Recent studies have revealed the essential role of retinol binding protein 4 (RBP4) in insulin resistance. However, the impact of RBP4 on aberrant lipogenesis, the common hepatic manifestation in insulin resistance states, and the underlying mechanism remain elusive. The present study was designed to examine the effect of RBP4 on sterol regulatory element-binding protein (SREBP-1) and hepatic lipogenesis. Treatment with human retinol-bound RBP4 (holo-RBP4) significantly induced intracellular triglyceride (TAG) synthesis in HepG2 cells and this effect is retinol-independent. Furthermore, RBP4 treatment enhanced the levels of mature SREBP-1 and its nuclear translocation, thereby increasing the expression of lipogenic genes, including fatty acid synthase (FAS), acetyl coenzyme A carboxylase-1 (ACC-1), and diacylglycerol O-acyltransferase 2 (DGAT-2). Stimulation of HepG2 cells with RBP4 strongly up-regulated the expression of transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator 1β (PGC-1β) at both the messenger RNA (mRNA) and protein levels. The transcriptional activation of PGC-1β is necessary and sufficient for the transcriptional activation of SREBP-1 in response to RBP4. The cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) was identified as the target transcription factor involved in the RBP4-mediated up-regulation of PGC-1β transcription as a result of phosphorylation on Ser133. Furthermore, in vivo RBP4 infusion induced SREBP-1c activation and consequently accelerated hepatic lipogenesis and plasma TAG in C57BL/6J mice, a phenomenon not observed in Ppargc1b knockout mice. Conclusion: These findings reveal a novel mechanism by which RBP4 achieves its effects on hepatic lipid metabolism. (HEPATOLOGY 2013;8:564-575)
Retinol binding protein 4 (RBP4), a protein that belongs to the lipocalin family, was initially known as a specific carrier for the delivery of retinol (vitamin A) in the circulation.[1, 2] It is encoded by the RBP4 gene, localized in chromosome 10q23-q24. Hepatocytes are regarded as the major source of RBP4 secretion under normal conditions; however, adipose tissue expresses a considerable amount of RBP4 and could make a substantial contribution to elevated serum RBP4 levels in insulin-resistant states. RBP4 is identified as a new adipokine suggested to link obesity with its comorbidities, especially insulin resistance, type 2 diabetes (T2D), and certain components of the metabolic syndrome. Serum RBP4 concentrations are elevated in subjects with impaired glucose tolerance, T2D, and correlate inversely with insulin sensitivity in nondiabetic subjects with a family history of T2D. Circulating RBP4 levels correlate with the degree of insulin resistance in these subjects and the relationship is independent of obesity. Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice induced insulin resistance. Conversely, heterozygous or homozygous RBP4 knockout mice had improved insulin sensitivity. Increased serum RBP4 decreased glucose transporter type 4 (Glut4) expression in adipocytes and induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes, thus contributing to the impairment of systemic insulin resistance. Recently, a high circulating level of RBP4 was demonstrated to associate with elevated liver fat accumulation in human studies. Blocking RBP4 expression in the liver was sufficient to reduce lipid droplets and ameliorate high fat diet-induced hepatic steatosis in C57BL/6 mice, which confirmed the potential role of RBP4 in the regulation of lipid metabolism in liver. However, the pathophysiological roles of RBP4 involved in the regulation of hepatic lipid metabolism and the underlying molecular mechanism has not yet been fully characterized.
Sterol regulatory element binding protein (SREBP) is known as a key lipogenic transcription factor controlling the biosynthesis of cholesterol, fatty acids, and triglyceride (TAG).[15, 16] Mammalian genomes have two separate SREBP genes (SREBF1 and SREBF2). SREBP-1 expression produces two different isoforms, SREBP-1a and -1c. These isoforms differ in their first exons due to the use of different transcriptional start sites for the SREBP-1 gene. SREBP-1c was also identified in rats as ADD-1. SREBP-1a and SREBP-1c preferentially stimulate the lipogenic process by activating genes involved in fatty acid and TAG synthesis. SREBP-2 encodes SREBP-2, which mainly controls cholesterol homeostasis by inducing genes required for cholesterol synthesis and uptake.[17, 18] The precursor of these three isoforms of SREBP (P-SREBP) is synthesized as an endoplasmic reticulum (ER) membrane-bound protein, which is transported from the ER to the Golgi and undergoes proteolytic processing to release the transcriptionally active nuclear form. The nuclear form of SREBP (N-SREBP) is translocated into the nucleus, where it binds to sterol regulatory elements (SREs) present in the promoters of target genes and activates the transcription of SREBP-responsive genes involved in lipogenic pathways, such as fatty acid synthase (FAS), acetyl coenzyme A carboxylase-1 (ACC-1), and diacylglycerol O-acyltransferase 2 (DGAT-2). Thus, the dysregulation of SREBP-1 contributes significantly to the pathogenic hepatic biosynthesis of fatty acids and the metabolism of TAG.
In this study we examined the effects of RBP4 on SREBP-1 activation and lipogenesis in vitro and in vivo. Our data reveal that RBP4 activates SREBP-1 through a peroxisome proliferator-activated receptor-γ coactivator 1β (PGC1β)-dependent pathway in HepG2 cells and contributes to increased hepatic lipogenesis in mice. Our findings highlight RBP4 as a potential target for therapeutic intervention in metabolic syndrome-related lipid disorders.
The present study uncovered novel findings that RBP4 promotes lipogenesis in hepatocytes by way of PGC-1β-dependent SREBP-1 activation both in vitro and in vivo. The following data support this conclusion: (1) RBP4 induces SREBP-1 activation by regulating SREBP-1 expression and nuclear active forms. (2) At cellular level, SREBP-1 activation by RBP4 promotes the transcription of target lipogenic genes, thereby stimulating de novo lipogenesis in HepG2 cells. (3) SREBP-1 is a direct target of PGC-1β and RBP4 increases ppargc1b transcription through CREB. (4) RBP4 stimulates the SREBP-1c and its target genes expression, resulting in hepatic triglyceride accumulation and VLDL secretion in C57BL/6J mice but not in Ppargc1b−/− mice. Thus, the induction of PGC-1β-dependent SREBP-1 activation may represent a molecular mechanism by which RBP4 regulates hepatic lipogenesis.
In the present study we found a positive dose-response effect of RBP4 (ranging from 0 to 80 μg/mL) in inducing hepatocyte lipogenesis. The stimulatory effect of RBP4 on lipogenesis was present at all doses, to a maximum at 80 μg/mL RBP4. Although different clinical studies reported different serum or plasma levels of RBP4 due to different measurement procedures and conditions, human plasma RBP4 levels in patients with metabolic syndrome usually range from ranged from ∼20 μg/mL to ∼90 μg/mL. A plasma concentration of 20 μg/mL RBP4 was reported in normal lean humans.[7, 8] Plasma concentrations of 40∼90 μg/mL RBP4 were the typical range documented in patients who were obese and diabetic individuals. Based on these reports, we estimate that our in vitro dose of RBP4 may encompass the clinically relevant range of serum RBP4 concentrations in patients with metabolic syndrome. In addition, this dose of RBP4 is consistent with those in the previous reports.[10, 33]
RBP4′s most well-defined function is to deliver retinol to tissues and since retinol has many important effects on lipid metabolism,[23, 24] it would not have been surprising if the action of RBP4 on hepatic lipid lipogenesis was retinol-dependent. However, we found that apo-RBP4 elicits lipid lipogenesis as robust as that of holo-RBP4 in HepG2 cells. Apo-RBP4 with retinol added back had similar effects to the original holo-RBP4, confirming that the retinol-stripping process did not alter the function of RBP4. Thus, RBP4 can elicit a lipogenic process from hepatocytes in a retinol-independent manner.
It is curious that RBP4 has such a robust effect on HepG2 cells because hepatocytes themselves are an important source of this protein. Actually, hepatocytes are regarded as the major source of RBP4 under normal physiological status; however, adipose tissue may be a major site of RBP4 synthesis in insulin-resistant states. Therefore, adipose tissue may play an endocrine role by increasing the circulating concentrations of RBP4. We thus speculate that RBP4 may affect hepatocyte function in both an autocrine and endocrine manner. In the autocrine model, the RBP secreted by hepatocytes affects hepatocyte function. Future studies are necessary to determine the relative contribution of the secretion of RBP4 from the liver and adipose tissue in the regulation of lipid synthesis in hepatocytes.
SREBPs are known to be important transcription factors and play a central role in lipid homeostasis; however, there is yet no evidence that links SREBP to the lipogenic effect of RBP4. Our present results reveal that in HepG2 cells, stimulation with human recombinant RBP4 did not affect the protein expression of SREBP-1, but rather reduced the nuclear mature form of SREBP-1, thus leading to a potent induction of its target genes as well as lipid accumulation in vitro. However, SREBP-2 predominantly regulates genes controlling cholesterol homeostasis, such as LDLR, HMG coenzyme A reductase, and squalene synthase[36, 37] was not affected in response to RBP4. These results are consistent with previous findings that hepatic overexpression of SREBP-1 induced lipogenesis. SREBP-1a and SREBP-1c mainly regulates the transcription of key enzymes associated with the biosynthesis of fatty acids and the lipogenic process, although in the HepG2 cells, there are more SREBP-1a isoforms than SREBP-1c, but only the SREBP-1c promoter is transcriptionally activated in response to RBP4 treatment. This is demonstrated by our results that the transcription level of SREBP-1c is greatly induced by RBP4 treatment, while SREBP-1a is not significantly changed. Thus, the induced protein levels of SREBP-1 under RBP4 treatment is mainly from induced SREBP-1c, not SREBP-1a. Therefore, SREBP-1c might be the primary isoform of RBP4 action. Deletion analysis of the SREBP-1c promoter further showed that activity of the WT SREBP-1c promoter, but not SRE or the LXRE deletion promoter, was induced by RBP4. This study indicates that RBP4 functions as a potential adipokine that controls SREBP-1c transcriptional activity through autoloop regulation by way of an SRE/LXRE motif-dependent mechanism. Further studies with the use of SREBP-1c knockout mice are necessary to prove this feedforward mechanism. On the contrary, RBP4 exerted less effect on SREBP-2 transcriptional activity, as demonstrated by nuclear SREBP-2-induced autoregulation and the transcription of HMG-CoA and LDLR. These results strengthened the conclusion that the induction of lipogenesis by RBP4 is mainly dependent on SREBP-1c.
We further found that PGC-1β is a critical effector downstream of RBP4, which mediates RBP4 effects on the induction of SREBP-1c and thus promotes hepatic lipogenesis. Originally, PGC-1β was identified as the closest homolog of PGC-1α and a cold-inducible coactivator that interacts with peroxisome proliferator-activated receptor gamma (PPARγ) in brown adipose tissue. PGC-1β is most highly expressed in tissues with high oxidative metabolism, such as brown adipose tissue, cardiac muscle, and skeletal muscle.[38, 39] PGC-1β coactivates the SREBP and LXR families of transcription factors, as it induces a broad program of lipid metabolism, including de novo lipogenesis and lipoprotein secretion.
Subsequent studies identified Ppargc1b as an essential regulator in the lipid synthesis pathway and a transcriptional coactivator of SREBP-1c induced by RBP4. Both loss- and gain-of-function experiments suggest that PGC-1β is involved in transcriptional activation of SREBP-1c in response to RBP4 treatment. The depletion of PGC-1β strongly abolished the inductive effects of RBP4 on lipogenic gene transcription. In contrast, the overexpression of PGC-1β potently enhanced RBP4-mediated lipogenic gene transcription. Thus, PGC-1β is primarily responsible for the lipogenesis effect of RBP4. Furthermore, we provide the novel findings that RBP4 stimulates Ppargc1b expression in HepG2 cells. RBP4 treatment increases PGC-1β mRNA expression in a dose- and time-dependent fashion in hepatocytes. RBP4 treatment was also found to increase PGC-1β protein expression. However, RBP4 had little effect on Ppargc1α, the other isoform of PGC-1. Several pieces of data link PGC-1β with the LXR pathway. PGC-1β coactivates LXR on both a synthetic reporter gene containing multimerized binding elements and an endogenous promoter in an LXR ligand-dependent manner. More important, PGC-1β is recruited to the promoter region of cytochrome P450 7A1 (CYP7A1) and ATP binding cassette A1 (ABCA1) and activates the expression of these LXR target genes. We show here that RBP4 increased the recruitment of PGC-1β to the LXREs of specific SREBP-1c target genes implicated in hepatic lipogenesis, leading to their up-regulation and enhanced de novo TAG synthesis. Thus, LXRE is permissive for lipogenesis by RBP4 in hepatocytes. Although studies in this field have not elucidated how LXR activates the pathways of lipid transport in hepatocytes, the ability of PGC-1β to modulate LXR target gene expression in cultured cells and in vivo suggests that PGC-1β elicits at least a proportion of this hyperlipidemia through the coactivation of LXR. Taken together, it is clear that PGC-1β couples these two important aspects of lipid metabolism in liver, i.e., lipid synthesis by way of the coactivation of the SREBPs and lipoprotein secretion by way of the coactivation of LXR and likely other transcription factors.
Next, we explored the potential underlying mechanism by which RBP4 augments PGC-1β transcription. CREB is a cellular transcription factor that binds to certain DNA sequences called CRE, thereby increasing or decreasing the transcription of downstream genes. Our study implicates the activation of CREB as a mechanism by which RBP4 increases PGC-1β expression. The ChIP assay revealed the direct binding of CRE to a noncanonical CRE motif upstream of the transcription initiation site of PGC-1β. This binding was enhanced by RBP4 treatment. Further studies indicate that CREB Ser133 is the critical target involved in the transcriptional induction of Ppargc1b induced by RBP4.
In conclusion, our findings further suggest that the development of future therapeutic intervention(s) to modulate RBP4 levels may serve to ameliorate hepatic abnormal lipogenesis and lipid accumulation related to obesity and diabetes.