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/EBP␤ mRNA. Coexpressed with human insulin receptor substrate-1 (IRS-1) in COS cells, SIK2 could phosphorylate Ser 794 of human IRS-1. Adenovirus-mediated overexpression of SIK2 in adipocytes elevated the level of phosphorylation at Ser 789 , the mouse equivalent of human Ser 794 . Moreover, the activity and content of SIK2 were elevated in white adipose tissues of db/db diabetic mice. These results suggest that highly expressed SIK2 in insulin-stimulated adipocytes phosphorylates Ser 794 of IRS-1 and, as a result, might modulate the efficiency of insulin signal transduction, eventually causing the insulin resistance in diabetic animals.The lipid metabolism in adipose tissues is under the control of two hormonal signaling pathways; insulin stimulates glucose uptake and lipogenesis, whereas cAMP, generated by exogenous stimuli like adrenalin and glucagon, stimulates lipolysis. If the balance between the two signaling systems becomes lost and the adipose tissues are exposed to hyperinsulinemia for a prolonged time, they gradually become resistant to insulin stimulation (1, 2). The insulin resistance occurring in tissues involved in biological fuel metabolism, such as adipose tissues, liver, and skeletal muscles, would finally cause disorders in energy metabolism of the whole body, such as obesity and type 2 diabetes (3, 4). Insulin receptor substrate (IRS) 1 proteins are key molecules of the insulin-signaling cascade (5); they are phosphorylated on tyrosine residues by the action of insulindependently activated insulin receptor kinase, and the tyrosine-phosphorylated IRS proteins trigger further intracellular cascades. Several investigators recently reported (6, 7) that IRS proteins, under certain non-physiological conditions, were phosphorylated on serine residues. The serine phosphorylation of IRS proteins would modulate the efficiency of the insulinsignaling cascade (8, 9) and eventually render the animals resistant to insulin stimulation (10, 11). Molecular identification of several protein kinases responsible for the serine phosphorylation of IRS proteins has been reported (12-24).Salt-inducible kinase (SIK) was first cloned from ...

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25‐Dx and membrane‐associated progesterone receptor. IZA was purified as a glutathione S‐transferase‐fused or His6‐fused protein, and its molecular properties were studied. The UV‐visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high‐spin type. Analysis of the heme indicated that it is of the b type. Site‐directed mutagenesis studies were performed to identify the amino‐acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99–K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21‐hydroxylation reaction was investigated in COS‐7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21‐hydroxylase activity.

PCR-coupled cDNA subtraction hybridization was adapted to identify the genes expressed in the adrenocortical tissues from high salt diet-treated rat. A novel cDNA clone, termed salt-inducible kinase (SIK), encoding a polypeptide (776 amino acids) with significant similarity to protein serine/ threonine kinases in the SNF1/AMPK family was isolated. An in vitro kinase assay demonstrated that SIK protein had autophosphorylation activity. Northern blot revealed that SIK mRNA levels were markedly augmented by ACTH treatment both in rat adrenal glands and in Y1 cells. SIK may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation.z 1999 Federation of European Biochemical Societies.

Background: Many types of fermented food are consumed in Japan. Although some are produced by plant-origin lactic-acid bacteria (LAB) fermentation, the physiological functions of such bacteria remain unclear. We therefore isolated LAB of plant origin from Kyoto pickles and determined the immunological activity of heat-killed preparations of plant-origin LAB. Methods: The Lactobacillus pentosus strain S-PT84 was selected from among 16 LAB of plant origin as the strongest interleukin (IL)-12-inducing strain. IL-12- and IL-10-inducing activities were determined with macrophages from BALB/c mice. The in vivo immunomodulating effect of S-PT84was determined with BALB/c mice fed S-PT84. The antiallergic activity of S-PT84 was examined in ovalbumin (OVA)/alum-administered BALB/c mice. Results: The L. pentosus strain S-PT84 induced production of both IL-12 and IL-10 in vitro. S-PT84 enhanced splenic natural-killer activity and modulated the T helper (Th) type 1/type 2 balance toward a Th1-dominant state. In the OVA-induced allergy model, orally administered S-PT84 lowered serum IgE levels and suppressed active cutaneous anaphylaxis reaction and splenic IL-4 production. IL-10 production from splenocytes of OVA-immunized mice was upregulated by feeding S-PT84. Conclusions: Despite heat-killing, S-PT84 exhibited antiallergic effects by modulating the Th1/Th2 balance and inducing regulatory T cells. The L. pentosus strain S-PT84, which is of plant origin and isolated from a traditional Japanese food, is expected to be useful for treatment of many immune diseases including allergies, tumors, infectious diseases and auto-immune diseases.

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The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

Previously we expressed rat 25-hydroxyvitamin D 3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D 3 and 1a, 25-dihydroxyvitamin D 3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335±343]. In this study, we demonstrate further oxidation by CYP24 including four-and six-step monooxygenation towards 25-hydroxyvitamin D 3 and 1a,25-dihydroxyvitamin D 3 , respectively. When the substrate 25-hydroxyvitamin D 3 was added to a culture of recombinant E. coli, four metabolites, 24,25-dihydroxyvitamin D 3 , 24-oxo-25-hydroxyvitamin D 3 , 24-oxo-23,25-dihydroxyvitamin D 3 and 24,25,26,27-tetranor-23-hydroxyvitamin D 3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D 3 . Furthermore, in-vivo and in-vitro metabolic studies on 1a,25-dihydroxyvitamin D 3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1a,25-dihydroxyvitamin D 3 into calcitroic acid which is known as a final metabolite of 1a,25-dihydroxyvitamin D 3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D 3 and 1a,25-dihydroxyvitamin D 3 .Keywords: CYP24; electron transfer; P450, vitamin D.During the last decade, many mammalian P450 species have been expressed in Escherichia coli cells [1±4] mainly for the purpose of overproduction of the P450s. A merit of the E. coli expression system is the low background as compared with eukaryotic expression systems; this allows characterization of the expressed P450. Complete genome sequence analysis of E. coli K12 suggested the absence of a P450 gene in the genome [5] and E. coli has no steroids in the cell membranes; these facts strongly suggest that the E. coli expression system is useful for enzymatic studies of steroidogenic P450s.Barnes et al.[2] reported the interesting finding that mammalian microsomal P450 can exhibit monooxygenase activity in E. coli cells. Electrons are transferred from NADPH through NADPH-flavodoxin reductase and flavodoxin to microsomal P450s. NADPH-flavodoxin reductase and flavodoxin contain a flavin adenine dinucleotide (FAD) and a flavin mononucleotide (FMN) molecule, respectively. Thus, these two enzymes function as an electron transfer system instead of a mammalian microsomal NADPH-P450 reductase which contains both FAD and FMN molecules. However, on mitochondrial P450s such as P450scc (CYP11A) [4] and P450c27 (CYP27) [6] no report showing monooxygenase activity in living E. coli cells has been published. In this report, we describe the presence of an electron transfer system for the mitochondrial P450s in E. coli cells.Previous studies in vitro using the membrane fraction of recombinant E. coli cells indicated that rat P450c24 (CYP24) is not only active in 24-hydroxylation but is also responsible for the subsequent two hydroxylation steps in the metabolis...

Cardiac aldosterone levels have not been evaluated in diastolic heart failure (DHF), and its roles in this type of heart failure remain unclear. This study aimed to detect cardiac aldosterone by use of a liquid chromatographic-mass spectrometric method and to assess the effects of mineralocorticoid receptor blockade on hypertensive DHF. Dahl salt-sensitive rats fed 8% NaCl diet from 7 wk (hypertensive DHF model) were divided at 13 wk into three groups: those treated with subdepressor doses of eplerenone (12.5 or 40 mg x kg(-1) x day(-1)) and an untreated group. Dahl salt-sensitive rats fed 0.3% NaCl diet served as controls. Cardiac aldosterone was detected in the DHF rats but not in the control rats, with increased ventricular levels of mineralocorticoid receptor. Cardiac levels of 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone were not different between the control and DHF rats, but the tissue level of corticosterone that has an affinity to mineralocorticoid receptor was 1,000 times as high as that of aldosterone. Aldosterone synthase activity and CYP11B2 mRNA were undetectable in the ventricular tissue of the DHF rats. Administration of eplerenone attenuated ventricular hypertrophy, ventricular fibrosis, myocardial stiffening, and relaxation abnormality, leading to the prevention of overt DHF. In summary, the myocardial aldosterone level increased in the DHF rats. However, its value was extremely low compared with corticosterone, and no evidence for enhancement of intrinsic myocardial aldosterone production was found. The upregulation of mineralocorticoid receptor may play a central role in the pathogenesis of DHF, and blockade of mineralocorticoid receptor is likely an effective therapeutic regimen of DHF.