III expression[END_REF][START_REF] Lacorte | Repression of apoC-III gene expression by TNFalpha involves C/EBPdelta/NF-IL6beta via an IL-1 independent pathway[END_REF]. Transcription factors found previously to mediate this process include the Activation Protein-1 (AP-1) proteins c-jun and activating transcription factor 2 (ATF-2) as well as the CAAT/Enhancer Binding Protein δ (C/EBPδ) [START_REF] Lacorte | Repression of apoC-III gene expression by TNFalpha involves C/EBPdelta/NF-IL6beta via an IL-1 independent pathway[END_REF][START_REF] Hadzopoulou-Cladaras | Transactivation of the ApoCIII promoter by ATF-2 and repression by members of the Jun family[END_REF]. Natural extinguishing of the acute phase response occurs in part due to the production of anti-inflammatory cytokines such as interleukin-10, interleukin-13, and Transforming Growth Factor β (TGFβ) [START_REF] Koj | Termination of acute-phase response: role of some cytokines and anti-inflammatory drugs[END_REF]. TGF-β and its signaling mediators, the Smad proteins, are potent anti-inflammatory cytokine in mammals [START_REF] Shull | Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease[END_REF][START_REF] Moustakas | Smad regulation in TGF-beta signal transduction[END_REF][START_REF] Massague | TGF-beta signal transduction[END_REF][START_REF] Ten Dijke | New insights into TGF-beta-Smad signalling[END_REF].

We have shown recently that TGFβ and its signal transducers Smad proteins transactivate the APOC3 gene promoter by interacting physically and functionally with HNF-4 that binds to the proximal APOC3 HRE (element B) [START_REF] Kardassis | SMAD proteins transactivate the human ApoCIII promoter by interacting physically and functionally with hepatocyte nuclear factor 4[END_REF][START_REF] Chou | Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4[END_REF]. We now show that the pro-inflammatory cytokine Tumor Necrosis Factor α (TNFα) antagonizes TGFβ for the regulation of APOC3 gene expression in hepatocytes. Inhibition of the APOC3 promoter by TNFα requires the participation of the NF-κB pathway which affects the DNA binding and transactivation potential of HNF-4.

MATERIALS AND METHODS

Materials

Indirect Immunofluorescence.

HepG2 cells or transfected COS-7 cells were seeded on glass coverslips, coated with 0.1% gelatin. Cells were washed three times on a slow rotating platform with PBS+/+ (PBS plus 0.9 mM CaCl 2 and 0.5 mM MgCl 2 ) and fixed with 3% p-formaldehyde in PBS +/+ for 5 min at room temperature. Cells were washed three times with PBS+/+ and permeabilized with 0.5% Triton X-100 in buffer 1

Western blotting

For Western blotting analysis of endogenous (HepG2) or transfected (HEK-293T) proteins, cells were trypsinized, pelleted by centrifugation at 2,000 rpm for 5 min, washed 1X with ice-cold phosphate-buffered saline, re-centrifuged as above and resuspended in lysis buffer (50mM Tris-HCl pH=7.4, 150mM Nacl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA) supplemented with protease inhibitors

(1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin and 1 µg/ml leupeptin).

Extracts were allowed to rotate at 4 o C for 30 min and centrifuged at 13,000 rpm for 5 min at 4 o C. Protein concentration was measured using the Dc Protein Assay kit and equal amounts were loaded on 10.5 % SDS-polyacrylamide gel electrophoresis followed by electrotransfer to Protran nitrocellulose transfer membrane 0,45 µm (S & S). Immunoblotting was performed using appropriate monoclonal or polyclonal antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence.

Chromatin immunoprecipitations

The chromatin immunoprecipitation assay was performed as described previously [START_REF] Koutsodontis | Inhibition of p53-mediated transcriptional responses by mithramycin A[END_REF] using chromatin from HepG2 cells and a rabbit polyclonal

DNA affinity precipitation

For DNA affinity precipitation, nuclear extracts from HepG2 cells that had been treated with TNFα (1,000u) for 24h or from untreated HepG2 cells were used. Spermidine, 100 µg/ml BSA, 0.02mM Zn acetate, 0.05% NP40 and 0.5mM DTT.

Dynabeads (Dynal

Each reaction included 30 µg of nuclear extracts, 3 µg of competitor poly dI/dC and the biotinylated oligonucleotide-coupled dynabeads that were prepared as describe above in a total reaction volume of 50 µl. Biotinylated oligonucleotides corresponding to the -92/-67 region of the human APOC3 promoter [START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF] and the -86/-70 region of the human p21 Cip1 promoter [START_REF] Koutsodontis | The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter[END_REF] were utilized. HNF-4 bound to the oligonucleotides was detected by SDS-PAGE and immunoblotting using a polyclonal anti-HNF-4 antibody.

RESULTS

Antagonistic effects between TGFβ and TNFα on the activity of the human

APOC3 promoter in HepG2 cells. Negative cross talk between the TGFβ and TNFα signaling pathways on the activity of the human APOC3 promoter was investigated by transactivation assays in human hepatoma-derived HepG2 cells.

For this purpose, HepG2 cells were transfected with a plasmid bearing the firefly luciferase reporter gene under the control of the promoter of the human apolipoprotein C-III gene between nucleotides -890 and +24 (APOC3 -890/+24-Luc) and were treated with increasing amounts of recombinant human TNFα (250, 500, 1000 units) for 24 hr. As shown in Fig. 1A, TNFα treatment inhibited the activity of the APOC3 promoter in a dose-dependent manner. This finding was in agreement with previous observations showing that TNFα inhibited the transcription of the human APOC3 gene in HepG2 cells [START_REF] Lacorte | Activation of CAAT enhancer-binding protein delta (C/EBPdelta) by interleukin-1 negatively influences apolipoprotein C-III expression[END_REF].

It was next shown that the inhibitory effect of TNFα on the APOC3 promoter could be overcome by the simultaneous treatment of HepG2 cells with increasing doses of human recombinant TGFβ (Fig. 1B) suggesting a negative cross talk between the two pathways operating on the APOC3 promoter.

Inhibition of APOC3 promoter activity by TNFα could also be abrogated by overexpression of Smad3 and Smad4 proteins which are the key effectors of the TGFβ pathway (Fig. 1C). In agreement with the data of Fig. 1B, treatment of HepG2 cells with TNFα (1,000 units) for 24 hrs caused a reduction in the steady state mRNA levels of the human APOC3 gene and this inhibition could be overcome by the simultaneous treatment of HepG2 cells with increasing doses (40 and 80pM) of TGFβ (Fig. 1D).

In conclusion, the data of Fig. 1 combined with our previous findings [START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF][START_REF] Lacorte | Repression of apoC-III gene expression by TNFalpha involves C/EBPdelta/NF-IL6beta via an IL-1 independent pathway[END_REF][START_REF] Kardassis | SMAD proteins transactivate the human ApoCIII promoter by interacting physically and functionally with hepatocyte nuclear factor 4[END_REF][START_REF] Chou | Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4[END_REF] indicate that the activity of the human APOC3 promoter in HepG2 cells can be modulated by positive and negative signals which are produced by anti-inflammatory and pro-inflammatory cytokines respectively. This is shown schematically in Figure 1E. promoter activity (Fig. 2C).

Inhibition of APOC3 promoter activity by

Next, the effect of selected NF-κB signaling effectors on APOC3 promoter activity in HepG2 cells was investigated by transactivation assays. Figure 2D shows that overexpression of IKKβ kinase inhibited APOC3 promoter activity in a dose-dependent manner and Fig. 2E shows that overexpression of the p65/relA subunit of NF-κB totally abolished APOC3 promoter activity whereas the p50 subunit had no effect. Finally, a hybrid p65 protein in which the Rel Homology Domain (RHD) of p65 was swapped by the corresponding domain of p50 (p50-p65 hyb) was as effective as full length p65 in inhibiting the APOC3 promoter suggesting that the inhibition is mediated by the C-terminal transactivation domain of p65.

As an additional confirmation of the inhibitory role played by NF-κB in APOC3 promoter regulation, the Latent Membrane Protein 1 (LMP1) of Epstein Barr Virus was utilized in transactivation assays. LMP1 is a protein homologous to TNFα receptor that has been shown previously to be a potent activator of the NF-κB pathway in various cell types [START_REF] Hatzivassiliou | Cellular signaling pathways engaged by the Epstein-Barr virus transforming protein LMP1[END_REF]. In a control experiment, it was established that LMP1 strongly activated a NF-κB-responsive promoter [(NF-κB) 3 ] in HepG2 cells and this activation was inhibited by the dominant negative IκB-ND mutant (Fig. 2F). More importantly, it was shown that a small amount of LMP1 was sufficient to fully block APOC3 promoter activity in HepG2 cells (Fig. 2G).

In summary, the findings of promoter that responds to TNFα, two truncated APOC3 promoter fragments were utilized in transactivation experiments in HepG2 cells. One mutant (APOC3 -686/+24) lacks the region between nucleotides -890 and -687 which includes the distal enhancer elements F-J (Fig. 3A). The second mutant (APOC3 -99/+24) contains the region between nucleotides -99 and +24 which includes only the proximal element B (Fig. 3A). Element B was shown previously to harbor an HRE that binds a selection of orphan and ligand-dependent nuclear receptors including HNF-4 [START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF]. Mutagenesis of this element abolished APOC3 promoter activity both in vitro and in vivo [START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF].

As shown in Fig. 3B, both APOC3 promoter mutants were inhibited by TNFα and LMP1 to approximately the same degree as the full length APOC3

promoter. The minimal region of the APOC3 promoter required for TNFα or LMP1-induced inhibition is the -99/+24 since further deletion of the APOC3 promoter to nucleotide -55 abolished both the constitutive and the TNFα-inhibited APOC3 promoter activity (data not shown).

The data of Figure 3 indicate that inhibition of the APOC3 promoter activity by TNFα could be mediated, at least in part, by interfering with the activity of nuclear receptors that bind to the proximal APOC3 HRE.

TNFα inhibits the HNF-4-mediated transactivation of the APOC3 and APOC2 promoters. Previous in vivo studies have established that HNF-4 is the single most important regulator of the expression APOC3 as well as of other apoliporpotein genes in hepatocytes [START_REF] Hayhurst | Hepatocyte Nuclear Factor 4α (Nuclear Receptor 2A1) Is Essential for Maintenance of Hepatic Gene Expression and Lipid Homeostasis[END_REF]. It was shown previously that HNF-4 binds with high affinity to the proximal HRE B of the APOC3 promoter in vitro [START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF]. The recruitment of HNF-4 to the APOC3 promoter in vivo was established here by a chromatin immunoprecipitation experiment. As shown in Fig. 4B, HNF-4 was recruited to the proximal APOC3 promoter region -230/-20 that includes HRE B but, surprisingly, it failed to bind to the distal region -880/-520 that harbors the distal HREs G and I.

It was then shown that HNF-4 strongly transactivated the APOC3 (-890/+24) promoter in HepG2 cells and that this transactivation was inhibited by TNFα or LMP1 expression (Fig. 4C). A similarly negative effect of TNFα and LMP1 on HNF-4-mediated transactivation was observed using the APOC2 promoter (Fig. 4D) that also contains a binding site for HNF-4 (HRE B, Fig. 4A).

To confirm the requirement of HNF-4 for the TNFα and LMP1-induced inhibition of the APOC3 promoter, transactivation experiments were performed in HEK-293T cells that lack endogenous HNF-4. As shown in Fig. 4E, TNFα or LMP1 had no effect on the activity of the APOC3 promoter in HEK-293T cells whereas they strongly inhibited the HNF-4 mediated transactivation of the same promoter.

As a negative control, the human APOA4 promoter (-700/+10) was utilized. This promoter cannot be transactivated by HNF-4 despite the presence of a HNF-4 binding site in the proximal region (Fig. 4A andE) [START_REF] Ktistaki | Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene[END_REF].

In summary, the findings of Figure 4 was not affected following treatment with TNFα for 24 hr (Fig. 5A). In a double immunofluorescence experiment, it was shown that, similar to TNFα, LMP1 had no effect on HNF-4 nuclear localization in HEK293T cells that had been cotransfected with both factors (Fig. 5B). Immunoblotting analysis clearly showed that neither TNFα nor LMP1 affected the stability of endogenous in HepG2 cells Fig. 5C) or exogenous HNF-4 in HEK-293T cells (Fig. 5D). The transcriptional activity of GAL4-HNF-4 was also inhibited by LMP1 as well as by different effectors of the TNFα and LMP1 pathways including the NIK, the IKKβ kinase and the p65/RelA subunit of NF-κB (Fig. 6C). Finally, it was shown that p65 inhibited not only the constitutive, but also the Smad-inducible transcriptional activity of HNF-4 (Fig. 6D). The latter findings suggested that the antagonistic effects between TGFβ and TNFα on the APOC3 promoter shown in To investigate potential competition between HNF-4 and NF-κB for common coactivators, the ability of PGC-1 to function as a coactivator of LMP1 signaling to NF-κB was examined. As shown in Fig. 8C, PGC-1 strongly coactivated LMP1 in a transactivation assay in which the artificial promoter (NF-κB) 3 was used suggesting that PGC-1 may act as a coactivator of LMP1 signaling to NF-κB. Competition between HNF-4 and LMP1 for PGC-1 was specific for the AF-2 domain of HNF-4 (compare GAL4-HNF-4 (48-455) lacking AF-1 in Fig. 8D with GAL4-HNF-4 (1-49) that contains only AF-1 in Fig. 8E). In contrast, CBP was equally active on both AF-1 and AF-2 domains (Fig. 8 D,E)

TNFα modifies the

The findings of Figure 8 suggested that TNFα and LMP1-induced inhibition of HNF-4 transcriptional activity could be accounted for, at least in part, by interference with the recruitment of coactivators that interact with both the AF-1 and AF-2 domains of HNF-4.

Discussion

The findings of this study indicate that pro-inflammatory cytokines such as TNFα that activate multiple signalling pathways including NF-κB can inhibit the expression of liver-specific genes by a novel mechanism that involves the modification of the DNA binding and the transactivation properties of a hormone nuclear receptor such as HNF-4. This mechanism seems to be important for the negative cross talk between the TNFα and the anti-inflammatory cytokine TGFβ on the expression of the apolipoprotein C-III gene during the acute phase response, a process by which organisms respond to various conditions such as infections, ischemic necrosis and trauma [START_REF] Koj | Termination of acute-phase response: role of some cytokines and anti-inflammatory drugs[END_REF]. This is not the first example of a negative cross talk between the two cytokines. TGFβ/TNFα antagonism regulates the expression of the collagen genes and is very important for the control of tissue homeostasis and repair [START_REF] Schiller | TGF-beta-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing[END_REF]. However, in the case of the collagen genes, the JNK MAPK pathway rather than nuclear receptors is involved in this antagonism [START_REF] Verrecchia | Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha[END_REF].

The participation of nuclear receptors in the mechanism of transcriptional inhibition of the APOC3 gene by TNFα was suggested by the results of transactivation experiments in which deletion mutants of the APOC3 promoter were utilized. These experiments showed that the proximal HRE (element B) was sufficient to mediate repression by TNFα (Fig. 3). Previous in vitro studies had established that this proximal HRE of the APOC3 promoter is of the DR1 type (Direct Repeat with 1 nucleotide spacing) and strongly binds HNF-4 as well as, ARP-1, EAR-2, EAR-3, and heterodimers of RXRα with RARα and less efficiently binds homodimers of RARα and heterodimers of RXRα with T3Rβ or PPARα [START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF]. Among the nuclear receptors that bind to this HRE, HNF-4 seems to play the prominent role as conditional inactivation of HNF-4 in the liver abolished the expression of the APOC3 gene [START_REF] Hayhurst | Hepatocyte Nuclear Factor 4α (Nuclear Receptor 2A1) Is Essential for Maintenance of Hepatic Gene Expression and Lipid Homeostasis[END_REF]. However, in vivo evidence for the recruitment of HNF-4 to this HRE as well as the HRE of the APOC3 enhancer in hepatic cells was missing. By performing a chromatin immunoprecipitation assay, we were able to demonstrate for the first time here the constitutive recruitment of HNF-4 to the proximal HRE of the APOC3 promoter in HepG2 cells (Fig. 4B). Surprisingly, HNF-4 was not found on the distal HRE of the APOC3 enhancer which was shown previously to bind HNF-4 in vitro and to be crucial for the expression of the APOC3 and APOA1 genes in vitro and in vivo (Fig. 4B) [START_REF] Ladias | Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3[END_REF][START_REF] Lavrentiadou | Binding specificity and modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependent nuclear receptors[END_REF][START_REF] Kan | A hormone response element in the human apolipoprotein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I and ApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenic mice[END_REF]. This finding could suggest that nuclear receptors other than HNF-4 are the predominant regulators of APOC3 gene via the distal HRE but this has to be confirmed by additional in vivo DNA binding experiments. The recruitment of nuclear receptors other than HNF-4 on the proximal HRE and their involvement in transcriptional responses to extracellular signal also need to be thoroughly investigated.

Using GAL4-HNF-4 fusion proteins and different NF-kB inducing agents such as TNFα and the LMP1 transforming protein of the Epstein Barr Virus, we provide strong evidence for the involvement of the NF-κB pathway in the nuclear functions of HNF-4. (Fig. 6). This could be explained by different, not necessarily mutually exclusive mechanisms: a) An indirect mechanism such as the transcriptional activation of an HNF-4 inhibitor by NF-κB. Such an inhibitor could be the small heterodimer partner (SHP) which was shown to physically interact with HNF-4 and inhibit its transcriptional activity [START_REF] Lee | The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression[END_REF]. This hypothesis is supported by the findings of Fig. 2E and that at least one of these proteins (PGC-1) may function as coactivator of NF-κB signaling (Fig. 8C).

In addition to the negative effect of the TNFα/NF-kB pathway on the transactivation properties of HNF-4, TNFα appears to modify the DNA binding properties of this transcription factor as well. As shown in Fig. 5E, HNF-4 present in nuclear extracts from HepG2 cells that had been pre-treated with TNFα has a lower affinity for the proximal APOC3 HRE than HNF-4 present in untreated cells.

This may be due to a modification such as phosphorylation of HNF-4 in the DNA Binding Domain that affects its affinity for DNA. In support of this hypothesis are previous studies which have shown that the DNA binding properties of HNF-4 can be modulated in a positive or a negative manner by modifications such as acetylation [START_REF] Soutoglou | Acetylation regulates transcription factor activity at multiple levels[END_REF] and phosphorylation which is induced by different signal transduction pathways involving MAP and JAK kinases [START_REF] De Fabiani | The negative effects of bile acids and tumor necrosis factor-alpha on the transcription of cholesterol 7alpha-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4: a novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors[END_REF][START_REF] Li | Hepatocyte nuclear factor 4 response to injury involves a rapid decrease in DNA binding and transactivation via a JAK2 signal transduction pathway[END_REF].

In summary, the present as well as previous studies from our group showed that the orphan nuclear receptor HNF-4 may have an important role in hepatic gene regulation not only as a constitutive transcriptional activator but also

  All reagents for cell culture including Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Trypsin-Ethylene Diamine Tetraacetic Acid (Tryprin-EDTA) and Phosphate-Buffered Saline (PBS) were purchased from Life Technologies. O-nitrophenyl-galactopyranoside (ONPG), phenylmethylsulfonyl fluoride, the anti-FLAG (M2) mouse monoclonal antibody and the anti-β tubulin mouse monoclonal antibody were purchased from Sigma. The anti-HNF-4 (C-19) goat polyclonal antibody and anti-NF-κB p65(A) rabbit polyclonal antibody were purchased from Santa Cruz Biotechnology. The anti-pERK and anti-ERK antibodies were gifts of Dr G. Mavrothalassitis (University of Crete, Heraklion, of Thessaloniki, Greece). The expression vector for the human PGC-1 coactivator was a kind gift of Dr Anastasia Kralli (Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA). The expression vector for the human CBP coactivator was a kind gift of Dr Ioannis Talianidis (Institute of Molecular Biology and Biotechnology of Crete, Heraklion). The expression vector for the human SRC3 coactivator was a kind gift of Dr Hinrich Gronemeyer (IGBMC, Illkrich, Strasbourg, France) Reverse Transcription PCR HepG2 cells were treated with 1,000 units of TNFα in the absence or in the presence of TGFβ (40 and 80pM) for 24 hr or left untrated, total RNAs were prepared and equal concentrations of RNAs were used for cDNA synthesis. The synthesis of cDNAs was performed using Superscript reverse transcriptase and random haxamers as primers. The cDNAs were used for PCR amplifications with primers corresponding to the human APOC3 cDNA. The sequences of the

  antibody for human HNF-4. Immunoprecipitated chromatin was analyzed by PCR using primers corresponding to the proximal (-233/-21) and distal (-882/-518) regions of the human APOC3 promoter. The proximal APOC3 promoter primers were: P1: 5' CAG GCC CAC CCC CAG TTC CTG AGC TCA 3'; P2: 5' CCT GTT TTA TAT CAT CTC CAG GGC AGC AGG C 3'. The distal APOC3 promoter primers were: D1: 5' AGT TGC TCC CAC AGC CAG GGG GCA GT 3'; D2: 5' TCT CAC AGC CCC TCC CAG CAC CTC CAT 3'. The products of the PCR amplifications (35 cycles) were analyzed by agarose gel electrophoresis and ethidium bromide staining.

  TNFα is mediated by NF-κB. To identify and characterize the signaling pathway(s) that (is) are responsible for the TNFα-induced inhibition of APOC3 promoter activity in HepG2 cells, two inhibitors that specifically block well characterized and distinct TNFα-induced signaling cascades were utilized: a mutant that blocks the cascade leading to NF-κB activation (a dominant negative, non-degradable form of the IκB inhibitor that keeps NF-κB in the cytoplasm, Iκ-ND) and a chemical inhibitor (the MEK1 inhibitor U0126) that blocks the cascade leading from TNFα to the Extracellular Signal Regulated Kinase (ERK). First, the activation of the above signaling cascades by TNFα in HepG2 cells was shown by well established assays such as the TNFα-induced p65 nuclear translocation (Fig.2A) and the TNFα-induced phosphorylation of ERK (Fig.2B). Then the effect of the two inhibitors on the constitutive as well as on the TNFα-inducible activity of the APOC3 promoter was evaluated. As shown in Fig.2C, inhibition of the APOC3 promoter by TNFα was totally abolished in the presence of the IκB-ND inhibitor. In fact, a strong activation of the APOC3 promoter was observed by the NF-κB inhibitor even in the absence of TNFα indicative of the existence of strong endogenous negative signals in HepG2 cells that constitutively inhibit APOC3 promoter activity via the NF-κB pathway. In contrast, the U0126 inhibitor of the MEK1/ERK pathway had only a minor positive effect on both the basal and the TNFα-inhibited APOC3

Figure 2

 2 indicate that activation of the NF-κB pathway in HepG2 cells by TNFα causes a strong inhibition of the APOC3 promoter activity. The molecular mechanism of this inhibition was the subject of the subsequent experiments presented below. The proximal Hormone Response Element of the APOC3 promoter is sufficient to mediate inhibition by TNFα. To identify the region of the APOC3

4 .

 4 indicate that TNFα and other NF-κB activating factors can inhibit the activity of a certain set of promoters harboring HNF-4 binding sites and that HNF-4 is required for this inhibition. TNFα does not affect the nuclear localization or the stability of HNF-To investigate further the mechanism of inhibition of HNF-4-mediated transactivation by TNFα, the effect of TNFα and LMP1 on HNF-4 nuclear localization and stability was examined. Using immunofluorescence analysis, it was shown that the constitutive nuclear accumulation of HNF-4 in HepG2 cells

DNA binding properties of HNF- 4 .

 4 It was next examined whether TNFα affected the DNA binding properties of HNF-4. For this purpose a DNA affinity precipitation experiment was performed using endogenous HNF-4 in HepG2 cells and a biotinylated double stranded oligonucleotide corresponding to the -92/-67 region of the APOC3 promoter that includes the proximal HRE. As shown in Fig.5E, HNF-4 bound efficiently to the proximal APOC3 (-92/-67) oligonucleotide and this binding was inhibited by TNFα. In control experiments it was shown that HNF-4 did not bind to the streptavidin dynabeads or to a double stranded synthetic oligonucleotide corresponding to the -86/-70 region of the human p21 Cip1 promoter[START_REF] Koutsodontis | The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter[END_REF].TNFα and NF-κB inhibit the transcriptional activation function of HNF-4 by modulating the activity of AF-1 and AF-2 domains.In the next series of experiments, the effects of the NF-κB pathway on nuclear HNF-4 functions was investigated using the well characterized GAL4 system. For this purpose, a fusion protein consisting of full length rat HNF-4a1 (aa 1-455) fused with the DNA Binding Domain of the yeast transactivator GAL4 (aa 1-147) (Fig.6A) was transiently expressed in HepG2 cells and its ability to transactivate the GAL4-responsive artificial promoter G5B (consisting of 5 tandem GAL4 binding sites) in the presence or in the absence of different NF-κB pathway activators and effectors was evaluated by luciferase assays. As shown in Fig.6B, TNFα strongly inhibited the transcriptional activity of GAL4-HNF-4 in HepG2 cells. This repression could be partially reversed by the dominant negative IκB-ND mutant but not by the MEK1 inhibitor U0126 in agreement with the findings of Fig.2C.

Fig. 1B could 4 ( 4 -

 44 Fig. 1B could be accounted for by antagonistic interactions between their

  which showed that inhibition of HNF-4 requires the transactivation domain of the p65/Rel subunit of NF-κB; β) Inhibition could be accomplished by interference of NF-κB with the recruitment or functions of different HNF-4 coactivators. HNF-4 has been shown to interact with members of the p160 family of coactivators including SRC-1 and GRIP1/TIF2 as well as with the histone acetyltransferase CREB-binding protein (CBP) and with PGC-1, a coactivator that plays important roles in hepatic gluconeogenesis[START_REF] Ruse | Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain[END_REF][START_REF] Sladek | Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1[END_REF][START_REF] Wang | SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4[END_REF][START_REF] Iordanidou | Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha[END_REF][START_REF] Dell | CREB-binding Protein Is a Transcriptional Coactivator for Hepatocyte Nuclear Factor-4 and Enhances Apolipoprotein Gene Expression[END_REF][START_REF] Soutoglou | Acetylation regulates transcription factor activity at multiple levels[END_REF][START_REF] Yoon | Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1[END_REF]. In agreement with this hypothesis are the data presented in Fig.8of this study which showed that inhibition of HNF-4 by TNFα or LMP1 was overcome by overexpression of different HNF-4 coactivators including CBP, SRC3 and PGC1 which interact with distinct transactivation domains of HNF-4 (AF-1 and AF-2)

Figure 1 :

 1 Figure 1: Antagonistic effects of pro-and anti-inflammatory cytokines on

Figure 2 :

 2 Figure 2: Inhibition of APOC3 promoter activity by TNFα is mediated by NF-κB. Panel A, TNFα activates NF-κB in HepG2 cells. HepG2 cells were serum

Figure 3 :

 3 Figure 3: The proximal Hormone Response Element of the APOC3

Figure 4 :

 4 Figure 4: TNFα inhibits the HNF-4-mediated transactivation of the APOC3

Figure 5 :Figure 6 :

 56 Figure 5: TNFα does not affect the nuclear localization or the stability but

Figure 7 : 4 ( 48 -

 7448 Figure 7: Both transactivation domains of HNF-4 (AF1 and AF2) are

Figure 8 : 4 (

 84 Figure 8: Inhibition of HNF-4 activity by TNFα may be accounted for by

ACKNOWLEDGEMENTS

We are grateful to Drs C. Stournaras, G. Mavrothalassitis, A. Eliopoulos, C. Tsatsanis (University of Crete), I. Talianidis (IMBB-FORTH, Heraklion), A. Gafencu (ICBP "N. Simionescu", Bucharest) and M. Hadzopoulou-Cladaras (Aristotelian University of Thessaloniki) for reagents, protocols and discussions. We thank Dr G. Koutsodontis for assistance in the chromatin immunoprecipitation experiment. This research was supported by a grant from the Greek Ministry of Development (PENED-2001) and internal funds from the Institute of Molecular Biology and Biotechnology of Crete.


Sergio Fernández-Pascual

Henrik Ortsäter

Martín Del Río

Peter Bergsten

Jorge 1 Tamarit-Rodriguez

Oxo-4-methylpentanoic acid directs the metabolism of GABA into the citric acid cycle in rat pancreatic islets

Keywords: methylpentanoic acid, GABA ( -aminobutyric acid), -amino acids, islets, glucose, L-leucine aminobutyric acid; OMP: Oxo-4-methylpentanoic acid;

Oxo-4-methylpentanoic acid (OMP) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-glutamine is predominantly converted to GABA in rat islets and L-leucine seems to promote its metabolism in the "GABA shunt" (Fernández-Pascual S. et al, Biochem. J. 379, 721-729, 2004). We have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. At variance with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the medium L-glutamine concentration. It was transaminated to L-leucine whose intracellular concentration did not change because it was very efficiently transported out of the islet cells. L-[U-14 C]Glutamine, 0.5 and 10.0 mM, conversion to 14 CO 2 was enhanced by 10 mM OMP within 30 and 70 %, respectively. Gabaculine (250 M), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not Lleucine or glucose stimulated respiration. It also suppressed OMP-induced decrease of islet GABA content and OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the "GABA shunt" generating 2oxoglutarate, in the branched-chain -amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the "GABA shunt" from -hydroxybutyrate to succinic acid production.

INTRODUCTION

L-Leucine is unique, amongst all the other natural -amino acids, in its ability to trigger a transient stimulation of insulin secretion [START_REF] Somers | Stimulus-secretion coupling of amino acid-induced insulin release VII. The B-cell memory for L-glutamine[END_REF] that becomes sustained in the presence of L-glutamine [START_REF] Panten | Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable analogue[END_REF]. The common explanation is that L-leucine activates allostericallycell glutamate dehydrogenase (GDH) [START_REF] Sener | L-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase[END_REF][START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF] and, in that way, favours L-glutamate oxidative metabolism in the citric acid cycle [START_REF] Malaisse | Insulin release: reconciliation of the receptor and metabolic hypothesis. Nutrient receptors in islet cells[END_REF]. It is then understandable that secretion is only sustained in the presence of L-glutamine which provides the substrate (Lglutamate) for GDH. By contrast, the deaminated derivative of L-leucine, oxo-4methylpentanoic acid (OMP), is capable of triggering a biphasic secretion of insulin in islets in the complete absence of L-glutamine [START_REF] Lenzen | 2-Oxocarboxylic acids and function of pancreatic islets in obese-hyperglycaemic mice. Insulin secretion in relation to 45 Ca 2+ uptake and metabolism[END_REF][START_REF] Hutton | Similarities in the stimulus-secretion coupling mechanisms of glucose-and 2-keto acid-induced insulin release[END_REF]. It has been argued that OMP might be transaminated in -cells to L-leucine which would activate GDH and make more substrate

(2-oxoglutarate) available for the Krebs-cycle [START_REF] Lenzen | Transamination of neutral amino acids and 2-keto acids in pancreatic B-cell mitochondria[END_REF].

However, this does not explain the strikingly different potency, and L-glutaminedependence, between the amino acid and the -keto acid as insulin secretagogues. A different secretagogue behaviour of L-leucine and OMP has also been reported in islets cultured at high glucose [START_REF] Gao | Distinguishing features of leucine and -ketoisocaproate sensing in pancreatic -cells[END_REF] or in islets from diabetes-susceptible mice [START_REF] Rabaglia | Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes susceptible mice[END_REF]. Therefore, it does not seem unreasonable to propose that OMP might stimulate insulin secretion by a different mechanism than L-leucine, not relying exclusively in its transamination to the -amino acid.

We have supplied evidence [START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF] indicating that L-leucine might facilitate the metabolism of L-glutamine-derived L-glutamate in the Krebs cycle and the "GABA-shunt": L-Glutamine-derived L-glutamate is predominantly metabolized to GABA by glutamate acid decarboxylase (GAD) in islets, this probably being the reason for it not acting as a secretagogue, and L-leucine is capable of decreasing the islet content of GABA elevated by glutamine. GABA may accumulate in synaptic-like microvesicles from which it may be released by exocytosis [START_REF] Braun | Regulated exocytosis of GABAcontaining synaptic-like microvesicles in pancreatic -cells[END_REF]. Alternatively, GABA might be transaminated to semialdehyde succinic acid (SSA) by GABA transaminase (GABA-T) [START_REF] Garry | Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell[END_REF] which would then be either oxidized to succinic acid by the SSA-dehydrogenase [START_REF] Blasi | Structure of human succinic semialdehyde dehydrogenase gene: identification of promoter region and alternatively processed isoforms[END_REF] or reduced to -hydroxybutyrate by a SSA-reductase [START_REF] Kelly | Novel homodimeric rat -hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins[END_REF]. This work was aimed at investigating the capacity of oxo-4-methylpentanoic acid to modify the metabolism of glutamine (glutamate), main GABA precursor(s), in isolated rat islets. For this purpose, the effect of oxo-4-methylpentanoic acid on islet glutamine oxidation, islet amino acid profile (islet content of GABA, L-glutamate, and L-aspartate), and the different enzyme activities of the "GABA-shunt" (GAD, GABA-T, SSA-dehydrogenase and SSAreductase) were explored.

EXPERIMENTAL Materials

Collagenase P was obtained from Roche Diagnostics S.L. (Barcelona, Spain). Bovine serum albumin (BSA) and most of the organic compounds were obtained from Sigma-Aldrich Química S.A. (Madrid, Spain). Rat insulin was from Linco Research, Inc. (St.

Charles, Missouri, U.S.A.). Na[ 125 I] was obtained from Amersham Iberica S.A.

(Madrid, Spain); D-[U- 14 C]glutamine and NaH 14 CO 3 were from New England Nuclear (Itisa Biomedica S.A:, Madrid, Spain) or American Radiolabeled Chemicals (Itisa Biomedica S.A., Madrid, Spain). Inorganic compounds and organic solvents were obtained from Merck Farma y Química S.A. (Madrid, Spain).

Methods

Insulin secretion

Islets were isolated from the pancreas of male Wistar-albino rats (250 g BW) by collagenase digestion. Insulin secretion was studied in perifused or incubated islets.

Two groups, each of 40 rat islets, were perifused in parallel and at a flow rate of 0.5 ml/min with Krebs-Ringer, buffered with 0.5 mM NaHCO 3 and 20 mM HEPES, supplemented with 0.5 % BSA (KRBH), and heated at 37 0 C. The perifusion protocol was similar in all the experiments. After a pre-perifusion period of 45 minutes under basal conditions (in the absence of nutrients), the perifusion medium was switched to one containing the test substances and maintained for the next 30 minutes. Finally, the medium was changed back to pre-perifusion conditions where it was maintained for the last 25 minutes. The perifusate was collected at 1 minute intervals during the last 60-70 minutes of perifusion and its insulin concentration radioimmunologically measured.

Alternatively, three batches each of ten islets were incubated in 1 ml of KRBH at 37 0 C for 120 minutes and the concentration of insulin released in the incubation medium was radioimmunologically measured. Pig insulin was radio-iodinated with Na 125 I [START_REF] Sykes | A new chamber for tissue culture[END_REF] and rat insulin was used as a standard in the radioimmunoassay of insulin. Insulin antiserum was kindly provided by Dr. Janove Sehlin from the Department of Medical Cell Biology at the University of Umeå (Sweden).

L-Glutamine oxidation

Oxidation of L-glutamine was measured as the production of 14 CO 2 from L-[U- 14 C]glutamine as previously described [START_REF] Ashcroft | The pentose cycle and insulin release in mouse pancreatic islets[END_REF] and corrected according to the recovery of NaH 14 CO 3 which was measured three times during each experiment.

Amino acid measurements

Islet -amino acids were separated by reverse-phase HPLC after precolumn derivatization with o-phthalaldialdehyde [START_REF] Jones | Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phtalaldialdehyde precolumn labeling procedure[END_REF] and quantified by fluorescence detection.

Islets (groups of 20) were incubated for 60 minutes at 37 0 C in 100 l of the same buffer as that used in perifusions. Incubations were stopped by placing the tubes containing the islets on ice. The medium was aspirated and the islets were washed twice with 100 l of cold PBS. Finally, 100 l of 35 % (w/v) 5-sulfosalicylic acid were added in order to extract islet amino acids. Final extracts were kept at -80 0 C until their amino acid content was determined. Twelve amino acids were regularly identified and separated in the extracts and they were measured with a sensitivity close to 1 pmol (Asp, Glu, Ser, Gln, His, Gly, Thr, Arg, Tau, Ala, Tyr, and GABA). Occasionally, the gradient elution profile of the chromatographic separation was changed in order to separate and quantify also L-leucine. The amount of protein was measured in the extracts with the method of Lowry [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] and BSA was used as the standard. In experiments aimed at investigating the release of amino acids, islets (groups of 30) were incubated for 60 minutes at 37 0 C in 70 l of the same buffer as that used in perifusions. After stopping the incubation on ice, 50 l of the supernatant was withdrawn and stored frozen until amino acid measurement was performed. The remaining medium was discarded and the islets were processed as described before for the measurement of their amino acid content.

Enzyme activities

SSA-dehydrogenase and SSA-reductase activities were measured as the production of NADH or the consumption of NADPH, respectively, in homogenates of rat islets or cerebellum, at 0.2 mM SSA. Tissue samples (islets or cerebellum) were homogenized in a homogenization buffer (50 mM NaHPO 4 and 2 mM EDTA, pH= 7.2) containing: leupeptin (31 g/ml), aprotinin (4 g/ml), PMSF (40 l of a 50 mM solution in DMSO /ml), -mercaptoethanol (0.7 l/ml), and Triton-X100 (0.5 %, vol/vol). Isolated islets were homogenized by sonication (four times, ten strokes at 10-20 % of the cycle and the minimum potency of a Branson sonifier 450); for extraction of cerebellum samples, a higher potency [START_REF] Malaisse | Insulin release: reconciliation of the receptor and metabolic hypothesis. Nutrient receptors in islet cells[END_REF] and frequency (50 %) of sonication were used. The homogenates were centrifuged at 40,000 rpm for 20 minutes and the supernatants stored at + 4 0 C until the measurement was performed. Enzyme activities were measured in the same buffer used for homogenization but supplemented with -mercaptoethanol (5 mM), oxamate (lactate dehydrogenase inhibitor, 2 mM), rotenone (5 M), Triton-X100 (0.5 %, vol/vol), BSA (5 mg/ml), and either NADPH (50 M) or NAD + (1 mM). The total volume was 500 l and the reaction was initiated by addition of 5 l of 20 mM SSA, after mixing 470 l of the buffer plus 25 l of islet homogenate (approximately 6 islets / l) or a convenient dilution of the cerebellum homogenate (10 l, at 250 mg/ml) in the cuvette. Absorbance at 340 nm evolved linearly during several minutes and its slope was calculated and taken as a measure of enzyme activity.

GABA transaminase activity of islet homogenates was measured as the formation of GABA from saturating concentrations (20 mM) of L-glutamate and succinic acid semialdehyde and as the formation of L-glutamate from saturating concentrations of GABA (20 mM) and 2-oxoglutarate (10 mM) during 15 minutes of incubation at 37 0 C.

The enzyme activity was extracted in the same homogenization buffer described above for the assay of SSA-related activities, supplemented with leupeptin (25 g/ml), aprotinin (25 g/ml), and PMSF (20 l/ ml of a 50 mM solution in DMSO). Isolated islets were sonicated (approximately 1 islet / l) under the conditions described above for the other islet enzyme activities. The enzyme activity was measured in the same buffer as described above for homogenization but supplemented with the adequate substrates. The reaction was stopped by the addition of 5-sulfosalicylic acid (3.5 % w/v, final concentration). The amounts of GABA or L-glutamate formed were measured by fluorescence detection after precolumn derivatization with o-phthalaldialdehyde and HPLC separation.

Oxygen tension measurements

Intra-islet oxygen tension was measured as previously described [START_REF] Ortsäter | Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets[END_REF]. Briefly, individual islets were attached to a poly-lysine coated coverslip, subsequently used as a floor in a Sykes-Moore perifusion chamber [START_REF] Sykes | A new chamber for tissue culture[END_REF] thermostated at 37 0 C. The perifusion medium was supplemented with 1 mg/ml albumin and contained (in mM): NaCl 125, KCl 5.9, MgCl

RESULTS

Insulin secretion

The secretagogue capacity of oxo-4-methylpentanoic acid (OMP) was explored in a dynamic system of perifused rat islets as well as in batch type incubations. Figure 1 shows that 10 mM OMP stimulates a biphasic secretion of insulin by itself. L-Glutamine (0.5 or 10 mM) slightly enhanced the height of the first phase but did not modify at all the second phase. However, no significant differences between the overall effects induced by each of the two L-glutamine concentrations compared with the control could be demonstrated. In islets pre-perifused for 45 minutes with 250 M gabaculine, the subsequent secretory response to 10 mM OMP in the continuous presence of the drug was significantly reduced (26 %, p<0.008) as compared with control islets (Figure 2). The amount of insulin released during the first phase of secretion (first ten minutes of stimulation) was not modified although the height of its peak (minute 20th) was significantly reduced (2.1 0.2, n=16, vs. 2.7 0.1 ng/ min x 40 islets, n=17; p<0.007). The overall amount of insulin secreted during the second phase (last twenty minutes of stimulation) was decreased by 33% (22.1 2.6, n=14, vs. 33.0 1.9 ng/ 40 islets, n=17; p<0.002) and its rate showed a trend to diminish progressively with time. In fact, in islets incubated with 10 mM OMP for 2 hours, 250 M gabaculine induced a greater decrease (-47%) of the secretory response (9.5 1.2, n=6, vs. 17.9 2.2 pg / min x islet, n=6; p< 0.008).

Islet amino acid profile

As reported earlier (4), changes of the extracellular L-glutamine concentration in the range 0.1 to 10.0 mM induce variations of the intracellular concentration of L-glutamine and other amino acids deriving from the amine, like L-glutamate, L-aspartate, and GABA. In order to compare their respective turnover rates and to investigate the effect of OMP, the evolution of islet amino acids contents (Asp, Glu, and GABA) was followed for three hours. The results (Figure 3) show that the turnover rate of islet GABA, calculated as the slope of the linear decrease of the amino acid with time, is higher than that of L-aspartate (4.6 vs. 2.6 pmol / g protein x hour) and L-glutamate

(not modified at all). L-Aspartate transamination to L-glutamate might compensate its decarboxylation to GABA and contribute to keep islet L-glutamate constant. OMP (10 mM) strongly decreased islet GABA content (-40%) and accelerated islet GABA turnover (Figure 3), calculated as the mean of the differences in amino acid content between control and OMP-stimulated islets during three hours, by 77% (8.1 vs. 4.6 pmol/ g protein x hour). Furthermore, OMP accelerated L-aspartate by 65% (4.4 vs. 2.6 pmol/ g protein x hour), and L-glutamate by 400% (2.5 vs. 0.5 pmol/ g protein x hour). OMP-induced acceleration of islet GABA turnover might be attributed either to its increased vesicular accumulation and exocytosis or to its accelerated metabolism in the "GABA-shunt".

In order to distinguish between these two alternatives and to check whether GABA release is dependent on its intracellular content, islets were pre-loaded for 1 hour with different L-glutamine concentrations (0, 0.5, and 10.0 mM) and then incubated with, or without (control islets), 10 mM OMP for a second hour in the absence of L-glutamine.

The latter, at millimolar concentrations, interfered with the chromatographic separation of amino acids in the incubation medium. Figure 4A reveals that the magnitude of GABA release from control islets depends on the pre-loading L-glutamine concentration in the incubation medium which also conditions a concentration-related increase of the islet GABA content (Fig. 4C). A similar pattern of variation was also observed for L-aspartate and L-glutamate (Fig 4A and4C). However, L-aspartate and GABA showed the greatest relative increases of their islet content after pre-loading at 10 mM L-glutamine (4-and more than 5-fold, repectively, versus 1.5 for Glu) whereas the relative elevation of their release rate (4-and 3-fold, respectively) was much lower than that of Glu and Gln (6.6-and 19.4-fold, respectively). However, islet L-glutamine content did not change significantly with the extracellular L-glutamine concentration maintained during the pre-loading phase. Taurine, one of the most abundant but metabolically inert amino acid in islets, did not show any significant variations of its islet content or release at the different L-glutamine concentrations, in contrast to GABA, L-glutamate or L-aspartate (results not shown).

Addition of 10 mM OMP modified substantially the islet content and release of amino acids after the different pre-loading L-glutamine concentrations (Fig. 4B and4D). The most striking modification of islet content was a close to 50% supression of GABA levels (Fig 6D) at every L-glutamine concentration whereas L-aspartate and Lglutamate contents were either slightly decreased or not modified at all, respectively.

Neither the islet content of L-glutamine, nor that of L-leucine (data not shown), varied with the extracellular concentration of the amine during preloading conditions. Even though 10 mM OMP did not modify the tissue concentration of L-leucine, it induced a strong release of the amino acid whose magnitude was dependent on the L-glutamine concentration to which islets were pre-exposed (Fig 4B ). By contrast, L-leucine release did not vary with the L-glutamine concentration and it was at least 6-fold lower (Fig. 4A) in control islets. Like in those islets not exposed to OMP during the second hour of incubation, the release of L-glutamine also augmented with the concentration of the amine used during preloading. Similarly, the release of GABA increased with the concentration of L-glutamine at which islets were preloaded and it was not substantially modified by 10 mM OMP: it only decreased GABA release after preloading with 10 mM L-glutamine (23.6 2.1, n=7, vs. 31.8 2.0 pmol/ g protein, n=8; p<0.02) (Fig. 4A and 4B). However, 10 mM OMP decreased consistently the release of L-aspartate within almost 2-fold irrespective of the L-glutamine concentration used during islet preloading.

As gabaculine considerably decreased OMP -stimulated insulin secretion (see Figure 2), it was checked whether the inhibition of GABA transaminase with 250 M gabaculine interfered with KIC-induced modification of islet GABA content. 10 mM OMP decreased by more than 40 % the islet content of GABA and within only 20 % that of L-aspartate but the content of L-glutamate remained constant (Figure 5). 250 mol/l Gabaculine completely blocked OMP-induced decrease of islet GABA but did not affect the content of other amino acids (Figure 5). Gabaculine (250 M) alone increased significantly the islet content of GABA (37.2 5.2, n=6, vs. 23.8 2.7 pmol/ g protein, n=6; p< 0.04). These results are compatible with the known specificity of gabaculine as a GABA-transaminase inhibitor and suggest that OMP -induced decrease of islet GABA content might be due to its increased metabolism in the "GABA shunt".

In order to strengthen the evidence that OMP-induced decrease of islet GABA content is not the result of its increased exocytotic release, the effect of 250 mol/l diazoxide was investigated: the drug did not modify the level of GABA (9.1 1.4, n=6, vs. 9. precursor of a competitive inhibitor of glutamate acid decarboxylase (GAD), decreased the rate of glutamine oxidation at 0.5 mM (by more than 30 %) but had no effect at 10 mM L-glutamine (Figure 6). In contrast, 250 M gabaculine, a known GABA transaminase inhibitor added simultaneously with the other substrates, did not modify the rate of glutamine oxidation in the presence of 10 mM OMP (Figure 6). Succinic acid dimethyl ester (SAD, 10 mM), a membrane permeable form of the dicarboxylic acid [START_REF] Mukala-Nsengu | Similar effects of succinic acid dimethyl ester and glucose on islet calcium oscillations and insulin release[END_REF], reduced, probably by isotopic dilution, the oxidation rate of 0.5 mM Lglutamine alone (4.6 0.2,n=6, vs. 6.2 0.4 pmol / islet x hour; p<0.005) and in the presence of 10 mM OMP (6.3 0.3,n=6, vs. 7.9 0.2 pmol / islet x hour,n=6; p<0.002).

Islet enzyme activities of the "GABA shunt"

The effect of OMP on the enzyme activities of the "GABA shunt" was investigated in search of the hypothetical mechanism responsible of an increased islet metabolism of GABA that might result in an increased substrate inflow (succinic acid) into the Krebs cycle. Since it is already known that OMP does not modify islet GAD activity (4) we Copyright 2006 Biochemical Society investigated the effect of OMP on GABA transaminase activity (GABA-T). It was measured in islet homogenates in either the amination and deamination directions. The maximum rate of GABA deamination was 2 to 3-fold higher than the amination rate.

OMP (10 mM) reduced GABA-T activity. The reduction in amination (-34%) being greater than that of deamination (-17%) (Table 1). Gabaculine (40 M), a known GABA-T inhibitor, reduced very strongly the rate of GABA amination (-90%) and proportionately less the deamination rate (-69%).

The products of GABA-T activity are L-glutamate and SSA. SSA may be further oxidized in the "GABA shunt" by a dehydrogenase that uses NAD + as cofactor and generates succinic acid plus NADH, both susceptible of being further oxidised in the Krebs cycle and the respiratory chain. Alternatively, SSA may be reduced by a specific reductase that uses NADPH as a cofactor and produces -hydroxybutyrate and NADP + .

Both enzyme activities (SSA-dehydrogenase and SSA-reductase) were measured in islet homogenates and cerebellum, the latter being a tissue rich in GABAergic neurons (Table 1). The specific enzyme activities were much higher in islets than in cerebellum:

SSA-dehydrogenase activity was almost 70-fold and SSA-reductase about 10-fold greater. Another important difference between the two tissues was that the SSAdehydrogenase/SSA-reductase activity ratio was close to 1 in islets and around 6 in cerebellum. The rates of both enzyme activities were several hundred fold greater than the measured GABA-T activity in islets. SSA-reductase activity was strongly reduced by 10 mM OMP in either islet (-76%) and cerebellum (-47%) homogenates whereas SSA-dehydrogenase activity was not affected at all (Table 1). None of the two enzyme activities was modified by 10 mM L-leucine in either cerebellum and islet homogenates (results not shown).

Islet respiration

Oxygen consumption gives information on the overall rate of tissue metabolism. It was measured in rat islets to confirm whether OMP was actually increasing the rate of metabolism in the "GABA shunt" investigating the effect of a GABA-T inhibitor, 250 M gabaculine, on both islet respiration and insulin release. The temporal kinetics of evolution of islet oxygen tension differs considerably according to the substrate supporting respiration: Oxygen tension falls abruptly when islets respire in the presence of either L-leucine and OMP whereas it diminishes more slowly in time when high glucose is the respiration substrate (Figure 7). After exposing islets for 30 minutes to gabaculine, 10 mM OMP-induced decrease of the oxygen tension was partially (-59%, p<0.016) suppressed (Figure 7A) whereas the stimulation of respiration by either 20 mM glucose or 10 mM L-leucine were not modified (Figures 7B and7C, respectively).

DISCUSSION

It is confirmed in this work that OMP stimulates by itself a biphasic secretion of insulin in rat perifused islets [START_REF] Lenzen | 2-Oxocarboxylic acids and function of pancreatic islets in obese-hyperglycaemic mice. Insulin secretion in relation to 45 Ca 2+ uptake and metabolism[END_REF][START_REF] Hutton | Similarities in the stimulus-secretion coupling mechanisms of glucose-and 2-keto acid-induced insulin release[END_REF] and that the presence of L-glutamine does not induce any overall potentiation of the secretory response. This means that, at variance with Lleucine ([,2,4], OMP is not dependent on extracellular L-glutamine to trigger a biphasic insulin secretion. It has been previously reported that OMP greatly increases the amount of L-leucine accumulating in incubated islets but no distinction was made between medium and islets [START_REF] Malaisse | The stimulus-secretion coupling of amino acid-induced insulin release. Metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets[END_REF]. Our results confirm that OMP increases L-leucine production in islets but they also show that the amino acid does not accumulate intracellularly and is very efficiently transported outward down its concentration gradient. At variance with other amino acids (GABA, L-aspartate and L-glutamate) which do not seem to permeate islet cells [START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF], 10 mM extracellular L-leucine increased several fold its islet content that remained, however, 5-fold lower than the levels of GABA, L-aspartate, L-glutamate and L-glutamine obtained at 10 mM extracellular L-glutamine [START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF]. These data support the view that L-leucine is transported through the plasma membrane of islet cells by a passive, mediated process. Failure to demonstrate any intracellular accumulation of Lleucine argues against the possibility that the allosteric activation of glutamate dehydrogenase is the main mechanism underlying OMP stimulation of insulin secretion.

However, it cannot be completely discarded that a L-leucine gradient was developed in any subcellular compartment (mitochondria), not detected in overall cell measurements, that might increase the -amino acid concentration to values compatible with a stimulation of glutamate dehydrogenase [START_REF] Bryla | Regulation of the glutamate dehydrogenase activity in rat islets of Langerhans and its consequence on insulin release[END_REF]. Similarly to L-leucine, L-glutamine seemed to be passively transported out of the islet cells down its concentration gradient and this process efficiently prevented its intracellular accumulation. Given that islet Lglutamine uptake may also limit its rate of metabolism [START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF], it seems plausible that it is transported trough the plasma membrane by a passive mediated mechanism with predominance over an active, Na + -dependent system. Copyright 2006 Biochemical Society

Islet GABA content exhibited the highest turnover rate among the amino acids that are synthesised from L-glutamine (Asp, Glu) and it is accelerated by OMP. The turnover rate of GABA is determined by the balance among its rate of synthesis and the sum of its exocytotic release and its metabolism. As OMP did not induce any substantial modification of GABA release but strongly decreased (close to 50%) its islet content, we conclude that the 4-oxo acid improves "GABA shunt" metabolism. Even though the release of GABA was not affected by OMP, it was positively related to its islet content itself depending on the extracellular L-glutamine concentration. This might simply reflect that a greater availability of the -amino acid results in its increased accumulation in the synaptic like microvesicles of the -cells. These data reinforce previously reported evidence indicating that GABA release from -cells is positively correlated with its content [START_REF] Smismans | Nutrient regulation of gammaaminobutyric acid release from islet beta cells[END_REF].

10 mM OMP increased 14 CO 2 -production from 0.5 and 10.0 mM L-[U-14 C]glutamine by almost 30 and 70 %, respectively. This increment might reflect an increased decarboxylation of L-glutamine-derived L-glutamate to GABA or an increased oxidation in the Krebs cycle. As OMP did not increase islet GAD activity and it strongly diminished islet GABA content, one is tempted to speculate that it favours the metabolism of GABA at any enzymatic step in the "GABA shunt". This is strengthened by the observation that 0.5 mM L-glutamine oxidation is significantly reduced by allylglycine, a precursor of a competitive inhibitor of GAD. The 2-oxoglutarate generated in the transamination of OMP to L-leucine, or in the proper metabolism of the 4-oxo acid [START_REF] Hutton | Similarities in the stimulus-secretion coupling mechanisms of glucose-and 2-keto acid-induced insulin release[END_REF][START_REF] Lenzen | Signal function of metabolism of neutral amino acids and 2-keto acids for initiation of insulin secretion[END_REF], might be used to trigger the transamination of GABA to SSA in the reaction catalyzed by GABA-T. An increased concentration of islet 2-oxoglutarate above a certain level has been demonstrated to lower the GABA content [START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF].

Alternatively, 2-oxoglutarate might be directly decarboxylated to succinyl-CoA in the citric acid cycle. The predominance of one of these two alternatives (metabolism in the "GABA shunt" versus the citric acid cycle) will depend on the ratio of the 2oxoglutarate K m -values for the two implicated enzymes (GABA-T and 2-oxoglutarate dehydrogenase) and on the tissue ratio of enzymes' activities. Moreover, if OMP acted as a competitive inhibitor of the 2-oxoglutarate dehydrogenase complex in islets as reported in rat brain [START_REF] Patel | Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain[END_REF], the 2-oxo acid would favour "GABA-shunt" metabolism over the direct oxidation of 2-oxoglutarate in the citric acid cycle.

Neither GABA-T, nor SSA-dehydrogenase activities were activated by OMP which, on the other hand, very strongly diminished SSA-reductase activity in both islets and cerebellum. However, L-leucine failed to modify either SSA-reductase or SSAdehydrogenase activity in islets and cerebellum homogenates. In islets, SSA-reductase activity, responsible of SSA conversion into -hydroxybutyrate, is of similar magnitude than SSA-dehydrogenase, the enzyme activity catalyzing the transformation of SSA into succinic acid. Therefore, OMP, at variance with L-leucine, might favour succinic acid production in the "GABA shunt" and ultimately facilitate the oxidation of GABA carbons in the Krebs cycle. In support of this conclusion, pre-incubation with gabaculine, a GABA-T inhibitor, partially suppressed the increased consumption of oxygen by islets induced by OMP whereas it had no effect on either glucose or Lleucine stimulated respiration. The inhibition of OMP-supported respiration (-59%) correlated with a 33 % (perifused islets) or 47% (incubated islets) decrease of the insulin secretory response to the 4-oxo acid. The greater suppression of respiration than secretion might be explained either by the existence of non-metabolic factors partially responsible of the secretory response to OMP or to unspecific metabolic effects of gabaculine exerted on both -and non-cells in the islets. Moreover, this partial and concomitant suppression of islet respiration and secretion by a relatively high extracellular concentration of gabaculine (250 M) might reflect the drug failure to reach effective intracellular concentrations that completely antagonize GABA-T activity and hence islet GABA usage. Alternatively, other metabolic pathways, like the oxidation of the 4-oxo acid itself and of the 2-oxoglutarate generated during its transamination to L-leucine, might also be contributing to the stimulation of secretion.

The balance between these two alternatives would be definitively settled if one could measure with a sufficiently sensitive method the effect of gabaculine on an end product of the "GABA-shunt" like -hydroxybutyric acid.

It is generally accepted that an increased glutamate oxidative metabolism in the citric acid cycle secondary to glutamate dehydrogenase activation by L-leucine predominantly contributes to the biphasic stimulation of insulin secretion in the presence of Lglutamine [START_REF] Sener | L-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase[END_REF][START_REF] Fernández-Pascual | Conversión into GABA ( -aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue[END_REF]. This mechanism might explain the hyperinsulinism observed in children with gain of function mutations of the glutamate dehydrogenase gene [27,28].

OMP, at variance with L-leucine, is capable of stimulating a biphasic secretion of insulin in the absence of L-glutamine. Moreover, it is efficiently transaminated to L-

2 1 . 2 ,

 12 CaCl 2 1.28, and HEPES 25, titrated to pH 7.4 with NaOH and thermostated at 37 0 C. Oxygen tension was measured by a modified Clark-type microelectrode, with its tip positioned inside the islets. A stereomicroscope was used to control the penetration depth of the electrode to approximately 50 m. After an equilibration period of 10 minutes, islets were perifused in the absence or presence of 250 M gabaculine for 30 minutes. Subsequently, either 10 mM OMP, 20 mM glucose or 10 mM L-leucine were introduced into the perifusion buffer in the continued absence or presence of 250 M gabaculine and oxygen tension was monitored for an additional 20 minutes. Statistics All the experimental data are here presented as the mean values ± SE, and the numbers of separate experiments are given in parentheses. Statistical comparisons were performed with non-paired, two-tailed Student's t tests.

4 1. 1 pmolFigure 6

 16 Figure 6 that 10 mM OMP increased significantly the rate of 14 CO 2 -production from 0.5 and 10 mM L-[U-14 C]glutamine by 27 and 66%, respectively. Allylglycine (10 mM), a

Biochemical Journal Immediate Publication. Published on 5 Figure 1 .

 51 Figure 1. Effects of 10 mM oxo-4-methylpentanoic acid (OMP) alone and in combination with L-glutamine, on insulin secretion by rat perifused islets.

Figure 2 .

 2 Figure 2. Effect of 250 M gabaculine, a GABA transaminase inhibitor, on the insulin response induced by 10 mM oxo-4-methylpentanoic acid (OMP) in perifused rat islets.

Figure 3 .

 3 Figure 3. Effect of 10 mM oxo-4-methylpentanoic acid (OMP) on the time evolution of the islet amino acid content in the absence of L-glutamine.

Figure 4 .

 4 Figure 4. Effect of 10 mM oxo-4-methylpentanoic acid (OMP) on islet amino acid content and release after pre-incubation with L-glutamine (Gln).

Figure 5 .

 5 Figure 5. Effect of oxo-4-methylpentanoic acid (OMP) and gabaculine on islet amino acid content in the absence of L-glutamine.

Figure 6 .

 6 Figure 6. Effect of 10 mM oxo-4-methylpentanoic acid (OMP) and 250 M gabaculine on islet L-glutamine oxidation.

Figure 7 .

 7 Figure 7. Islet oxygen tension of rat islets perifused in the presence oxo-4methylpentanoic acid (OMP), glucose or L-leucine alone or in combination with gabaculine.

  Fig 1

  Fig. 5

Fig. 7 Biochemical

 7 Fig. 7

Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060173

Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060173

Table 1 .

 1 Effect of oxo-4-methylpentanoic acid (OMP) and gabaculline (GCUL) on semialdehyde succinic acid (SSA) reductase and dehydrogenase activities and GABAtransaminase (GABA-T) activity of rat islet and cerebellum homogenates.SSA-dehydrogenase and SSA-reductase activities of islets and cerebellum homogenates were measured as the production of NADH or the consumption of NADPH, respectively, in homogenates of rat islets or cerebellum, at 0.2 mM SSA. GABA-T activity of islet homogenates was measured as the formation of GABA from saturating concentrations of L-glutamate and succinic acid semialdehyde (20 mM) and as the formation of L-glutamate from saturating concentrations of GABA (20 mM) and 2oxoglutarate (10 mM). (N.S., non significant)

		ISLETS			CEREBELLUM
		mean ± SEM	n	p	mean ± SEM	n	p
	CONTROL	4312,0 ± 190,3	6	--	62,0 ± 6,5	6	--
	10 mM OMP	1046,0 ± 118,8	6	<0,001	33,2 ± 5,1	6	<0,01
		SSA DEHYDROGENASE activity (nmol NADH/min per mg prot)
		ISLETS			CEREBELLUM
		mean ± SEM	n	p	mean ± SEM	n	p
	CONTROL	3754,7 ± 419,5	6	--	366,6 ± 38,3	6	--
	10 mM OMP	4137,2 ± 285,0	5	N.S.	359,7 ± 40,8	6	N.S.
		GABA-T

SSA REDUCTASE activity (nmol NADPH/min per mg prot)

activity (pmol/15 min per islet) DEAMINATION VELOCITY AMINATION VELOCITY

		mean ± SEM	n	p	mean ± SEM	n	p
	CONTROL	50,7 ± 1,9	6	--	19,7 ± 1,1	14	--
	10 mM OMP	42,1 ± 1,7	6	<0,01	13,1 ± 1,0	6	<0,005
	40 µM GCUL	15,5 ± 1,5	6	<0,001	2,1 ± 0,3	5	<0,001

Biochemical Journal Immediate Publication. Published on 5 Jul 2006 as manuscript BJ20060173 Copyright 2006 Biochemical Society

ACKNOWLEDGMENTS

This work was supported by grants (BFI 2001-1916) from Ministerio de Ciencia y Tecnología (Spain), Instituto de Salud Carlos III (RGDM G03/212), Madrid (Spain), Swedish Medical Research Council (72X-14019), European Foundation for the Study of Diabetes, Swedish Diabetes Association and Novo Nordisk Foundation. Sergio Fernández-Pascual and Ines Hernández-Fisac were supported by a four-year fellowship of the Ministerio de Educación y Ciencia, Madrid, Spain, and J. Pizarro Delgado by the Instituto de Salud Carlos III (RGDM G03/212), Madrid (Spain).