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

Chiefari E, Paonessa F, Iiritano S, Le Pera I, Palmieri D, Brunetti G, Lupo A, Colantuoni V, Foti D, Gulletta E, De Sarro G, Fusco A, Brunetti A - BMC Biol. (2009)

Bottom Line: These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Catanzaro, Italy. echiefari@libero.it

ABSTRACT

Background: We 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.

Results: We 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.

Conclusion: These 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.

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RBP4 gene expression is induced by HMGA1. (Top) Mouse RBP4-Luc reporter vector (2 μg) was transfected into HepG2 and Hepa1 cells plus increasing amounts (0, 0.5, or 1 μg) of HMGA1 expression plasmid. Data represent the means ± standard errors for three separate experiments; values are expressed as factors by which induced activity increased above the level of Luc activity obtained in transfections with RBP4-Luc reporter vector plus the empty expression vector, which is assigned an arbitrary value of 1. (Middle) HMGA1 expression plasmid was transfected into HepG2 and Hepa1cells. After 6 h of transfection, the cells were treated with anti-HMGA1 (100 pmol), siRNA, or a non-targeting control siRNA, and endogenous RBP4 mRNA expression was measured 48 to 96 h later. Western blots of HMGA1 in each condition are shown in the autoradiograms. (Bottom) ChIP of the RBP4 promoter gene in HepG2 and Hepa1 cells, either untreated or pretreated with HMGA1 siRNA. ChIP was done using an anti-HMGA1 specific antibody (Ab).
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Figure 1: RBP4 gene expression is induced by HMGA1. (Top) Mouse RBP4-Luc reporter vector (2 μg) was transfected into HepG2 and Hepa1 cells plus increasing amounts (0, 0.5, or 1 μg) of HMGA1 expression plasmid. Data represent the means ± standard errors for three separate experiments; values are expressed as factors by which induced activity increased above the level of Luc activity obtained in transfections with RBP4-Luc reporter vector plus the empty expression vector, which is assigned an arbitrary value of 1. (Middle) HMGA1 expression plasmid was transfected into HepG2 and Hepa1cells. After 6 h of transfection, the cells were treated with anti-HMGA1 (100 pmol), siRNA, or a non-targeting control siRNA, and endogenous RBP4 mRNA expression was measured 48 to 96 h later. Western blots of HMGA1 in each condition are shown in the autoradiograms. (Bottom) ChIP of the RBP4 promoter gene in HepG2 and Hepa1 cells, either untreated or pretreated with HMGA1 siRNA. ChIP was done using an anti-HMGA1 specific antibody (Ab).

Mentions: We first performed experiments to see whether HMGA1 had a role in activating the mouse RBP4 gene promoter at the transcriptional level. To test this possibility, HepG2 human hepatoma cells and mouse Hepa1 hepatoma cells were cotransfected transiently with mouse RBP4-Luc reporter plasmid plus increasing amounts of the HMGA1 expression vector. As shown in Figure 1, overexpression of HMGA1 considerably increased RBP4-Luc activity in both cell types and this effect occurred in a dose-dependent manner. Consistent with these results, RBP4 mRNA abundance was increased in cells overexpressing HMGA1 and was reduced in cells pretreated with siRNA targeting HMGA1 (Figure 1), indicating that activation of the RBP4 gene requires HMGA1. These data were substantiated by chromatin immunoprecipitation (ChIP) assay, showing that binding of HMGA1 to the endogenous RBP4 locus was increased in whole, intact HepG2 and Hepa1 cells naturally expressing HMGA1, and was decreased in cells exposed to siRNA against HMGA1 (Figure 1). Based on these results, in addition to previous observations indicating that cAMP, or agents which elevate intracellular cAMP, increase RBP transcript levels [16], we were interested to see whether a functional link could be established between cAMP, HMGA1, and RBP4. To this end, we first confirmed and extended the observation made by Jessen and Satre [16] that RBP is induced by cAMP in Hepa1 cells. As measured by Northern blot analysis of total RNA (Figure 2), RBP4 mRNA increased ≈ 5-fold over the basal level in Hepa1 cells treated with 0.5 mM 8-bromo cAMP (Br-cAMP), a standard concentration for c-AMP induction experiments [16]. As shown in Figure 2, RBP4 mRNA levels increased starting at 3 h, peaking at 24 h and then declining, suggesting a transient transcriptional stimulation. To establish whether HMGA1 was required for basal and cAMP-dependent RBP4 transcription, we transfected the HMGA1 expression vector in Hepa1 cells treated or not with Br-cAMP and RBP4 protein levels were analyzed 48 h later by Western blot. As shown in Figure 2, RBP4 protein expression was enhanced in cells overexpressing HMGA1 and even further in cells treated with cAMP, in which an increase in HMGA1 protein expression was simultaneously observed, suggesting that induction of RBP4 by cAMP may occur, at least in part, through activation of endogenous HMGA1 expression. This hypothesis was supported by the fact that RBP4 was reduced in cAMP treated cells in which endogenous levels of HMGA1 were specifically lowered by transfecting cells with HMGA1 antisense expression plasmid (Figure 2). However, further experiments are needed to fully explain the role of cAMP on HMGA1 expression. The functional significance of HMGA1 in RBP4 gene expression was confirmed in transient transcription assays in Hepa1 (and differentiated 3T3-L1, data not shown) cells, in which overexpression of HMGA1 caused an increase in both basal and cAMP-induced Luc activity from the mouse RBP-Luc reporter plasmid (Figure 3). This effect was substantiated in HEK-293 cAMP-responsive cells, a cell line ideally suited for studying the effects of HMGA1 on transcription since it does not express appreciable levels of this protein. As shown in Figure 3, in support of the role that HMGA1 plays in the context of RBP4 gene, the direct effect of cAMP was less effective in promoting RBP4 transcription in HEK-293 cells expressing low levels of HMGA1, becoming considerably higher in cells with forced expression of HMGA1.


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

Chiefari E, Paonessa F, Iiritano S, Le Pera I, Palmieri D, Brunetti G, Lupo A, Colantuoni V, Foti D, Gulletta E, De Sarro G, Fusco A, Brunetti A - BMC Biol. (2009)

RBP4 gene expression is induced by HMGA1. (Top) Mouse RBP4-Luc reporter vector (2 μg) was transfected into HepG2 and Hepa1 cells plus increasing amounts (0, 0.5, or 1 μg) of HMGA1 expression plasmid. Data represent the means ± standard errors for three separate experiments; values are expressed as factors by which induced activity increased above the level of Luc activity obtained in transfections with RBP4-Luc reporter vector plus the empty expression vector, which is assigned an arbitrary value of 1. (Middle) HMGA1 expression plasmid was transfected into HepG2 and Hepa1cells. After 6 h of transfection, the cells were treated with anti-HMGA1 (100 pmol), siRNA, or a non-targeting control siRNA, and endogenous RBP4 mRNA expression was measured 48 to 96 h later. Western blots of HMGA1 in each condition are shown in the autoradiograms. (Bottom) ChIP of the RBP4 promoter gene in HepG2 and Hepa1 cells, either untreated or pretreated with HMGA1 siRNA. ChIP was done using an anti-HMGA1 specific antibody (Ab).
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Figure 1: RBP4 gene expression is induced by HMGA1. (Top) Mouse RBP4-Luc reporter vector (2 μg) was transfected into HepG2 and Hepa1 cells plus increasing amounts (0, 0.5, or 1 μg) of HMGA1 expression plasmid. Data represent the means ± standard errors for three separate experiments; values are expressed as factors by which induced activity increased above the level of Luc activity obtained in transfections with RBP4-Luc reporter vector plus the empty expression vector, which is assigned an arbitrary value of 1. (Middle) HMGA1 expression plasmid was transfected into HepG2 and Hepa1cells. After 6 h of transfection, the cells were treated with anti-HMGA1 (100 pmol), siRNA, or a non-targeting control siRNA, and endogenous RBP4 mRNA expression was measured 48 to 96 h later. Western blots of HMGA1 in each condition are shown in the autoradiograms. (Bottom) ChIP of the RBP4 promoter gene in HepG2 and Hepa1 cells, either untreated or pretreated with HMGA1 siRNA. ChIP was done using an anti-HMGA1 specific antibody (Ab).
Mentions: We first performed experiments to see whether HMGA1 had a role in activating the mouse RBP4 gene promoter at the transcriptional level. To test this possibility, HepG2 human hepatoma cells and mouse Hepa1 hepatoma cells were cotransfected transiently with mouse RBP4-Luc reporter plasmid plus increasing amounts of the HMGA1 expression vector. As shown in Figure 1, overexpression of HMGA1 considerably increased RBP4-Luc activity in both cell types and this effect occurred in a dose-dependent manner. Consistent with these results, RBP4 mRNA abundance was increased in cells overexpressing HMGA1 and was reduced in cells pretreated with siRNA targeting HMGA1 (Figure 1), indicating that activation of the RBP4 gene requires HMGA1. These data were substantiated by chromatin immunoprecipitation (ChIP) assay, showing that binding of HMGA1 to the endogenous RBP4 locus was increased in whole, intact HepG2 and Hepa1 cells naturally expressing HMGA1, and was decreased in cells exposed to siRNA against HMGA1 (Figure 1). Based on these results, in addition to previous observations indicating that cAMP, or agents which elevate intracellular cAMP, increase RBP transcript levels [16], we were interested to see whether a functional link could be established between cAMP, HMGA1, and RBP4. To this end, we first confirmed and extended the observation made by Jessen and Satre [16] that RBP is induced by cAMP in Hepa1 cells. As measured by Northern blot analysis of total RNA (Figure 2), RBP4 mRNA increased ≈ 5-fold over the basal level in Hepa1 cells treated with 0.5 mM 8-bromo cAMP (Br-cAMP), a standard concentration for c-AMP induction experiments [16]. As shown in Figure 2, RBP4 mRNA levels increased starting at 3 h, peaking at 24 h and then declining, suggesting a transient transcriptional stimulation. To establish whether HMGA1 was required for basal and cAMP-dependent RBP4 transcription, we transfected the HMGA1 expression vector in Hepa1 cells treated or not with Br-cAMP and RBP4 protein levels were analyzed 48 h later by Western blot. As shown in Figure 2, RBP4 protein expression was enhanced in cells overexpressing HMGA1 and even further in cells treated with cAMP, in which an increase in HMGA1 protein expression was simultaneously observed, suggesting that induction of RBP4 by cAMP may occur, at least in part, through activation of endogenous HMGA1 expression. This hypothesis was supported by the fact that RBP4 was reduced in cAMP treated cells in which endogenous levels of HMGA1 were specifically lowered by transfecting cells with HMGA1 antisense expression plasmid (Figure 2). However, further experiments are needed to fully explain the role of cAMP on HMGA1 expression. The functional significance of HMGA1 in RBP4 gene expression was confirmed in transient transcription assays in Hepa1 (and differentiated 3T3-L1, data not shown) cells, in which overexpression of HMGA1 caused an increase in both basal and cAMP-induced Luc activity from the mouse RBP-Luc reporter plasmid (Figure 3). This effect was substantiated in HEK-293 cAMP-responsive cells, a cell line ideally suited for studying the effects of HMGA1 on transcription since it does not express appreciable levels of this protein. As shown in Figure 3, in support of the role that HMGA1 plays in the context of RBP4 gene, the direct effect of cAMP was less effective in promoting RBP4 transcription in HEK-293 cells expressing low levels of HMGA1, becoming considerably higher in cells with forced expression of HMGA1.

Bottom Line: These 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Catanzaro, Italy. echiefari@libero.it

ABSTRACT

Background: We 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.

Results: We 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus