Pref-1 mRNA Research Articles

Knockdown of adiponectin promotes the adipogenesis of goat intramuscular preadipocytes

Intramuscular fat (IMF) content determined by the intramuscular preadipocytes differentiation has a huge influence on the sensory quality traits of meats. It was reported that the adiponectin (ADIPOQ) gene could promote adipocytes differentiation, but the underlying molecular and functional characterization of the ADIPOQ for regulating goat IMF deposition remained unknown. Herein, the knockdown of ADIPOQ was mediated by siRNAs during goat intramuscular preadipocytes differentiation. Also, the qRT-PCR technique was performed to detect the mRNA levels of target genes in multiply experiment groups. These results showed that the ADIPOQ was expressed more than ∼400 folds in subcutaneous adipose tissue compared to that of heart tissue, and the mRNA level of ADIPOQ reached a peak at Hour 60 during the differentiation process, while at Hour 36 did ADIPOR1 and ADIPOR2. Moreover, the knockdown of ADIPOQ promoted the intramuscular preadipocytes differentiation and accelerated the lipid accumulation in the mature adipocytes with down-regulating the ADIPOR1 and preadipocyte factor 1 (Pref-1) mRNA levels and up-regulating the mRNA expression levels of the CAAT/enhancer-binding proteins (C/EBPs) and transcription factor peroxisomal proliferator-activated receptor γ (PPARγ), etc. Our study will provide a new opposite insight that the inhibition of ADIPOQ expression during intramuscular preadipocytes differentiation promotes goat IMF deposition.

The effect of TGFβ1 on thermogenic markers is dependent on the degree of adipocyte differentiation.

Transforming growth factor β (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at the different stages of adipocyte differentiation was unclear. In the present study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFβ on CEBP’s, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1α, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ, PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment stage decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP’s by TGFβ and CCN2. The addition of rhTGFβ1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue.

A link between adipogenesis and innate immunity: RNase-L promotes 3T3-L1 adipogenesis by destabilizing Pref-1 mRNA.

Ribonuclease L (RNase-L) is an endoribonuclease well known for its roles in innate immunity. Recently it has been shown to regulate several cellular functions by modulating the levels of specific mRNAs. In this study, we investigated whether RNase-L may regulate adipocyte functions. We showed that knockdown of RNase-L reduced 3T3-L1 adipocyte differentiation and lipid accumulation. After mRNA profiling, we found that upregulation of Pref-1 mRNA, an inhibitory regulator of adipogenesis, could explain the reduced adipocyte differentiation with RNase-L downregulation. The signaling molecules downstream to Pref-1, including focal adhesion kinase, extracellular signal-regulated kinases and SRY-box 9, were activated by RNase-L suppression. The presence of Pref-1 mRNA was detected in the mRNP complexes precipitated by anti-RNase-L antibody. Moreover, the Pref-1 mRNA decay rate was raised by elevated RNase-L ribonuclease activity. Finally, in stable cell clones with RNase-L silencing, suppression of Pref-1 mRNA by specific siRNA partially recovered the adipocyte differentiation phenotype. Consistent with our findings, meta-analysis of 45 public array datasets from seven independent studies showed a significant negative relationship between RNase-L and Pref-1 mRNA levels in mouse adipose tissues. Higher RNase-L and lower Pref-1 mRNAs were found in the adipose tissues of high-fat diet mice compared to those of ND mice. In line with this, our animal data also showed that the adipose tissues of obese rats contained higher RNase-L and lower Pref-1 expression in comparison to that of lean rats. This study demonstrated that Pref-1 mRNA is a novel substrate of RNase-L. RNase-L is involved in adipocyte differentiation through destabilizing Pref-1 mRNA, thus offering a new link among RNA metabolism, innate immunity and adipogenesis in obesity progression.

Effect of the anatomical site on telomere length and pref-1 gene expression in bovine adipose tissues

Adipose tissue growth is associated with preadipocyte proliferation and differentiation. Telomere length is a biological marker for cell proliferation. Preadipocyte factor-1 (pref-1) is specifically expressed in preadipocytes and acts as a molecular gatekeeper of adipogenesis. In the present study, we investigated the fat depot-specific differences in telomere length and pref-1 gene expression in various anatomical sites (subcutaneous, intramuscular and visceral) of fattening Wagyu cattle. Visceral adipose tissue expressed higher pref-1 mRNA than did subcutaneous and intramuscular adipose tissues. The telomere length in visceral adipose tissue tended to be longer than that of subcutaneous and intramuscular adipose tissues. The telomere length of adipose tissue was not associated with adipocyte size from three anatomical sites. No significant correlation was found between the pref-1 mRNA level and the subcutaneous adipocyte size. In contrast, the pref-1 mRNA level was negatively correlated with the intramuscular and visceral adipocyte size. These results suggest that anatomical sites of adipose tissue affect the telomere length and expression pattern of the pref-1 gene in a fat depot-specific manner.

Expression of pref-1/dlk-1 is regulated by microRNA-143 in 3T3-L1 cells.

Preadipocyte factor 1 (Pref-1), also known as a delta-like 1 protein, is a transmembrane and secreted protein containing the epidermal growth factor-like repeat. Pref-1 inhibits adipocyte differentiation by activating the ERK1/2 pathway. MicroRNAs, a new class of small noncoding RNAs of 20-24 nucleotides, act as negative regulators of gene expression and result in mRNA degradation or translational repression. MicroRNA-143 (miR-143) is known to induce adipocyte differentiation; however, miR-143 targets in the regulation of adipocyte differentiation remain unknown. In this study, we investigated whether pref-1 is a miR-143 target to regulate adipogenesis. After the induction of adipocyte differentiation the level of miR-143 was increased, whereas the expression of pref-1 mRNA was decreased. The pref-1 protein level was also down-regulated in preadipocytes ectopically expressing miR-143, and recovered by miR-143 inhibitor. The binding region for miR-143 was predicted to be located between positions 247 and 252 in the 3'-UTR of pref-1. The luciferase activity of the vector containing the wild-type 3'-UTR of pref-1 was decreased by 65% in cells transfected with miR-143 mimic compared to that of the corresponding control. In contrast, the activity of the pref-1 mutant cells was not affected by the treatment with miR-143 mimic. The ectopic expression of miR-143 mimic suppressed the phosphorylation of ERK1/2 induced by pref-1 in 3T3-L1 cells. However, the suppressed phosphorylation was restored by miR-143 inhibitor. Taken together, these data suggest that miR-143 promotes adipogenesis by directly modulating the pref-1 expression in adipocytes.

Mechanical stretch inhibits adipogenesis and stimulates osteogenesis of adipose stem cells

A reciprocal relationships between osteogenesis and adipogenesis has been observed in vitro and in vivo, and mechanical stretch has been believed to be a regulating factor of osteo-adipogenic axis differentiation of mesenchymal stem cells. In this study, rat adipose stem cells (ASCs) were isolated and cultured in adipogenic or normal medium. Their exposure to cyclic mechanical stretch (2000 με, 1 Hz) in the presence of adipogenic medium decreased mRNA and protein level of PPAR-γ, and increased Runx2 mRNA and protein levels as well as Pref-1 mRNA level, compared to static samples. ASCs cultured in normal medium without adipogenic induction did not show any significant change in mRNA expression of PPAR-γ, Runx2, nor Pref-1 irrespective of mechanical loading. Stretching induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) during the induction period. It was concluded that mechanical stretch inhibited adipogenesis and stimulated osteogenesis of these ASCs in the presence of adipogenic medium and that ERK1/2 activation may be involved in the mechanical stress-induced trans-differentiation.

Suppression of lamin A/C by short hairpin RNAs promotes adipocyte lineage commitment in mesenchymal progenitor cell line, ROB-C26

Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

γ‐secretase inhibitor induces adipogenesis of adipose‐derived stem cells by regulation of Notch and PPAR‐γ

To determine the inhibitory effect and mechanism of Notch signalling on adipogenesis of mouse adipose-derived stem cells (mASCs). Varied concentrations of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT) were added to mASCs 3 days before adipogenic induction with insulin-containing differentiation medium. The process of adipogenesis and ability of lipid droplet accumulation were analysed using oil red-O staining. The Notch signalling pathway (Notch-1, -2, -3, -4, Hes-1 and Hey-1) and adipogenesis-related factors (PPAR-gamma, DLK-1/Pref-1 and Acrp) were tested using real-time PCR, Western blot analysis and immunofluorescence staining assays. We demonstrated that Notch-2-Hes-1 signalling pathway was inhibited dose-dependently by DAPT in mASCs. In addition, transcription of PPAR-gamma was promoted by DAPT before adipogenic induction, while inhibitor of adipogenesis DLK-1/Pref-1 was further depressed. At early stages of differentiation (2-4 days), adipogenesis in mASCs was advanced and significantly enhanced in 5 and 10 mum DAPT pre-treated cases. On day 4, in differentiated mASCs cases with DAPT pre-treatment, we also found promotion of activation of de-PPAR-gamma and depression of HES-1, DLK-1/Pref-1 mRNA and protein expression. We conclude that blocking Notch signalling with DAPT enhances adipogenesis of differentiated mASCs at an early stage. It may be due to depression of DLK-1/Pref-1 and promotion of de-PPAR-gamma activation, which work through inhibition of Notch-2-Hes-1 pathway by DAPT.

Transcriptional activation of pref-1 by E2F1 in 3T3 L1 cells

The E2F gene family appears to regulate the proliferation and differentiation of events that are required for adipogenesis. Pref-1 is a transmembrane protein that inhibits adipocyte differentiation in 3T3-L1 cells. In this study, we found that the expression of pref-1 is regulated by the transcription factor E2F1. The expression of pref-1 and E2F1 was strongly induced in preadipocytes and at the late differentiation stage. Using luciferase reporter assay, ChIP assay and EMSA, we found that the -211/-194 region of the pref-1 promoter is essential for the binding of E2F1 as well as E2F1-dependent transcriptional activation. Knockdown of E2F1 reduced both pref-1 promoter activity and the level of pref-1 mRNA. Taken together, our data suggest that transcriptional activation of pref-1 is stimulated by E2F1 protein in adipocytes.

Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome

To determine whether adipocyte storage capacity influences the onset and severity of type 2 diabetes and other components of the metabolic syndrome, we made normal and db/db mice resistant to obesity by overexpressing leptin receptor-b on the aP2-Lepr-b promoter. On a 4% diet, these mice have no phenotype, but on a 60% fat diet, they resist diet-induced obesity because constitutive adipocyte-specific overexpression of Lepr-b prevents obesity via the antilipogenic autocrine/paracrine action of leptin on adipocytes. After 8 months on the same 60% fat diet, body fat of transgenic mice was 70% below WT controls. Cardiac and liver fat was elevated in the transgenics, and their hyperinsulinemia was more marked, suggesting greater insulin resistance. The aP2-Lepr-b transgene also prevented obesity in db/db mice; at 10 weeks of age their body fat was half that of the db/db mice. This lack of obesity was attributable to reduced expression of sterol regulatory element binding protein-1c and its target lipogenic enzymes in adipose tissue and a 6-fold increase in Pref-1 mRNA. Severe diabetes was present in transgenics at 4 weeks of age, 10 weeks before db/db controls. Echocardiographic evidence of cardiomyopathy appeared at 10 weeks, weeks before the db/db mice. Histologically, loss of beta cells and myocardial fibrosis was present in the transgenic group at least 6 weeks before the db/db mice. These results suggest that the expression level of genes that regulate the adipogenic response to overnutrition profoundly influences the age of onset and severity of diet-induced type 2 diabetes and co-morbidities.

Differentiation Arrest by Hypoxia

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.

Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone-induced differentiation have been evaluated for the first time in 3T3-L1 preadipocytes. Unlike control cells, oxLDL-treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor-1 (Pref-1) mRNA expression at late times. By silencing Pref-1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref-1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref-1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.

Azoxymethane-induced pre-adipocyte factor 1 (Pref-1) functions as a differentiation inhibitor in colonic epithelial cells.

Inbred mice differ dramatically in their sensitivity to the colon carcinogen, azoxymethane (AOM). Identifying genes associated with this differential susceptibility in mice may ultimately reveal molecular mechanisms responsible for colon carcinogenesis. A cDNA array approach was taken to study gene expression changes induced by AOM in the colons of sensitive (A/J) and resistant (AKR) mice. Among the genes represented on the array, pre-adipocyte factor 1 (Pref-1), associated previously with suppression of adipocyte differentiation, was induced specifically by AOM in the distal colons of sensitive A/J mice (5.4-fold). Reverse transcription-PCR followed by sequence analysis revealed the presence of four alternative splice variants of Pref-1 mRNA in the colon. The potential significance of Pref-1 in colon tumorigenesis was explored in colon cancer cells infected with a retroviral construct containing the major splice variant. Over-expression of Pref-1A in HT-29 cells led to a marked resistance to butyrate-induced differentiation and growth inhibition. Our data indicate that Pref-1, a protein that suppresses differentiation and promotes colonocyte growth, may account in part for the sensitivity of A/J mice to AOM-induced carcinogenesis. In addition, detection of Pref-1 in a human colon tumor cell line suggests that it may also participate in human colon tumorigenesis.

Bezafibrate Reduces mRNA Levels of Adipocyte Markers and Increases Fatty Acid Oxidation in Primary Culture of Adipocytes

The molecular mechanisms by which peroxisome proliferator-activated receptor (PPAR) activation by fibrates reduces fat deposition and improves insulin sensitivity are not completely understood. We report that exposure of a rat primary culture of adipocytes for 24 h to the PPAR activator bezafibrate increased the mRNA levels of crucial genes involved in peroxisomal and mitochondrial beta-oxidation. The mRNA levels of the peroxisomal beta-oxidation rate-limiting enzyme acyl-CoA oxidase and of the muscle-type carnitine palmitoyl transferase I (M-CPT-I), which determines the flux of mitochondrial beta-oxidation, increased by 1.6-fold (P < 0.02) and 4.5-fold (P = 0.001), respectively. These changes were accompanied by an increase in the transcript levels of the uncoupling protein-2 (UCP-2; 1.5-fold induction; P < 0.05) and UCP-3 (3.7-fold induction; P < 0.001), mitochondrial proteins that reduce ATP yield and may facilitate the oxidation of fatty acids. Furthermore, bezafibrate increased the mRNA levels of the fatty acid translocase (2-fold induction; P < 0.01), suggesting a higher fatty acid uptake into adipocytes. In agreement with these changes, bezafibrate caused a 1.9-fold induction (P < 0.02) in 9,10-[(3)H]palmitate oxidation. Moreover, bezafibrate reduced the mRNA expression of several adipocyte markers, including PPARgamma (30% reduction; P = 0.05), tumor necrosis factor-alpha (33% reduction; P < 0.05), and the ob gene (26% reduction). In contrast, adipocyte fatty acid binding protein mRNA levels increased (1.5-fold induction; P < 0.01), pointing to a mobilization of fatty acids to mitochondria and peroxisomes. The reduction of the adipocyte markers caused by bezafibrate was accompanied by an increase in the mRNA levels of the preadipocyte marker Pref-1 (1.6-fold induction; P < 0.01). Some of the changes observed in the primary culture of rat adipocytes also were studied in the epididymal white adipose tissue of bezafibrate-treated rats for 7 days. In vivo, M-CPT-I mRNA levels increased (4.5-fold induction; P = 0.001) in epididymal white adipose tissue of bezafibrate-treated rats. Similarly, fatty acid translocase (2.6-fold induction; P = 0.002) and Pref-1 (5.6-fold induction) mRNA levels increased, although differences in the latter were not significant because of huge individual variations. These results indicate that exposure of adipocytes to bezafibrate, independent of its hepatic effects, increases the degradation of fatty acids, reducing their availability to synthesize triglycerides. As a result, some degree of dedifferentiation of adipocytes to preadipocyte-like cells is achieved. These changes may be involved in the reduction in fat depots and in the improvement of insulin sensitivity observed after bezafibrate treatment.

Genomic Organization and Genetic Mapping of the Bovine PREF-1 Gene

As a potential regulator of nutrient partitioning in beef cattle, we have cloned and genetically mapped the bovine PREF-1 gene. A full-length PREF-1 cDNA was isolated by iterative purification from a mixed-tissue cDNA library to which adipose contributed mRNA. Analysis of partial cDNAs from this library revealed that the 3′-terminal exon of the bovine PREF-1 mRNA is spliced in a manner analogous to its murine ortholog. However, we have also detected a PREF-1 splice form apparently unique to cattle. Aside from this alternative selection of a splice donor in the bovine fifth exon, the exon/intron junctions of the bovine PREF-1 gene recapitulate those observed for mice. The sequences proximal to the bovine PREF-1 transcription start site are homologous to the mouse PREF-1 promoter. Importantly, the sequence experimentally identified as critical to PREF-1 “suppression in adipocyte differentiation” is conserved in the bovine gene. The bovine PREF-1 gene was mapped to the telomeric end of BTA 21 by virtue of a physically linked microsatellite with seven alleles and 285 informative meiosis.

Transcriptional Repression of pref-1by Glucocorticoids Promotes 3T3-L1 Adipocyte Differentiation

Pref-1 is an epidermal growth factor-like domain-containing transmembrane protein that is cleaved to generate a soluble factor. It is abundant in 3T3-L1 preadipocytes but absent in mature adipocytes. Constitutive expression of pref-1 or the addition of its ectodomain inhibits adipogenesis. We find that the pref-1 gene is an early target of dexamethasone, a component of the dexamethasone/methylisobutylxanthine differentiation mixture used routinely for adipoconversion. The time course of the decrease in pref-1 mRNA by dexamethasone reflected the pref-1 mRNA half-life determined by actinomycin D treatment. Nuclear run-on assays showed that dexamethasone attenuates pref-1 transcription. We demonstrate a correlation between pref-1 down-regulation and adipoconversion by varying the time period and concentration of dexamethasone. Increasing the dexamethasone treatment from 2 to 4 days resulted in a time-dependent pref-1 down-regulation and increased differentiation as measured by adipocyte marker mRNAs. The dexamethasone concentration between 1 and 10 nM showed a dose-dependent decrease in pref-1 mRNA and an enhancement of adipogenesis. To test the hypothesis that dexamethasone initiation of adipoconversion may be via down-regulation of pref-1, we lowered endogenous pref-1 mRNA levels by stably transfecting 3T3-L1 preadipocytes with antisense pref-1. At 1 microM, antisense cells had enhanced adipose conversion; a similar degree of differentiation occurred with 2 nM dexamethasone, a concentration that does not support differentiation of control 3T3-L1 cells. We conclude that dexamethasone-mediated repression of pref-1 contributes to the mechanisms whereby glucocorticoids promote adipogenesis.

Growth Hormone and Prolactin Stimulate the Expression of Rat Preadipocyte Factor-1/Δ-Like Protein in Pancreatic Islets: Molecular Cloning and Expression Pattern during Development and Growth of the Endocrine Pancreas*

GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte factor-1 (Pref-1, or delta-like protein (Dlk)]. The size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA content of islets from adult rats was only 1% of that in neonatal islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant rats from day 12 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day 14 and the other at term, whereafter it decreased to nonpregnant levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine GH or ovine PRL. During the development of pancreas from embryonic day 12 (E12) to postnatal day 4, we observed a 2-fold increase in Pref-1 mRNA on E17 and a 5-fold increase at birth, followed by a rapid decline on postnatal day 4. Pref-1 immunoreactivity was found in a subpopulation of insulin cells of neonatal islets of Langerhans. At an early embryonal stage (E13), most cells of the pancreatic anlage were Pref-1 positive, becoming predominantly restricted to the insulin-producing cells during development. In conclusion, these findings suggest that Pref-1 is involved in both differentiation and growth of beta-cells.

TNF -Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPAR without Effects on Pref-1 Expression

Tumor necrosis factor α (TNFα) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNFα inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor γ (PPARγ) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARγ and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARγ occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNFα-mediated inhibition of adipocyte differentiation, PPARγ messenger RNA (mRNA) expression stays at low levels. In contrast, TNFα treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors[ such as TNFα, basic fibroblast growth factor, transforming growth factor β, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARγ mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNFα to fully differentiated adipocytes results in the rapid disappearance of PPARγ protein expression and the rapid loss of PPARγ DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNFα-generated signals, whereas PPARγ expression remains sensitive to TNFα at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPARgamma without effects on Pref-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.