Retinol-binding Protein Gene Expression Research Articles

The Effects of Soyferment-Tempeh on Lipid Profile, Retinol-Binding Protein 4 (RBP4), and Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression in Type 2 Diabetic Mice

Diabetes Type 2 can cause oxidative stress leading to the accumulation of reactive oxygen species. Soyferment-Tempeh, a fermented soybean product with aerobic and anaerobic R. oligosporus incubation has a high antioxidant content of isoflavones that can regulate oxidative stress in diabetes. In this study, we evaluate the effects of Soyferment-Tempeh on lipid profile, Retinol-Binding Protein 4 (RBP4), and Phosphoenolpyruvate Carboxykinase (PEPCK) gene expression in type 2 diabetic mice. A total of 30 mice with the age of eight weeks were divided into six groups as follows: A) nondiabetic, B) diabetic mice, C) diabetic mice with metformin, D), E), and F) diabetic mice with Soyferment-Tempeh doses of 10, 20, or 40mg/100g body weight (BW), respectively, were administered treatments orally by gavages. Blood was collected for assessment of blood glucose level, and lipid profile before and after 3 weeks of the administration. After sacrificing the mice, livers were used for RBP4 and PEPCK gene expression assessment. Supplementation with three different doses of Soyferment-Tempeh in streptozotocin-induced diabetic mice for 21 days significantly (p<0.001) reduced blood glucose level, total cholesterol, triglycerides, low-density lipoprotein level, atherogenic index, and increased high-density lipoprotein level. There was a significant decrease in RBP4 gene expression in the Soyferment-Tempeh of dose 10mg/100g BW treatment groups (p<0.05), while the PEPCK gene expression did not significantly differ (p>0.05). These results demonstrate that supplementation with Soyferment-Tempeh decreases blood glucose level, atherogenic index, improves lipid profile, and decreases RBP4.

Transcriptional regulation of retinol binding protein 4 by Interleukin-6 via peroxisome proliferator-activated receptor α and CCAAT/Enhancer binding proteins

Interleukin-6 (IL-6) is a major mediator of the acute phase response (APR) that regulates the transcription of acute phase proteins (APPs) in the liver. During APR, the plasma levels of negative APPs including retinol binding protein 4 (RBP4) are reduced. Activation of the IL-6 receptor and subsequent signaling pathways leads to the activation of transcription factors, including peroxisome proliferator-activated receptor alpha (PPARα) and CCAAT/enhancer binding protein (C/EBP), which then modulate APP gene expression. The transcriptional regulation of RBP4 by IL-6 is not fully understood. Therefore, this study aimed to elucidate the molecular mechanisms of PPARα and C/EBP isoforms in mediating IL-6 regulation of RBP4 gene expression. IL-6 was shown to reduce the transcriptional activity of RBP4, and functional dissection of the RBP4 promoter further identified the cis-acting regulatory elements that are responsible in mediating the inhibitory effect of IL-6. The binding sites for PPARα and C/EBP present in the RBP4 promoter were predicted at −1079 bp to −1057 bp and −1460 bp to −1439 bp, respectively. The binding of PPARα and C/EBPs to their respective cis-acting elements may lead to antagonistic interactions that modulate the IL-6 regulation of RBP4 promoter activity. Therefore, this study proposed a new mechanism of interaction involving PPARα and different C/EBP isoforms. This interaction is necessary for the regulation of RBP4 gene expression in response to external stimuli, particularly IL-6, during physiological changes.

Strong Correlation Between CTLA-4 and LEF1 Gene Expression Levels in CLL: Targeting of the Wnt/β-Catenin Pathway May Adversely Affect CTLA-4 Expression and Function

Recently published clinical trials have confirmed the effectiveness of anti-CD38 monoclonal antibody therapy in myeloma. Furthermore, in vitro studies of chronic lymphocytic leukaemia (CLL) cells suggest that CD38 expression can be enhanced by treatment with retinoid derivatives and thus may enhance the cytotoxic effects of anti-CD38 therapy. However, retinoids have been shown to have diverse effects on cellular function and we have previously shown that the retinoid drug acitretin upregulates CD38 expression while also reducing cell homing to the chemokine CXCL12 in primary CLL cells.To investigate possible key mechanisms for these effects, we purified CD20+ B cells from the peripheral blood of 20 CLL patients (9 previously treated, 11 untreated) and, using flow cytometry, measured percentage cell surface expression of CD38 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152). We also measured gene expression levels of the key retinoid receptor, stimulated by retinoic acid 6 (STRA6) and it's agonist, retinol-binding protein 4 (RBP4), as well as CTLA-4, cyclin D1 (CCND1) and the transcription factors, lymphoid enhancer factor 1 (LEF1) and signal transducer and activator of transcription 3 (STAT3) using RT-PCR. GAPDH was used as a reference gene.Mean percentage surface expression of CD38 and CTLA-4 was 21.96% and 45.25% respectively. Mean ΔCT gene expression levels of CCND1, CTLA-4, LEF1 and STAT3 were 12.03, 5.57 , 5.99 and 8.98 respectively. RBP4 and STRA6 gene expression levels were undetectable in all 20 patients. Gene expression of LEF1 showed significant correlations with CTLA-4 (rs=0.572, p=0.008), CCND1 (rs=0.61, p=0.004) and STAT3 (rs=0.587, p=0.006). There was also a significant correlation between gene expression of CCND1 and of STAT3 (r =0.499, p=0.025). No significant correlations were found between percentage surface expression of CTLA-4 and gene expression levels of either CTLA-4 or of LEF1. A weak negative correlation between percentage surface expression of CTLA-4 and of CD38 was not statistically significant. Comparing untreated and previously treated patients, there was no significant difference in gene expression levels of CTLA-4 and of LEF1 or in surface expression of CTLA-4.The failure to detect RBP4 and STRA6 gene expression in unstimulated peripheral blood CLL cells is evidence against an autocrine retinoid effect in CLL, although upregulation of STRA6 gene expression following stimulation by retinoids might be anticipated. The Wnt signalling pathway has been shown to be active in CLL, including aggressive disease subtypes, highlighting the potential benefits in targeting this pathway. Intriguingly, CTLA-4 expression, although found to be the most highly induced gene following treatment with recombinant Wnt-3a in melanoma cell lines, is associated with a favourable outcome in CLL, possibly by inhibiting cell proliferation and survival. In contrast, expression of LEF1, which is a direct target of the Wnt signalling pathway, is associated with disease progression in CLL. Our finding that CTLA-4 and LEF1 gene expression levels are strongly correlated suggests that further investigation of the relationship between CTLA-4 and the Wnt/β-Catenin pathway in CLL is required and that targeting of the Wnt/β-catenin pathway may have unwanted consequences on CTLA-4 expression and function. DisclosuresQuinn:Celgene: Honoraria; Janssen Cilag: Honoraria.

Effect of endurance training on retinol-binding protein4 gene expression and its protein level in adipose tissue and the liver in diabetic rats induced by a high-fat diet and streptozotocin.

The present study was designed to investigate from which tissues the decrease in retinol-binding protein 4 (RBP4) expression could contribute to the improvement of serum RBP4 and insulin resistance (IR) after endurance training. Male 7-week-old Wistar rats were randomly assigned into four groups including control (C), trained (T), diabetic control (DC) and trained diabetic (TD). At 8weeks-of-age, diabetes was induced by a high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ; 35mg/kg). Rats in the T and TD groups carried out a 7-week exercise program on a motorized treadmill (15-20m/min for 20min/day for 5weeks), whereas the C and DC remained sedentary in their cages. Tissues gene expression and protein levels of RBP4 were assessed by using real-time polymerase chain reaction and western blot, respectively, while serum RBP4 was measured using an enzyme-linked immunosorbent assay kit. Exercise significantly improved IR and reduced serum concentration of RBP4 in the TD group. This reduction of serum RBP4 was accompanied by decreased RBP4 protein expression in visceral fat tissue. In contrast, exercise had no significant effect on RBP4 expression in liver and subcutaneous fat tissue in the TD group. Exercise also significantly decreased RBP4 gene expression in visceral fat tissue and muscle, whereas the effect of exercise on liver RBP4 messenger ribonucleic acid expression was not significant. The present study showed that the mechanism for RBP4 reducing the effect of endurance training could involve decreased RBP4 messenger ribonucleic acid expression and its protein level in adipose tissue in STZ-induced diabetic rats.

Retinol binding protein‐4 circulating levels were higher in nonalcoholic fatty liver disease vs. histologically normal liver from morbidly obese women

We aimed to analyze the retinol binding protein-4 (RBP4) messenger RNA (mRNA) expression profiles in adipose tissues and liver of morbidly obese (MO) women with or without nonalcoholic fatty liver disease (NAFLD), and to study the relationships with other pro- and anti-inflammatory adipokines in vivo and in vitro. We performed a cross-sectional analysis of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and liver samples from four lean and 45 MO women with or without NAFLD by enzyme-linked immunosorbent assay and real-time reverse transcription-PCR. We also studied RBP4 expression in HepG2 hepatocytes under various inflammatory stimuli. Circulating RBP4 levels were higher in MO women, and specifically, in MO subjects with NAFLD compared with normal liver controls (lean and MO). RBP4 liver expression was higher in nonalcoholic steatohepatitis (NASH)-moderate/severe than in NASHmild. Overall RBP4 gene expression was higher in liver than in adipose tissues. Among them, the higher expression corresponded to SAT. VAT expression was lower in the MO cohort. In HepG2, RBP4 mRNA expression was reduced by tumor necrosis factor (TNF)-α and increased by adiponectin treatment. The results obtained in MO women with NAFLD, brings up the use of RBP4 and other adipokines as a panel of noninvasive molecular biomarkers when NAFLD is suspected. Further studies are needed with other obesity groups.

Associations between Tissue Visfatin/Nicotinamide, Phosphoribosyltransferase (Nampt), Retinol Binding Protein-4, and Vaspin Concentrations and Insulin Resistance in Morbidly Obese Subjects

Visfatin/Nampt, vaspin, and retinol binding protein-4 (RBP-4) play an important role in insulin resistance. The objectives of this study were to measure visfatin/Nampt, vaspin, and RBP-4 concentrations in blood, liver, muscle, subcutaneous, omental, and mesenteric adipose tissues in morbidly obese subjects and investigate their relationship to insulin resistance. Blood and tissue samples were collected from 38 morbidly obese subjects during Roux-en-Y surgery. Insulin resistance biomarkers were measured using standard kits. Visfatin/Nampt, vaspin, and RBP-4 gene expression levels in tissues were measured using real-time PCR. Their protein concentrations in blood and tissues were measured using ELISA kits. Diabetic subjects had significantly higher homeostasis model of assessment-insulin resistance and age and lower blood HDL-cholesterol concentrations than nondiabetic and prediabetic subjects. Diabetic and prediabetic subjects had significantly higher blood concentrations of visfatin/Nampt and vaspin than nondiabetic subjects. Liver RBP-4 concentrations were positively associated with blood glucose concentrations. Blood insulin resistance biomarker levels were positively associated with visfatin/Nampt concentrations in omental adipose tissue and liver, and vaspin concentrations in mesenteric adipose tissue. In conclusion, the correlations of visfatin/Nampt, vaspin, and RBP-4 with insulin resistance are tissue dependent.

Bovine hepatic and adipose retinol-binding protein gene expression and relationship with tumor necrosis factor-α

Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-α in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at −21, −4, +1, +7, and +21 relative to parturition and frozen in liquid N2. Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and β-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to β-actin, and significance was determined at P<0.05. Serum samples (−21, −4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-α was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-α (r=0.60). Within adipose tissue, expression of RBP and TNF-α was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-α. Although TNF challenge caused an upregulation of hepatic TNF-α expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-α transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-α) via induction of transcription factor nuclear factor-interleukin 6.

Visfatin and RBP4 gene expression levels in different adipose tissues and insulin resistance

Background As an endocrine organ, adipose tissue produces and secretes lots of hormones and adipokines that play important roles in metabolism and inflammation [1]. Varied adipose tissues may affect the inflammation distinctively, mainly because of the different secretion levels of the adipokines. Adipokines like visfatin and retinol binding protein-4 (RBP4) may have important roles in insulin sensitivity and glucose uptake to the cell [2,3]. Our research focuses on different tissue secretions of visfatin and RBP4 and their inflammatory properties.

Peroxisome Proliferator-Activated Receptors-α and -γ, and cAMP-Mediated Pathways, Control Retinol-Binding Protein-4 Gene Expression in Brown Adipose Tissue

Retinol binding protein-4 (RBP4) is a serum protein involved in the transport of vitamin A. It is known to be produced by the liver and white adipose tissue. RBP4 release by white fat has been proposed to induce insulin resistance. We analyzed the regulation and production of RBP4 in brown adipose tissue. RBP4 gene expression is induced in brown fat from mice exposed to cold or treated with peroxisome proliferator-activated receptor (PPAR) agonists. In brown adipocytes in culture, norepinephrine, cAMP, and activators of PPARγ and PPARα induced RBP4 gene expression and RBP4 protein release. The induction of RBP4 gene expression by norepinephrine required intact PPAR-dependent pathways, as evidenced by impaired response of the RBP4 gene expression to norepinephrine in PPARα-null brown adipocytes or in the presence of inhibitors of PPARγ and PPARα. PPARγ and norepinephrine can also induce the RBP4 gene in white adipocytes, and overexpression of PPARα confers regulation by this PPAR subtype to white adipocytes. The RBP4 gene promoter transcription is activated by cAMP, PPARα, and PPARγ. This is mediated by a PPAR-responsive element capable of binding PPARα and PPARγ and required also for activation by cAMP. The induction of the RBP4 gene expression by norepinephrine in brown adipocytes is protein synthesis dependent and requires PPARγ-coactivator-1-α, which acts as a norepinephine-induced coactivator of PPAR on the RBP4 gene. We conclude that PPARγ- and PPARα-mediated signaling controls RBP4 gene expression and releases in brown adipose tissue, and thermogenic activation induces RBP4 gene expression in brown fat through mechanisms involving PPARγ-coactivator-1-α coactivation of PPAR signaling.

Retinol-binding protein 4 (RBP4) protein expression is increased in omental adipose tissue of severely obese patients.

Visceral fat has been linked to insulin resistance and type 2 diabetes mellitus (T2DM); and emerging data links RBP4 gene expression in adipose tissue with insulin resistance. In this study, we examined RBP4 protein expression in omental adipose tissue obtained from 24 severely obese patients undergoing bariatric surgery, and 10 lean controls (4 males/6 females, BMI = 23.2 +/- 1.5 kg/m(2)) undergoing elective abdominal surgeries. Twelve of the obese patients had T2DM (2 males/10 females, BMI: 44.7 +/- 1.5 kg/m(2)) and 12 had normal glucose tolerance (NGT: 4 males/8 females, BMI: 47.6 +/- 1.9 kg/m(2)). Adipose RBP4, glucose transport protein-4 (GLUT4), and p85 protein expression were determined by western blot. Blood samples from the bariatric patients were analyzed for serum RBP4, total cholesterol, triglycerides, and glucose. Adipose RBP4 protein expression (NGT: 11.0 +/- 0.6; T2DM: 11.8 +/- 0.7; lean: 8.7 +/- 0.8 arbitrary units) was significantly increased in both NGT (P = 0.03) and T2DM (P = 0.005), compared to lean controls. GLUT4 protein was decreased in both NGT (P = 0.02) and T2DM (P = 0.03), and p85 expression was increased in T2DM subjects, compared to NGT (P = 0.03) and lean controls (P = 0.003). Regression analysis showed a strong correlation between adipose RBP4 protein and BMI for all subjects, as well as between adipose RBP4 and fasting glucose levels in T2DM subjects (r = 0.76, P = 0.004). Further, in T2DM, serum RBP4 was correlated with p85 expression (r = 0.68, P = 0.01), and adipose RBP4 protein trended toward an association with p85 protein (r = 0.55, P = 0.06). These data suggest that RBP4 may regulate adiposity, and p85 expression in obese-T2DM, thus providing a link to impaired insulin signaling and diabetes in severely obese patients.

The cAMP-HMGA1-RBP4 system: a novel biochemical pathway for modulating glucose homeostasis

BackgroundWe previously showed that mice lacking the high mobility group A1 gene (Hmga1-knockout mice) developed a type 2-like diabetic phenotype, in which cell-surface insulin receptors were dramatically reduced (below 10% of those in the controls) in the major targets of insulin action, and glucose intolerance was associated with increased peripheral insulin sensitivity. This particular phenotype supports the existence of compensatory mechanisms of insulin resistance that promote glucose uptake and disposal in peripheral tissues by either insulin-dependent or insulin-independent mechanisms. We explored the role of these mechanisms in the regulation of glucose homeostasis by studying the Hmga1-knockout mouse model. Also, the hypothesis that increased insulin sensitivity in Hmga1-deficient mice could be related to the deficit of an insulin resistance factor is discussed.ResultsWe first show that HMGA1 is needed for basal and cAMP-induced retinol-binding protein 4 (RBP4) gene and protein expression in living cells of both human and mouse origin. Then, by employing the Hmga1-knockout mouse model, we provide evidence for the identification of a novel biochemical pathway involving HMGA1 and the RBP4, whose activation by the cAMP-signaling pathway may play an essential role for maintaining glucose metabolism homeostasis in vivo, in certain adverse metabolic conditions in which insulin action is precluded. In comparative studies of normal and mutant mice, glucagon administration caused a considerable upregulation of HMGA1 and RBP4 expression both at the mRNA and protein level in wild-type animals. Conversely, in Hmga1-knockout mice, basal and glucagon-mediated expression of RBP4 was severely attenuated and correlated inversely with increased Glut4 mRNA and protein abundance in skeletal muscle and fat, in which the activation state of the protein kinase Akt, an important downstream mediator of the metabolic effects of insulin on Glut4 translocation and carbohydrate metabolism, was simultaneously increased.ConclusionThese results indicate that HMGA1 is an important modulator of RBP4 gene expression in vivo. Further, they provide evidence for the identification of a novel biochemical pathway involving the cAMP-HMGA1-RBP4 system, whose activation may play a role in glucose homeostasis in both rodents and humans. Elucidating these mechanisms has importance for both fundamental biology and therapeutic implications.

Retinol Binding Protein 4 Expression in Humans: Relationship to Insulin Resistance, Inflammation, and Response to Pioglitazone

Retinol binding protein 4 (RBP4) was recently found to be expressed and secreted by adipose tissue, and was strongly associated with insulin resistance. The aim was to determine the relationship between RBP4 and obesity, insulin resistance, and other markers of insulin resistance in humans. RBP4 mRNA levels in adipose tissue and muscle of nondiabetic human subjects with either normal or impaired glucose tolerance (IGT) were studied, along with plasma RBP4. RBP4 gene expression was also measured in adipose tissue fractions, and from visceral and sc adipose tissue (SAT) from surgical patients. The study was conducted at University Hospital and General Clinical Research Center. Insulin sensitivity (S(I)) was measured, and fat and muscle biopsies were performed. In IGT subjects, these procedures were performed before and after treatment with metformin or pioglitazone. The relationship between RBP4 expression and obesity, S(I), adipose tissue inflammation, and intramyocellular lipid level, and response to insulin sensitizers was measured. RBP4 was expressed predominantly from the adipocyte fraction of SAT. Although SAT RBP4 expression and the plasma RBP4 level demonstrated no significant relationship with body mass index or S(I), there was a strong positive correlation between RBP4 mRNA and adipose inflammation (monocyte chemoattractant protein-1 and CD68), and glucose transporter 4 mRNA. Treatment of IGT subjects with pioglitazone resulted in an increase in S(I) and an increase in RBP4 gene expression in both adipose tissue and muscle, but not in plasma RBP4 level, and the in vitro treatment of cultured adipocytes with pioglitazone yielded a similar increase in RBP4 mRNA. RBP4 gene expression in humans is associated with inflammatory markers, but not with insulin resistance. The increase in RBP4 mRNA after pioglitazone treatment is unusual, suggesting a complex regulation of this novel adipokine.

Insulin Resistance in Liver Cirrhosis Is Not Associated With Circulating Retinol-Binding Protein 4

Retinol-binding protein 4 (RBP4) has been identified as a novel adipokine mediating systemic insulin resistance, and elevated serum RBP4 indicates overt or impending insulin resistance in lean, obese, and type 2 diabetic subjects. As insulin resistance is present in nearly all patients with liver cirrhosis, we evaluated RBP4 in patients with chronic liver disease (CLD). Serum RBP4 was measured in 111 CLD patients. Ninety-nine age- and sex-matched healthy blood donors served as control subjects. RBP4 gene expression was also quantified in normal and cirrhotic rat liver. In CLD patients, serum RBP4 was significantly reduced compared with healthy control subjects and closely correlated with the stage of liver cirrhosis. CLD patients without cirrhosis showed normal RBP4 concentrations, which correlated with serum glucose and insulin secretion and inversely correlated with insulin sensitivity. In patients with Child A-C liver cirrhosis, however, RBP4 was not correlated with glucose metabolism or other adipokines, such as adiponectin or resistin, but closely linked to the hepatic biosynthetic capacity, fibrotic changes in liver histology, or clinical complications such as portal hypertension. In an animal model of experimental cirrhosis, hepatic RBP4 gene expression decreased in cirrhotic liver. RBP4 appears, unlike in obesity or type 2 diabetes, not to be a relevant systemic factor in the pathogenesis of insulin resistance in liver cirrhosis. Liver function has a tremendous impact on RBP4 levels, and future studies will need to take liver function into account when examining serum RBP4 levels.

Insulin resistance in liver cirrhosis is not associated with circulating Retinol Binding Protein 4

OBJECTIVE—Retinol-binding protein 4 (RBP4) has been identified as a novel adipokine mediating systemic insulin resistance, and elevated serum RBP4 indicates overt or impending insulin resistance in lean, obese, and type 2 diabetic subjects. As insulin resistance is present in nearly all patients with liver cirrhosis, we evaluated RBP4 in patients with chronic liver disease (CLD). RESEARCH DESIGN AND METHODS—Serum RBP4 was measured in 111 CLD patients. Ninety-nine age- and sex-matched healthy blood donors served as control subjects. RBP4 gene expression was also quantified in normal and cirrhotic rat liver. RESULTS—In CLD patients, serum RBP4 was significantly reduced compared with healthy control subjects and closely correlated with the stage of liver cirrhosis. CLD patients without cirrhosis showed normal RBP4 concentrations, which correlated with serum glucose and insulin secretion and inversely correlated with insulin sensitivity. In patients with Child A-C liver cirrhosis, however, RBP4 was not correlated with glucose metabolism or other adipokines, such as adiponectin or resistin, but closely linked to the hepatic biosynthetic capacity, fibrotic changes in liver histology, or clinical complications such as portal hypertension. In an animal model of experimental cirrhosis, hepatic RBP4 gene expression decreased in cirrhotic liver. CONCLUSIONS—RBP4 appears, unlike in obesity or type 2 diabetes, not to be a relevant systemic factor in the pathogenesis of insulin resistance in liver cirrhosis. Liver function has a tremendous impact on RBP4 levels, and future studies will need to take liver function into account when examining serum RBP4 levels.

Steroid regulation of retinol-binding protein in the ovine oviduct.

Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17beta (E2), progesterone (P4), E2+P4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P4 alone or in combination with E2 significantly reduced steady-state RBP mRNA levels compared to those in E2-treated animals. Oviductal explants from E2- and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E2 and E2+P4 treatments. This study demonstrates that P4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.

Induction of Mouse Retinol Binding Protein Gene Expression by Cyclic AMP in Hepa 1–6 Cells

Retinol binding protein (RBP) is the primary circulating transport molecule for retinol, facilitating its transport to target tissues and influencing target cell uptake. Specific signals and molecular mechanisms that regulate RBP gene expression are poorly understood. Using the mouse hepatoma cell line (Hepa 1–6), we examined the role of cAMP in the molecular regulation of RBP. Dibutyryl cAMP (dbcAMP) or the adenylate cyclase activator, forskolin, increased RBP mRNA levels >6-fold at 24 h. Increases in RBP mRNA were dose dependent over the range of 10 μM–1 mM for dbcAMP and 0.5–10 μM for forskolin. 8-Bromo cAMP, a nonhydrolyzable analog, over the range of 0.01–0.5 mM, increased RBP mRNA levels 9.2-fold at 24 h. Induction of RBP transcripts by analogs also resulted in a comparable increase in intracellular RBP protein. Cycloheximide (10 μg/ml) did not prevent cAMP-mediated induction of RBP mRNA, indicating thatde novoprotein synthesis is not required for cAMP-mediated induction of RBP transcription.These studies demonstrate that cAMP, or agents which elevate intracellular cAMP, increase RBP transcript levels. The time course and extent of RBP mRNA induction and the resultant increase in RBP protein support the concept that cAMP regulation of RBP gene expression may be physiologically relevent. Given the ubiquitous nature of cAMP as a second messenger, and the several mechanisms by which cAMP regulates gene expression, studies are in progress to define molecular mechanisms by which cAMP regulates RBP gene expression.

Retinol-Binding Protein: Immunolocalization of Protein and Abundance of Messenger Ribonucleic Acid in Conceptus and Maternal Tissues during Pregnancy in Pigs1

Retinol-binding protein (RBP) is a major secretory product of liver as well as uterine endometrium and periimplantation conceptuses of pigs. The present study examined distribution of RBP protein and abundance of RBP mRNA in pig maternal and conceptus tissues throughout pregnancy. A porcine liver RBP cDNA clone was isolated and used as probe in these studies. Northern blot analysis detected a 1.0-1.1-kb transcript in conceptus, trophoblast, yolk sac, placenta, endometrium, myometrium, oviduct, and numerous fetal tissues. Slot blot analyses of RBP mRNA abundance in endometrium, myometrium, conceptus, trophoblast, chorioallantoic placenta, and embryo/fetus indicated differential RBP gene expression in each tissue. Immunocytochemical localization studies indicated the presence of immunoreactive RBP in endometrial surface and glandular epithelium, circular and longitudinal muscle of myometrium, parenchymal cells of fetal liver, and proximal convoluted tubules of fetal kidney. Results suggest an integrated system of RBP gene expression and protein secretion that allows for transport of retinol from the uterine endometrium to the periimplantation conceptus during early pregnancy and to the fetal-placental unit throughout pregnancy in pigs.

Early gestational expression of retinol-binding protein mRNA by the ovine conceptus and endometrium.

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol-binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two-fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue-specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression.

Retinol-binding protein gene expression in cyclic and pregnant endometrium of pigs, sheep, and cattle.

Retinol-binding protein (RBP) is a major secretory product of conceptuses and endometrium of the large domestic animals. The present study examined the abundance of RBP mRNA in cyclic and early pregnant endometrium of pigs, sheep, and cattle and confirmed the presence of RBP mRNA in preiimplantation conceptuses of the large domestic animals. Northern blot analysis, using porcine liver RBP cDNA as the probe, detected a 1.0-1.1-kb transcript in conceptuses and endometrium collected during the preiimplantation period of pregnancy in pigs (Day 15), sheep (Day 16), and cows (Day 18). Slot-blot analysis of RBP mRNA in endometrium of pigs, sheep, and cows indicated differential RBP gene expression across days and statuses in pigs and sheep and across days alone in cows. In pigs, RBP gene expression was low to undetectable at Days 0, 5, and 10 of the estrous cycle and Day 10 of pregnancy. Levels of RBP mRNA increased (p < 0.06) from Days 10 to 12 and further (p < 0.001) to Day 15 across both statuses. At Day 18, RBP mRNA levels decreased (p < 0.01) in cyclic pigs but remained elevated in endometrium of pregnant pigs. In sheep, levels of RBP mRNA increased between Days 10 and 12 and 14 in both cyclic and pregnant ewes; however, between Days 14 and 16, RBP mRNA levels remained elevated in cyclic endometrium but decreased (p < 0.01) in endometrium of pregnant ewes. In a second experiment, RBP mRNA levels increased (p < 0.01) between Days 0-5 and Days 13-15 of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Extinction of retinol-binding protein gene expression in somatic cell-hybrids: identification of the target sequences

The Retinol-Binding Protein (RBP) is expressed primarily in the liver. The regulatory elements involved in its tissue-specific expression have been identified and mapped to the 5' flanking region of the RBP gene. In this paper heterokaryons and somatic cell-hybrids have been produced and analysed in order to demonstrate that the RBP gene is subject to extinction and to identify the target sequences of this phenomenon. We show here that the gene is extinguished in fusions of hepatoma with a variety of cells of different species and embryonic lineages. The repression is not due to loss of the gene and occurs also when chromosome 10, where the gene is located, is inherited from the expressing parental cell-type. Hybrid clones were transfected with constructs carrying DNA segments of different lengths from the 5' flanking region of the RBP gene fused to a reporter gene. We demonstrate that extinction takes place also on an exogenous RBP-CAT gene, mimicking the phenomenon observed with the endogenous gene in its chromosomal location. Moreover, we identify and map the target sequences of the putative extinguishing function. Our data thus show that extinction of RBP is mediated through the DNA segment that is involved in its tissue-specific expression.