Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Centre de Recherche en Cancérologie de Lyon

Université de Lyon

Université Lyon 1

Centre Léon Bérard

Android obesity is often associated with a metabolic syndrome characterized, in particular, by a type 2 diabetes and cardiovascular problems. This could be induced by an excess of local production of glucocorticoids (GC) by adipose tissue (or other tissues). This production of GC by its target tissues depends on the 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) enzyme. Our aim was to characterize some mechanisms which control the expression of the human 11betaHSD1 gene (hHSD11B1) in preadipocytes. By using different luciferase constructs containing fragments of the hHSD11B1 promoter, we demonstrate that two members of the CCAAT/enhancer-binding protein family, C/EBPalpha and C/EBPbeta, are required for the basal transcriptional activity of HSD11B1 in 3T3-L1 preadipocyte cells. This effect depends on the binding of each isoform to specific binding sites. Mutation of either one of these sites induced a 40-50% decrease of the constitutive activity of the hHSD11B1 promoter. A forskolin treatment of 3T3-L1 preadipocyte cells induced an increased endogenous expression of HSD11B1. By transfection studies using the hHSD11B1 luciferase constructs, it appears that C/EBPbeta was strongly involved in this induction, as the forskolin stimulation was suppressed after mutation of the C/EBPbeta binding site. Part of the mechanism involved the increase of nuclear C/EBPbeta protein levels induced by forskolin and a phosphorylation step associated with an enhanced binding of the transcription factor to its site. These data indicate that members of the C/EBP family control intracellular levels of GC in preadipocytes via the regulation of the constitutive and cAMP-dependent expressions of HSD11B1.

CCAAT/enhancer-binding proteins (C/EBPs) regulate the basal and cAMP-induced transcription of the human 11beta-hydroxysteroid dehydrogenase encoding gene in adipose cells.

The aldo-keto reductase 1B7 (AKR1B7) encodes an aldose-reductase that has been reported as a detoxification enzyme until now. We have demonstrated that AKR1B7 is differently expressed in various mouse white adipose tissues depending on their location. Its expression is associated with a higher ratio of preadipocytes vs. adipocytes. The cells that express AKR1B7 did not contain lipid droplets, and the expression level of akr1b7 was very low in mature adipocytes. We have defined the role of AKR1B7 in adipogenesis using either primary cultures of adipose stromal cells (containing adipocyte precursors) or the 3T3-L1 cell line. Under the same differentiation conditions, adipose stromal cells from tissues that expressed AKR1B7 had a decreased capacity to accumulate lipids compared with those that did not express it. Moreover, the overexpression of sense or antisense AKR1B7 in 3T3-L1 preadipocytes inhibited or accelerated, respectively, their rate of differentiation into adipocytes. In vivo experiments demonstrated that AKR1B7-encoding mRNA expression decreased in adipose tissues from mice where obesity was induced by a high-fat diet. All these results attributed for the first time a novel role to AKR1B7, which is the inhibition of adipogenesis in some adipose tissues.

A novel inhibitory protein in adipose tissue, the aldo-keto reductase AKR1B7: its role in adipogenesis.

The objectives of this study were to identify potential alterations in gene expression of melanocortin-4 receptor (MC4-R), proopiomelanocortin (POMC), and Agouti-related protein (AgRP) in mouse hypothalamus under a chronic peripheral infusion of leptin or at early (8 weeks) and advanced (16 weeks) phases of diet-induced obesity. Control or diet-induced obesity mice (8 or 16 weeks of high-fat diet) were either treated or not treated with leptin. Metabolic features were analyzed and expression of the genes of interest was measured by quantitative reverse transcriptase-PCR (RT-qPCR) and western blot. We reported that in control mice, but not in obese mice, leptin infusion induced an increase in POMC mRNA level as well as in MC4-R mRNA level suggesting that leptin could act directly and/or through alpha-melanocyte-stimulating hormone (alpha-MSH). This hypothesis was reinforced after in vitro studies, using the mouse hypothalamic GT1-7 cell line, since both leptin and Norleucine(4), D-Phenylalanine(7)-alpha-MSH (NDP-alpha-MSH) treatments increased MC4-R expression. After 8 weeks of high-fat diet, nondiabetic obese mice became resistant to the central action of leptin and their hypothalamic content of POMC and AgRP mRNA were decreased without modification of MC4-R mRNA level. After 16 weeks of high-fat diet, mice exhibited more severe metabolic disorders with type 2 diabetes. Moreover, hypothalamic expression of MC4-R was highly increased. In conclusion, several alterations of the melanocortin system were found in obese mice that are probably consecutive to their central resistance to leptin. Moreover, when the metabolic status is highly degraded (with all characteristics of a type 2 diabetes), other regulatory mechanisms (independent of leptin) can also take place.

Leptin infusion and obesity in mouse cause alterations in the hypothalamic melanocortin system.

To study the consequences of maternal obesity during gestation and suckling periods on metabolic features and expression of genes belonging to the melanocortinergic system, we developed Diet-Induced-Obesity (DIO) in mice fed high-fat-diet (HFD). After weaning, F1-descendants were fed the same diet than dams up to 16 weeks or received a 2-week standard chow at several time points. From birth, F1-DIO displayed higher body weight than F1-control. Hyperinsulinemia, hypertriglyceridemia, hyperleptinemia were detected from P10 and fasting hyperglycaemia from 2 week-post-weaning. From late gestation to 16-week-post-weaning the expression of MC4-R gene and/or the POMC/AgRP ratio was increased, suggesting an activation of this pathway to compensate the deleterious effects of HFD. Standard chow replacement at weaning normalized metabolic status but a partial recovery was obtained for later changes. Concomitant variations in the expression of the melanocortinergic genes were observed. Therefore, early nutritional intervention could override the impact of maternal and postnatal over-nutrition.

Metabolic and melanocortin gene expression alterations in male offspring of obese mice.

Transcriptional intermediary factor 1Î³ (TIF1Î³; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripotency, embryonic development, and tumor suppression. TIF1Î³ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1Î³ inactivation cooperates with the Kras(G12D) oncogene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras(G12D); Tif1Î³(lox/lox) mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1Î³ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic carcinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1Î³ and Smad4 signaling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras(G12D); Smad4(lox/lox); Tif1Î³(lox/lox) (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1Î³ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1Î³ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs.

Tif1Î³ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway.

NUPR1 (nuclear protein 1), also called P8 (molecular mass 8 kDa) or COM1 (candidate of metastasis 1), is involved in the stress response and in cancer progression. In the present study, we investigated whether human NUPR1 expression was regulated by TGFÎ² (transforming growth factor Î²), a secreted polypeptide largely involved in tumorigenesis. We demonstrate that the expression of NUPR1 was activated by TGFÎ² at the transcriptional level. We show that this activation is mediated by the SMAD proteins, which are transcription factors specifically involved in the signalling of TGFÎ² superfamily members. NUPR1 promoter analysis reveals the presence of a functional TGFÎ²-response element binding the SMAD proteins located in the genomic DNA region corresponding to the 5'-UTR (5'-untranslated region). Altogether, the molecular results of the present study, which demonstrate the existence of a TGFÎ²/SMAD/NUPR1 activation cascade, open the way to consider and investigate further a new mechanism enabling TGFÎ² to promote tumorigenesis by inducing stress resistance.

The human NUPR1/P8 gene is transcriptionally activated by transforming growth factor Î² via the SMAD signalling pathway.

Alk4 is a type I receptor that belongs to the transforming growth factor-beta (TGF-Î²) family. It takes part in the signaling of TGF-Î² ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult-tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed-allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre-mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen-inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue-specific contexts.

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues.

Pdx1-Cre; LSL-KRAS(G12D) mice develop premalignant pancreatic ductal lesions that can possibly progress spontaneously to pancreatic ductal adenocarcinoma (PDAC). Although Pdx1-Cre is expressed in the embryonic endoderm, which gives rise to all pancreatic lineages, the possible consequences of KRAS(G12D) expression in the endocrine compartment have never been finely explored.

Johann Gout

INSERM U1052,
CNRS UMR5286

Centre de Recherche en Cancérologie de Lyon

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Johann Gout

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Centre de Recherche en Cancérologie de Lyon

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

INSERM U1052,
CNRS UMR5286