RBP4 mRNA Research Articles

P163 PI(4,5)P2 alleviates colitis by inhibiting intestinal epithelial cell pyroptosis through NNMT-mediated RBP4 m6A modification

Abstract Background Lipid metabolism disorder is a critical feature of Crohn’s disease (CD). We previously reported that phosphatidylinositol (PI), and its derivative phosphatidylinositol bisphosphate (PIP2) were significantly associated with CD activity. The underlying mechanisms remained unknown. We aimed to explore the function of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the major PI derivative, in modulating CD pathogenesis. Methods Lipidomics was used to analyse the relationship between CD activity and PI or PIP2. The mucosal expression of PI(4,5)P2 in patients with CD was measured by immunofluorescence. Dextran sodium sulfate (DSS)-induced colitis mice and lipopolysaccharide (LPS)-induced Caco-2 cell model were established to explore the function and mechanism of PI(4,5)P2. MeRIP and mRNA sequencing were applied to further reveal the mechanism of PI(4,5)P2. Results Lipid PIP2 was substantially negatively associated with disease activity and high-sensitivity C-reactive protein. PI(4,5)P2 expression was downregulated in the inflamed mucosa of patients with CD. PI(4,5)P2 alleviated mice colitis, with improvements in survival rate, colon length, body weight, and disease activity index. PI(4,5)P2 also alleviated DSS-induced tissue damage, tight junction loss, and intestinal epithelial cell (IEC) pyroptosis. In the in vitro LPS-induced cell model, PI(4,5)P2 inhibited pyroptosis, NLRP3, and caspase-1 expression, and reduced IL-18, IL-1β, and lactate dehydrogenase (LDH) secretion. PI(4,5)P2 mediated NNMT upregulation in mice and Caco-2 cells. NNMT knockdown restricted the inhibitory effect of PI(4,5)P2 on IEC pyroptosis. NNMT inhibited the stability of RBP4 mRNA via the m6A modification to prevent pyroptosis upon PI(4,5)P2 treatment. Conclusion PI(4,5)P2 inhibited IEC pyroptosis through NNMT-mediated RBP4 m6A modification, which ameliorating colitis. PI(4,5)P2 may be a therapeutic target for CD.

Ovariectomy Increases Circulating Retinol-Binding Protein Concentrations Independently of Sex-Dependent Differences in Retinol Concentrations in Rats

BackgroundThere is a sex-dependent difference in blood retinol and RBP concentrations, and plasma RBP is associated with insulin resistance. ObjectivesWe aimed to clarify sex-dependent variations in body concentrations of retinol and RBPs and their association with sex hormones in rats. MethodsPlasma and liver retinol concentrations and hepatic mRNA and plasma concentrations of RBP4 were analyzed in 3- and 8-wk-old male and female Wistar rats before and after sexual maturity (experiment 1) and in orchiectomized male Wistar rats (experiment 2) and ovariectomized female Wistar rats (experiment 3). Furthermore, the mRNA and protein concentrations of RBP4 in adipose tissue were measured in ovariectomized female rats (experiment 3). ResultsThere were no sex-dependent differences in liver retinyl palmitate and retinol concentrations; however, the plasma retinol concentration was significantly higher in male rats than that in female rats after sexual maturity. Furthermore, the plasma retinol concentrations did not differ between the ovariectomized or orchiectomized rats and the control rats. Plasma Rbp4 mRNA concentrations were higher in male rats than those in female rats but not in castrated and control rats, a change consistent with plasma retinol concentration. Plasma RBP4 concentrations were also higher in male rats than those in female rats; however, unlike liver Rbp4 gene expression, plasma RBP4 concentrations were 7-fold higher in the ovariectomized rats than those in the control rats. Moreover, the Rbp4 mRNA concentrations in inguinal white adipose tissue was significantly higher in the ovariectomized rats than those in the control rats and correlated with plasma RBP4 concentrations. ConclusionsHepatic Rbp4 mRNA is higher in male rats through a sex hormone–independent mechanism, which may contribute to sex differences in blood retinol concentrations. Furthermore, ovariectomy leads to an increase in adipose tissue Rbp4 mRNA and blood RBP4 concentrations, which may contribute to insulin resistance in ovariectomized rats and postmenopausal women.

Retinol-binding protein 4 mRNA translation in hepatocytes is enhanced by activation of mTORC1.

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated Rbp4 mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, Rbp4 mRNA translation was low. In response to refeeding, Rbp4 mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted Rbp4 mRNA translation and expression of the protein. Rbp4 mRNA abundance was not increased by either experimental manipulation. Enhanced Rbp4 mRNA translation was associated with activation of the kinase mechanistic target of rapamycin in complex 1 (mTORC1) and enhanced phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the Rbp4 mRNA 5'-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the Rbp4 5'-UTR prevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of Rbp4 mRNA translation in the liver and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 upregulates Rbp4 mRNA translation to promote RBP4 synthesis.NEW & NOTEWORTHY RBP4 plays a critical role in metabolic disease, yet relatively little is known about the mechanisms that regulate its production. Herein, we provide evidence for translational control of RBP4 synthesis. We demonstrate that activation of the nutrient-sensitive kinase mTORC1 promotes hepatic Rbp4 mRNA translation. The findings support the possibility that targeting Rbp4 mRNA translation represents an alternative to current therapeutic interventions that lower serum RBP4 concentration by promoting urinary excretion of the protein.

Cbl downregulation increases RBP4 expression in adipocytes of female mice.

Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally.

Hepatocytes Are the Principal Source of Circulating RBP4 in Mice.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Sequence analysis and expression regulation of rbp4 by 9-cis-RA in Megalobrama amblycephala.

Retinol-binding protein 4 (rbp4) is mainly synthesized in the liver, where it binds retinol and then enters the bloodstream, delivering retinol to cells. The full-length cDNA coding rbp4 was cloned from Megalobrama amblycephala. The amino acid sequence showed strong homology with the homologues of other vertebrates, and all structural and functional domains were highly conserved. The mRNA levels in different tissues and development stages detected by quantitative real-time PCR revealed that M. amblycephala rbp4 was highly expressed in liver (P < 0.001), but the lower levels were also detected in eyes, kidney, intestine, and spleen. During the different development stages, the rbp4 mRNA appeared until 28 hours post-fertilization (hpf), underwent a slight drop, and then gradually increased after 50 hpf. In addition, the promoter sequence of M. amblycephala rbp4 was obtained using thermal asymmetric interlaced PCR. Two single nucleotide polymorphism sites (-385A>G and -329C>T) were found in the promoter. Transfection with recombinant plasmids of two different haplotypes (GT, AC) showed that 9-cis-retinoic acid (RA) increased the promoter activity, but the AC haplotype was more sensitive to RA.

Expression of Serum Retinol Binding Protein and Transthyretin within Mouse Gastric Ghrelin Cells

Ghrelin is an orexigenic peptide hormone produced mainly by a distinct group of dispersed endocrine cells located within the gastric oxyntic mucosa. Besides secreted gene products derived from the preproghrelin gene, which include acyl-ghrelin, desacyl-ghrelin and obestatin, ghrelin cells also synthesize the secreted protein nesfatin-1. The main goal of the current study was to identify other proteins secreted from ghrelin cells. An initial gene chip screen using mRNAs derived from highly enriched pools of mouse gastric ghrelin cells demonstrated high levels of serum retinol-binding protein (RBP4) and transthyretin (TTR), both of which are known to circulate in the bloodstream bound to each other. This high expression was confirmed by quantitative RT-PCR using as template mRNA derived from the enriched gastric ghrelin cell pools and from two ghrelin-producing cell lines (SG-1 and PG-1). RBP4 protein also was shown to be secreted into the culture medium of ghrelin cell lines. Neither acute nor chronic caloric restriction had a significant effect on RBP4 mRNA levels within stomachs of C57BL/6J mice, although both manipulations significantly decreased stomach TTR mRNA levels. In vitro studies using PG-1 cells showed no effect on RBP4 release of octanoic acid, epinephrine or norepinephrine, all of which are known to act directly on ghrelin cells to stimulate ghrelin secretion. These data provide new insights into ghrelin cell physiology, and given the known functions of RBP4 and TTR, support an emerging role for the ghrelin cell in blood glucose handling and metabolism.

Interleukin-1β Downregulates RBP4 Secretion in Human Adipocytes

Aims/hypothesisThe excessive accumulation of adipose tissue in the obese state is linked to an altered secretion profile of adipocytes, chronic low-grade inflammation and metabolic complications. RBP4 has been implicated in these alterations, especially insulin resistance. The aim of the present study was to determine if a local inflammatory micro-environment in adipose tissue regulates RBP4 expression and secretion.MethodsHuman SGBS and primary adipocytes cultured with conditioned media from human THP-1 macrophages were used as an in vitro model for adipose inflammation. Adipocytes were exposed to recombinant TNF-α, IL-1β, IL-6 or IL-8. In addition, coexpression of IL-1β and RBP4 was measured in adipose tissue samples from 18 healthy females. RBP4 expression was studied by quantitative PCR and ELISA.ResultsRBP4 mRNA expression and secretion was significantly reduced upon incubation with macrophage-conditioned media in SGBS adipocytes and human primary adipocytes. Out of several factors studied we identified IL-1β as a new factor regulating RBP4. IL-1β significantly downregulated RBP4 mRNA and secretion in a time- and dose-dependent manner. IL-1β mediated its inhibitory effects on RBP4 expression via IL-1 receptor and NF-κB, as incubation with the IL-1 receptor blocking antibody and the NF-κB inhibitors CAPE and SC-514 reversed its effect. Most interestingly, RBP4 mRNA was negatively correlated with IL-1β mRNA in subcutaneous adipose tissue.ConclusionsAdipose tissue inflammation as found in the obese state might lead to a downregulation in local RBP4 levels. IL-1β was identified as a major factor contributing to the decrease in RBP4. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to inflammatory processes in adipose tissue.

Retinol-binding protein 4 as a risk factor for cholesterol gallstone formation

Retinol-binding protein 4 (RBP4) is closely associated with a variety of abnormal glycolipid metabolism diseases such as insulin resistance, obesity, diabetes mellitus, and metabolic syndrome. Part 1 Twenty-four male C57/B6 mice were randomly divided into a normal control group (group C, n = 8), a gallstone group (group G, n = 8), and a medication group (group M, n = 8). Serum RBP4 levels were measured by ELISA. RBP4 expression in liver and adipose tissue was detected by western blotting. The transcription of RBP-4 mRNA, PPAR-γ mRNA in the liver was measured using real-time PCR. The incidences of gallstone formation were 0/8 (group C), 7/8 (group G), and 2/8 (group M) (p < 0.01). Group G had significantly higher body weight, adipose mass, fasting glucose (FG), serum RBP4, total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) than group C. In group M, pioglitazone decreased body weight, adipose mass, levels of serum RBP4, FG, TC, and TG, and it increased levels of HDL-C. Pioglitazone downregulated RBP4 expression in hepatic and adipose tissue and prevented decreases in PPAR-γmRNA levels induced by lithogenic diet. Part 2 Metabolism indices, including serum RBP4, FG, TC, TG, HDL-C, low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) of 110 human cholesterol gallstone patients and 73 healthy controls were collected for further analysis. Patients with gallstones had elevated levels of serum RBP4, FG, TC, TG, ALT, and AST, and had decreased HDL-C levels compared to those of healthy controls. Elevated RBP4 is associated with the morbidity of cholesterol gallstones and metabolic syndrome. RBP4 may play an important role in the course of cholesterol gallstone formation.

Differences in retinoid uptake and metabolism causes altered paracrine signaling in endometriosis

Decidualization alters molecular pathways in the endometrium to permit successful embryo implantation. Paracrine factors, including retinoids, secreted from progesterone-treated endometrial stromal cells (HESC), act on epithelium to alter steroid hormone response. These factors are absent in endometriotic stromal cells (OSIS). We noted significant differences in retinoid uptake, metabolism and action in endometriotic tissue and stromal cells compared to normal endometrium. Here we investigate retinoid signaling during decidualization in HESC and OSIS. Basic science. Primary HESC and OSIS were isolated, cultured and treated with decidualization cocktail over 14d. Protein and mRNA expression of genes involved in retinoid metabolism and trafficking (STRA6, CRBP1, ALD1A2, CRABP2, FABP5, RARα, RXRα, PPARβ/δ and CYP26B1) were examined with RT-PCR and western blotting. Decidualization markers (PRL and IGFBP1) were examined with RT-PCR and ELISA. RBP4 secretion was measured with ELISA. siRNA was used to silence genes. HESC and OSIS expressed genes involved in retinoid uptake and metabolism (n=5). Decidualization significantly reduced expression of genes responsible for RA uptake and shuttling to the nucleus (P<0.05). However, mRNA and protein expression of CRBP1, a retinol intracellular carrier protein, increased, as did RBP4 mRNA. Secreted RBP4 was detected by ELISA in media from decidualized HESC. In OSIS, there was neither an increase in CRBP1 mRNA nor protein expression with decidualization, and secreted RBP4 was not detected. Interestingly, silencing CRBP1 in HESC caused an increase in PRL mRNA expression and secretion. Decidualization alters RA trafficking to favor paracrine over intacrine signaling. RBP4 secretion may be an important paracrine signal between stroma and nearby epithelial cells. This signaling is blunted in endometriosis and may help explain its altered decidualization.

Effects of variation in retinol binding protein 4 gene and adipose specific expression of gestational diabetes in Beijing, China

ObjectiveTo investigate the clinical features of GDM in China and the effects of RBP4 genetic variants, and also to identify RBP4 expression changes in mRNA and protein levels. Research design and methods1595 Chinese pregnant women were included in this study. Four known RBP4 single nucleotide polymorphisms (SNPs) were genotyped in 505 cases and 687 controls. Expression levels of adipose specific RBP4 mRNA and protein were detected in 41 samples of subcutaneous adipose tissue. ResultsThe estimated indices of insulin resistance were gradually increased from NGT, GIGT to GDM. Two single SNPs were associated with GDM (rs3758539 G vs A, OR=1.446, P=0.009; rs3758539 GG vs AG+AA, OR=1.532, P=0.006; rs12265684C vs G, OR=1.296, P=0.038) and a haplotype of 3 common SNPs [G-G-T] was increased in subjects with GDM and GIGT (OR=1.322, 95% CI 1.054–1.659, P=0.016). RBP4 mRNA expression in adipose tissue of GDM patients was significantly increased in comparison to control subjects (1.438±0.187 vs 1.034±0.062, p=0.025). ConclusionThis finding suggests that impaired insulin sensitivity has an early onset in mild gestational intolerance. Two single SNPs were associated with GDM in the case-control study while a haplotype of 3 common SNPs [G-G-T] was increased in glucose intolerance subjects.

Alterations in systemic concentrations of progesterone during the early luteal phase affect RBP4 expression in the bovine uterus

Systemic progesterone affects the timing and duration of uterine endometrial gene and protein expression and has significant effects on conceptus development. The objective of the present study was to examine how changes in progesterone concentrations during the early luteal phase affect retinol-binding protein (RBP4) mRNA and protein concentrations in the uterus. Endometrial tissue and uterine flushings were recovered on Days 7 and 13 of the oestrous cycle in heifers with high, normal and low progesterone concentrations. RBP4 mRNA and protein concentrations were higher (P<0.05) on Day 13 compared with Day 7 in heifers with high and control progesterone concentrations. However, there was no difference in RBP4 protein concentrations between Days 7 and 13 in heifers with low progesterone (P>0.05). On Day 7, although heifers with low progesterone had lower RBP4 mRNA expression compared with controls (P<0.05) there was no difference in protein concentrations between treatment groups. On Day 13, RBP4 mRNA was 2-fold higher (P<0.001) in heifers with high and control progesterone compared with their low-progesterone counterparts and RBP4 protein concentrations were over 2-fold higher (P<0.001) in heifers with high compared to low progesterone. In conclusion, progesterone modulates uterine RBP4 mRNA and protein abundance in a time- and concentration-dependent manner.

Suppression of retinol-binding protein 4 with RNA oligonucleotide prevents high-fat diet-induced metabolic syndrome and non-alcoholic fatty liver disease in mice

Conflicting data have been reported regarding the role of retinol-binding protein (RBP4) in insulin resistance, obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this study, we used pharmacological methods to investigate the role of RBP4. RNA oligonucleotide against RBP4 (anti-RBP4 oligo) was transfected into 3T3-L1 adipocytes. RT-PCR analysis showed that RBP4 mRNA expression decreased by 55% (p<0.01) compared with control cells. Validated RNA oligo was used in an in vivo study with high fat diet (HFD) fed — mice. 14weeks of HFD feeding increased RBP4 expression (associated with elevated serum levels measured with immunoblotting and ELISA) by 56% in adipose tissue (p<0.05) and 68% in the liver (p<0.01). Adipose RBP4 levels were significantly reduced after 4weeks treatment with anti-RBP4 oligo (25mg/kg, p<0.01) and rosiglitazone (RSG, 10mg/kg, p<0.05) compared with scrambled RNA oligo (25mg/kg) treated mice. Only anti-RBP4 oligo significantly inhibited RBP4 protein (p<0.01) and mRNA expression (p<0.01) in the liver and reduced serum RBP4 levels. Anti-RBP4 oligo and RSG showed comparable effects on impaired glucose tolerance, hyperinsulinaemia and hyperglycaemia. Anti-RBP4 oligo significantly enhanced adipose-GLUT4 expression (p<0.01) but did not increase muscle-GLUT4. Both RSG and anti-RBP4 oligo significantly reduced hepatic phosphoenolpyruvate carboxykinase expression (both p<0.05). Histological analysis revealed that anti-RBP4 oligo ameliorated hepatic steatosis and reduced lipid droplets associated with normalized liver function. Histological and pharmacological results of this study indicate that RBP4 is not only an adipocytokine, but also a hepatic cytokine leading to metabolic syndrome, NAFLD and type 2 diabetes.

Expression and regulation of retinol binding protein 4 mRNA in human adipose tissue in obese and type 2 diabetics

To study the RBP4 mRNA expression between subcutaneous and visceral adipose tissue in obese and type 2 diabetic patients and to investigate the factors that influence RBP4 mRNA expression in Human visceral adipose tissue. 9 individuals with normal weight normal glucose regulation subjects, 9 obesity subjects and 9 type 2 diabetes subjects were enrolled. All of the subjects were prepared to undergone an operation because of nondiabetes disease. Subcutaneous and visceral adipose tissue were taken out as soon as cultured. RT-PCR and Real-time PCR were used to assay the relative expression of RBP4 mRNA. RBP4 mRNA level in visceral adipose tissue of obesity group was (2.10 +/- 1.84), and that of type 2 diabetes group was (1.54 +/- 0.46), both were significantly higher than that in normal weight normal glucose group (0.75 +/- 0.28, P < 0.01, P < 0.05). RBP4 mRNA level in subcutaneous adipose tissue of the three groups were (1.05 +/- 0.15 vs 0.99 +/- 0.14 vs 1.13 +/- 0.07), no difference among them(P > 0.05). Insulin, dexamethasone, pioglitazone, free fatty acids can significantly increase RBP4 mRNA expression, compared with the control group, respectively, have an increase of 2.13 times, 0.84 times, 2.04 times, 4.88 times; however, tumor necrosis factor-alpha can significantly lower RBP4 mRNA level, compared with the control group decreased by 38%. RBP4 mRNA expression in visceral adipose tissue were significantly higher in obesity and type 2 diabetes subjects. In vitro system, RBP4 gene expression in visceral adipose tissue of normal weight normal glucose subjects was regulated by insulin, dexamethasone, pioglitazone, palmitic acid and TNF-alpha, such factors were also participated in the pathophysiological process of insulin resistance and type 2 diabetes.

Retinol-binding protein 4 in twins: regulatory mechanisms and impact of circulating and tissue expression levels on insulin secretion and action.

OBJECTIVE—Retinol-binding protein (RBP) 4 is an adipokine of which plasma levels are elevated in obesity and type 2 diabetes. The aims of the study were to identify determinants of plasma RBP4 and RBP4 mRNA expression in subcutaneous adipose tissue (SAT) and skeletal muscle and to investigate the association between RBP4 and in vivo measures of glucose metabolism.RESEARCH DESIGN AND METHODS—The study population included 298 elderly twins (aged 62–83 years), with glucose tolerance ranging from normal to overt type 2 diabetes, and 178 young (aged 25–32 years) and elderly (aged 58–66 years) nondiabetic twins. Peripheral and hepatic insulin sensitivity was assessed by a euglycemic-hyperinsulinemic clamp, and β-cell function was estimated from an intravenous glucose tolerance test.RESULTS—The influence of environmental versus genetic factors in the regulation of plasma RBP4 increased with age. Plasma RBP4 was elevated in type 2 diabetes and increased with duration of disease. Plasma RBP4 correlated inversely with peripheral, but not hepatic, insulin sensitivity. However, the association disappeared after correction for covariates, including plasma adiponectin. Plasma retinol, and not RBP4, was inversely associated with insulin secretion. SAT RBP4 expression correlated positively with GLUT4 expression and inversely with glucose tolerance. Skeletal muscle RBP4 expression reflected intramuscular fat, and although it was suppressed by insulin, no association with insulin sensitivity was evident. RBP4 expression was not associated with circulatory RBP4.CONCLUSIONS—In conclusion, our data indicate that RBP4 levels in plasma, skeletal muscle, and fat may be linked to insulin resistance and type 2 diabetes in a secondary and noncausal manner.

Role of retinol-binding protein 4 in the pathogenesis of Type 2 diabetes

Retinol-binding protein (RBP)4 is a new adipocytokine that has been associated with insulin resistance. Both RBP4 mRNA expression in adipocytes and serum RBP4 levels are elevated in adipose-specific glucose transporter 4-knockout mice, and elevated circulating RBP4 levels cause insulin resistance by inhibiting phosphatidylinositol 3 kinase activity in skeletal muscle and increasing phosphoenolpyruvate carboxylase expression in liver. Several clinical cross-sectional studies have shown a significant negative association between circulating RBP4 levels and insulin sensitivity evaluated by the glucose clamp method, but it is unclear if RBP4 is associated with insulin resistance in humans because of many conflicting results. Drugs such as rosiglitazone, exercise and weight loss have been shown to decrease circulating RBP4 levels and improve insulin resistance, but contradictory results have been found in other studies. In addition, a recent clinical study has suggested that RBP4 is more closely related to visceral adiposity than subcutaneous adiposity. In summary, RBP4 is an adipocytokine that is especially associated with visceral fat and may be associated with insulin resistance in humans. However, this association remains uncertain and whether or not RBP4 is a new target for treatment of Type 2 diabetes remains to be determined.

Serum Retinol-Binding Protein Is More Highly Expressed in Visceral than in Subcutaneous Adipose Tissue and Is a Marker of Intra-abdominal Fat Mass

Intra-abdominal fat is associated with insulin resistance and cardiovascular risk. Levels of serum retinol-binding protein (RBP4), secreted by fat and liver cells, are increased in obesity and type 2 diabetes (T2D). Here we report that, in 196 subjects, RBP4 is preferentially expressed in visceral (Vis) versus subcutaneous (SC) fat. Vis fat RBP4 mRNA was increased approximately 60-fold and 12-fold in Vis and SC obese subjects respectively versus lean subjects, and approximately 2-fold with impaired glucose tolerance/T2D subjects versus normoglycemic subjects. In obese subjects, serum RBP4 was increased 2- to 3-fold, and serum transthyretin, which stabilizes RBP4 in the circulation, was increased 35%. Serum RBP4 correlated positively with adipose RBP4 mRNA and intra-abdominal fat mass and inversely with insulin sensitivity, independently of age, gender, and body mass index. RBP4 mRNA correlated inversely with GLUT4 mRNA in Vis fat and positively with adipocyte size in both depots. RBP4 levels are therefore linked to Vis adiposity, and Vis fat may be a major source of RBP4 in insulin-resistant states.

Retinol‐binding protein‐4 attenuates insulin‐induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes

Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.

Acute exercise increases serum retinol binding protein 4 concentrations

Serum retinol binding protein (sRBP4) levels are increased in insulin resistance and type 2 diabetes. RBP4 enhances hepatic glucose output (HGO) in cultured hepatocytes. During acute exercise, increased HGO balances a rise in muscle glucose uptake. We hypothesized that treadmill running in rats might increase sRBP4 concentrations. RBP4 was measured in sera of female SD rats in the sedentary state, after 60 minutes of exercise, or after 60 minutes of rest following exercise. RBP4 mRNA was detected in muscle, albeit at lower levels than in liver or visceral adipose tissue. Relative to the sedentary state, exercise increased sRBP4 concentrations (mean ± SEM, 100 ± 9.7 vs. 130 ± 9.2 AU; p < 0.05) and stimulated an ~4-fold increase of RBP4 mRNA in gastrocnemius muscle which declined toward sedentary levels 60 min after exercise cessation. RBP4 mRNA in liver and adipose tissue remained unchanged. The surprising finding that RBP4 mRNA is expressed in muscle and selectively induced in muscle by exercise suggests the possibility that exercise may stimulate synthesis and secretion of RBP4 from muscle. Therefore, RBP4 may be a novel muscle-secreted signal contributing to fuel homeostasis during exercise. SUPPORT: NIH T32 DK07260-29, DK068626, 43051, 69624