Nanao Horike

AMP-activated Protein Kinase Activation Increases Phosphorylation of Glycogen Synthase Kinase 3β and Thereby Reduces cAMP-responsive Element Transcriptional Activity and Phosphoenolpyruvate Carboxykinase C Gene Expression in the Liver

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3β (GSK3β) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3β within 15 min. Stimulation with AICAR or the GSK3β inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3β to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3β(S9G) but not wild-type GSK3β. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar. Furthermore, AMPK-mediated GSK3β phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3β is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

Adipose-specific Expression, Phosphorylation of Ser794 in Insulin Receptor Substrate-1, and Activation in Diabetic Animals of Salt-inducible Kinase-2

Salt-inducible kinase (SIK), first cloned from the adrenal glands of rats fed a high salt diet, is a serine/threonine protein kinase belonging to an AMP-activated protein kinase family. Induced in Y1 cells at an early stage of ACTH stimulation, it regulated the initial steps of steroidogenesis. Here we report the identification of its isoform SIK2. When a green fluorescent protein-fused SIK2 was expressed in 3T3-L1 preadipocytes, it was mostly present in the cytoplasm. When coexpressed in cAMP-responsive element-reporter assay systems, SIK2 could repress the cAMP-responsive element-dependent transcription, although the degree of repression seemed weaker than that by SIK1. SIK2 was specifically expressed in adipose tissues. When 3T3-L1 cells were treated with the adipose differentiation mixture, SIK2 mRNA was induced within 1 h, the time of induction almost coinciding with that of c/EBPβ mRNA. Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser794 of human IRS-1. Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser789, the mouse equivalent of human Ser794. Moreover, the activity and content of SIK2 were elevated in white adipose tissues ofdb/db diabetic mice. These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser794 of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals.

Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3-/- embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

SIK2 Is a Key Regulator for Neuronal Survival after Ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis

Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex. The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx). From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain. Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm. The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function. The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported. When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism. Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.

Salt-inducible kinase (SIK) isoforms: their involvement in steroidogenesis and adipogenesis.

The cloning of salt-inducible kinase-1 (SIK1) that was specifically expressed in the adrenal glands of high-salt diet-fed rats led to subsequent cloning of adipose-specific SIK2 and rather ubiquitous SIK3. The three enzymes constitute a novel serine/threonine kinase subfamily, a member of AMP-activated protein kinase (PKA) family. Physiological roles of SIK1 and SIK2 have been investigated. The SIK1 transcript was expressed very early in the ACTH-stimulated Y1 cells, even before the expression of transcripts for CYP11A and StAR protein. Forced expression of SIK1 inhibited the ACTH-dependent expression of CYP11A- and StAR protein-genes. Cotransfection assays employing CRE-reporter gene showed that SIK1 could repress the PKA-dependent activation of CRE by acting on the bZIP domain of the CRE-binding protein (CREB), though the target site of SIK1-mediated phosphorylation has yet to be determined. ACTH/PKA-dependent nucleocytoplasmic shuttling of SIK1 took place in Y1 cells, implying that the intracellular movement of SIK1 might be a physiologically important determining factor for regulation of steroidogenic gene expression in the early phase of ACTH-stimulation. The SIK2 gene was expressed in 3T3-L1 cells at a very early stage of adipogenesis. SIK2 could phosphorylate Ser-794 of human insulin-receptor-substrate-1 (IRS-1) in vitro as well as in vivo. In addition, the SIK2 activity in db/db mice adipose tissues was significantly higher than that in wild-type adipose. These results strongly suggest that SIK2 may play important role(s) in modulating the insulin-signaling cascade of adipocytes, and thus, may be involved in the development of insulin resistance. Taken together, these results suggest that the SIK isoforms regulate hormonal signal transduction in both adrenal and adipose tissues.

ACTH-induced Nucleocytoplasmic Translocation of Salt-inducible Kinase IMPLICATION IN THE PROTEIN KINASE A-ACTIVATED GENE TRANSCRIPTION IN MOUSE ADRENOCORTICAL TUMOR CELLS

Salt-inducible kinase (SIK), a serine/threonine protein kinase expressed at an early stage of adrenocorticotropic hormone (ACTH) stimulation in Y1 mouse adrenocortical tumor cells, repressed the cAMP-responsive element (CRE)-dependent gene transcription by acting on the basic leucine zipper domain of the CRE-binding protein (Doi, J., Takemori, H., Lin, X.-z., Horike, N., Katoh, Y., and Okamoto, M. (2002)J. Biol. Chem. 277, 15629–15637). The mechanism of SIK-mediated gene regulation has been further explored. Here we show that SIK changes its subcellular location after the addition of ACTH. The immunocytochemical and fluorocytochemical analyses showed that SIK was present both in the nuclear and cytoplasmic compartments of resting cells; when the cells were stimulated with ACTH the nuclear SIK moved into the cytoplasm within 15 min; the level of SIK in the nuclear compartment gradually returned to the initial level after 12 h. SIK translocation was blocked by pretreatment with leptomycin B. A mutant SIK whose Ser-577, the cAMP-dependent protein kinase (PKA)-dependent phosphorylation site, was replaced with Ala could not move out of the nucleus under stimulation by ACTH. As expected, the degree of repression exerted by SIK on CRE reporter activity was weak as long as SIK was present in the cytoplasmic compartment. The same was true for the SIK-mediated repression of a steroidogenic acute regulatory (StAR) protein-gene promoter, which contained a CRE-like sequence at −95 to −85 bp. These results suggest that in the ACTH-stimulated Y1 cells the nuclear SIK was PKA-dependently phosphorylated, and the phosphorylated SIK was then translocated out of the nuclei. This intracellular translocation of SIK, a CRE-repressor, may account for the time-dependent change in the level of ACTH-activated expression of the StAR protein gene.

Resistin-like Molecule β Activates MAPKs, Suppresses Insulin Signaling in Hepatocytes, and Induces Diabetes, Hyperlipidemia, and Fatty Liver in Transgenic Mice on a High Fat Diet

Resistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELMβ were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELMβ on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELMβ overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELMβ transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELMβ, suggesting the insulin resistance-inducing effect of RELMβ to be direct. Furthermore, it was shown that RELMβ acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELMβ transgenic mice. In conclusion, RELMβ, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELMβ may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELMβ is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents.

Peptidyl-prolyl Cis/Trans Isomerase NIMA-interacting 1 Associates with Insulin Receptor Substrate-1 and Enhances Insulin Actions and Adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.

Phosphorylation of the CREB-specific coactivator TORC2 at Ser307 regulates its intracellular localization in COS-7 cells and in the mouse liver

The CREB-specific coactivator TORC2 (also known as CRTC2) upregulates gluconeogenic gene expression in the liver. Salt-inducible kinase (SIK) family enzymes inactivate TORC2 through phosphorylation and localize it in the cytoplasm. Ser(171) and Ser(275) were found to be phosphorylated in pancreatic beta-cells. Calcineurin (Cn) is proposed as the Ser(275) phosphatase, because its inhibitor cyclosporin A (CsA) stabilizes phospho-Ser(275) and retains TORC2 in the cytoplasm. Because the regulation of dephosphorylation at Ser(171) has not been fully clarified, we performed experiments with a range of doses of okadaic acid (OA), an inhibitor of PP2A/PP1, and with overexpression of various phosphatases and found that PP1 functions as an activator for TORC2, whereas PP2A acts as an inhibitor. In further studies using TORC2 mutants, we detected a disassociation between the intracellular distribution and the transcription activity of TORC2. Additional mutant analyses suggested the presence of a third phosphorylation site, Ser(307). The Ser(307)-disrupted TORC2 was constitutively localized in the nucleus, but its coactivator activity was normally suppressed by SIK1 in COS-7 cells. CsA, but not OA, stabilized the phosphogroup at Ser(307), suggesting that differential dephosphorylation at Ser(171) and Ser(307) cooperatively regulate TORC2 activity and that the nuclear localization of TORC2 is insufficient to function as a coactivator. Because the COS-7 cell line may not possess signaling cascades for gluconeogenic programs, we next examined the importance of Ser(307) and Ser(171) for TORC2s function in mouse liver. Levels of phosphorylation at Ser(171) and Ser(307) changed in response to fasting or fed conditions and insulin resistance of the mouse liver, which were modified by treatment with CsA/OA and by overexpression of PP1/PP2A/Cn. These results suggest that multiple phosphorylation sites and their phosphatases may play important roles in regulating TORC2/CREB-mediated gluconeogenic programs in the liver.