Essential role in vivo of upstream stimulatory factors for a normal dietary response of the fatty acid synthase gene in the liver.

In the liver, transcription of several genes encoding lipogenic and glycolytic enzymes, in particular the gene for fatty acid synthase (FAS), is known to be stimulated by dietary carbohydrates. The molecular dissection of the FAS promoter pointed out the critical role of an E box motif, located at position -65 with respect to the start site of transcription, in mediating the glucose- and insulin-dependent regulation of the gene. Upstream stimulatory factors (USF1 and USF2) and sterol response element binding protein 1 (SREBP1) were shown to be able to interact in vitro with this E box. However, to date, the relative contributions of USFs and SREBP1 ex vivo remain controversial. To gain insight into the specific roles of these factors in vivo, we have analyzed the glucose responsiveness of hepatic FAS gene expression in USF1 and USF2 knock-out mice. In both types of mouse lines, defective in either USF1 or USF2, induction of the FAS gene by refeeding a carbohydrate-rich diet was severely delayed, whereas expression of SREBP1 was almost normal and insulin response unchanged. Therefore, USF transactivators, and especially USF1/USF2 heterodimers, seem to be essential to sustain the dietary induction of the FAS gene in the liver.

Differential roles of upstream stimulatory factors 1 and 2 in the transcriptional response of liver genes to glucose.

USF1 and USF2 are ubiquitous transcription factors of the basic helix-loop-helix leucine zipper family. They form homo- and heterodimers and recognize a CACGTG motif termed E box. In the liver, USF binding activity is mainly accounted for by the USF1/USF2 heterodimer, which binds in vitro the glucose/carbohydrate response elements (GlRE/ChoRE) of glucose-responsive genes. To assign a physiological role of USFs in vivo, we have undertaken the disruption of USF1 and USF2 genes in mice. We present here the generation of USF1-deficient mice. In the liver of these mice, we demonstrate that USF2 remaining dimers can compensate for glucose responsiveness, even though the level of total USF binding activity is reduced by half as compared with wild type mice. The residual USF1 binding activity was similarly reduced in the previously reported USF2 -/- mice in which an impaired glucose responsiveness was observed (Vallet, V. S., Henrion, A. A., Bucchini, D., Casado, M. , Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.

Glucose-dependent liver gene expression in upstream stimulatory factor 2 -/- mice.

Upstream stimulatory factors (USF) 1 and 2 belong to the Myc family of transcription factors characterized by a basic/helix loop helix/leucine zipper domain responsible for dimerization and DNA binding. These ubiquitous factors form homo- and heterodimers and recognize in vitro a CACGTG core sequence termed E box. Through binding to E boxes of target genes, USF factors have been demonstrated to activate gene transcription and to enhance expression of some genes in response to various stimuli. In particular, in the liver USF1 and USF2 have been shown to bind in vitro glucose/carbohydrate response elements of glycolytic and lipogenic genes and have been proposed, from ex vivo experiments, to be involved in their transcriptional activation by glucose. However, the direct involvement of these factors in gene expression and nutrient gene regulation in vivo has not yet been demonstrated. Therefore, to gain insight into the specific role of USF1 and USF2 in vivo, and in particular to determine whether the USF products are required for the response of genes to glucose, we have created, by homologous recombination, USF2 -/- mice. In this paper, we provide the first evidence that USF2 proteins are required in vivo for a normal transcriptional response of L-type pyruvate kinase and Spot 14 genes to glucose in the liver.

Secretogranin II (SgII) distribution and processing studies in human normal and adenomatous anterior pituitaries using new polyclonal antibodies.

Studies concerning the identification of Secretogranin II (SgII) and its processed forms in human pituitary remain scarce since no anti-human SgII antisera has been available. In the present report, a specific hSgII antiserum was used in immunohistochemistry experiments to determine the distribution of SgII in normal anterior pituitaries and pituitary adenomas (5 gonadotroph, 3 non-functioning and 5 mammotroph tumors). In normal pituitaries SgII was detected in gonadotrophs, thyrotrophs and corticotrophs but was absent from somatotrophs and mammotrophs. In tumor tissues, the SgII protein was found in gonadotroph and non-functioning adenomas but not in the mammotroph tumors. Northern blot analyses demonstrated the same 2.5 kb SgII mRNA species in all types of tumors as in normal anterior pituitaries. In Western blotting experiments, apart from the 97 K polypeptide. SgII antiserum detected two lower Mr proteins, 46 K and 31 K. These were observed in gonadotroph and in non-functioning adenomas and were absent from the mammotroph adenomas. Four new antisera were raised against sequential regions of SgII (N-terminal, two internal and C-terminal sequences). Western blotting experiments revealed that both the 46 K and 31 K polypeptides arose from the second half (C-terminal) of the molecule, thus suggesting that SgII may be processed by cleavage of short N-terminal polypeptides not detected in our conditions. Our results indicate that SgII may represent not only a valuable histological marker for non-functioning pituitary adenomas, but also a pertinent tool to study the proteolytic processing mechanisms in various neuroendocrine tumors.

Immunohistochemical study of secretogranin II in 62 neuroendocrine tumours of the digestive tract and of the pancreas in comparison with other granins.

An immunohistochemical study on 62 cases of gastrointestinal and pancreatic neuroendocrine tumours was performed in order to test the interest and specificity of a new anti-human secretogranin II antibody and compared to the other granin markers (chromogranin A and chromogranin B). Specific immunoglobulins were purified by affinity chromatography from an antiserum raised against a recombinant secretogranin II. Gastric tumours did not express secretogranin II, and ileal and appendiceal tumours only rarely expressed it (1/10 and 1/7 respectively), unlike the other classic neuroendocrine members of the granin family. In duodenal and pancreatic tumours, secretogranin II was detected when the other granins were also expressed. On the other hand, all rectal tumours expressed secretogranin II (7/7), frequently in the absence of chromogranin A. Though the granins expressed in tumoral tissue and in adjoining non tumoral tissue are mostly related, strong secretogranin II positivity occurred in 4 tumours while mucosa was secretogranin II negative. These observations highlight the interest for associating another antigranin such as secretogranin II to the classical markers of neuroendocrine tumours.

Early detection of secretogranin-II (SgII) in the human fetal pituitary: immunocytochemical study using an antiserum raised against a human recombinant SgII.

Secretogranin-II (SgII) is a protein contained within secretory granules of mainly gonadotrophs. The purpose of this study was to determine whether SgII immunoreactivity (SgII-IR) in the human fetal pituitary was temporally related to gonadotropin immunoreactivity. A specific antihuman SgII antiserum was thus required. A complementary DNA clone with an open reading frame for human (h) SgII was synthesized by reverse transcription-polymerase chain reaction from pituitary total RNA. This clone was used to obtain the SgII polypeptide (-9 to 152) as a fusion protein, in a heterologous expression prokaryotic system. Antisera against the fusion protein were raised in rabbits and checked for specificity and sensitivity through Western blotting. Human fetal pituitaries from week 6 of gestation onward were used for immunocytochemical studies. Consecutive semithin sections were treated with the specific antisera against hSgII, beta-endorphin, and hPRL and with monoclonal antibodies to hCG alpha, hLH, and hFSH. SgII immunoreactivity appeared at week 8 and was restricted to pituitary cells expressing beta-endorphin (100% colocalization). At week 9, FSH-positive cells did not contain SgII. From week 10, gonadotrophs progressively exhibited SgII-IR, up to 50% of that in FSH-containing cells at week 26. The granin was never found in PRL cells whatever the stage of development. The present data demonstrate that SgII-IR is detected very early in fetal life; however, the positive cells are not gonadotrophs, but corticotrophs. Within gonadotrophs, SgII appears subsequent to hormones. At birth, more than 90% of SgII-IR cells are represented by corticotrophs and gonadotrophs.