FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance.

Obesity, hyperlipidemia, and insulin resistance are common forerunners of type 2 diabetes mellitus. We have identified the human winged helix/forkhead transcription factor gene FOXC2 as a key regulator of adipocyte metabolism. Increased FOXC2 expression, in adipocytes, has a pleiotropic effect on gene expression, which leads to a lean and insulin sensitive phenotype. FOXC2 affects adipocyte metabolism by increasing the sensitivity of the beta-adrenergic-cAMP-protein kinase A (PKA) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Increased FOXC2 levels, induced by high fat diet, seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance--a likely consequence hereof would be protection against type 2 diabetes.

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers.

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.

Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis.

Epithiospecifier protein (ESP), a ferrous ion dependent protein, has a potential role in regulating the release of elemental sulphur, nitriles, isothiocyanates and cyanoepithioalkanes from glucosinolates. Two classes of ESP polypeptides were purified with molecular masses of 39 and 35 kDa, and we show that the previously reported instability was conditionally dependent. The 39 kDa polypeptide was made up of two distinct isozymes (5.00, 5.14) whilst several were present for the 35 kDa form of ESP (5.40-5.66). An anti-ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and strongly with a polypeptide corresponding to the 35 kDa ESP form in Crambe abyssinica, but did not detect any ESP in Sinapis alba or Raphanus sativus. A cytochrome P-450 mediated iron dependent epoxidation type mechanism is suggested for ESP.

Method of transfection affects the cAMP-mediated induction of the RIIbeta subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium.

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3',5'-cyclic adenosine monophosphate in Sertoli cells.

The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP alpha, beta, delta, and zeta messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP beta mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP beta mRNA was induced approximately 50-fold, C/EBP delta mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP beta and delta were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP beta, delta, and zeta by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP beta antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP beta expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.