Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response element-driven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin- and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.
Introduction
Glucose homeostasis is regulated by the opposing actions of insulin and glucagon (,
2
, 3
), and glucose production in the liver is controlled primarily by gluconeogenesis (4
). The regulation of hepatic gluconeogenesis involves the transcriptional regulation of key metabolic enzymes, including PEPCK 6
and G6Pase. The gluconeogenic program is largely regulated at the level of transcription and the process is coordinated by CREB via its direct binding to the cAMP-response element (CRE) site on the promoter of PEPCK, G6Pase, or PGC-1α (PPARγ coactivator-1α) (The abbreviations used are: PEPCK
phosphoenolpyruvate caboxykinase
G6Paseglucose-6-phosphatase
AMPKAMP-activated protein kinase
SHPsmall heterodimer partner
CREBcAMP-responsive element-binding protein
Adadenovirus
CREcAMP-response element
CREBCRE-binding protein
CBPCREB-binding protein
ERendoplasmic reticulum
PKAprotein kinase A
CA-AMPKconstitutively active AMPK
AICAR5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside
siSHPsiRNA specific to mouse SHP
siCRTC2siRNA specific to mouse CRTC2.
5
).Metformin has been shown to activate AMP-activated protein kinase (AMPK) via an LKB1-dependent mechanism (
6
). AMPK is a serine/threonine kinase that functions as an intracellular energy sensor and has been implicated in the modulation of glucose and fatty acid metabolism (7
). AMPK is activated by physiological stimuli, including exercise, muscle contraction, and hormones, such as adiponectin and leptin, as well as by physiological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock conditions (8
, 9
). In the liver, activation of AMPK suppresses hepatic gluconeogenesis acutely by direct phosphorylation of its substrates, including CREB-binding protein (CBP) (10
), CRTC2 (11
), and GSK3β (glycogen synthase kinase 3β) (12
). Recent studies also suggest that AMPK induces SHP gene expression and inhibits hepatic gluconeogenic gene expression in animal models (13
, 14
).Orphan nuclear receptor SHP (NR0B2) lacks a typical nuclear receptor DNA-binding domain and is expressed predominantly in the liver, whereas nominal expression is also detected in the heart, lung, pancreas, spleen, kidney, smooth muscle, testis, and ovary (
15
, 16
). SHP is a transcriptional repressor of a number of nuclear receptors and transcription factors, including estrogen receptor (ER) (17
, 18
), ER-related receptor (19
), glucocorticoid receptor (20
), androgen receptor (21
), forkhead transcription factor FoxA2 (HNF3) (22
), HNF4 (hepatocyte nuclear factor 4) (23
), HNF6 (hepatocyte nuclear factor 6) (24
), CCAAT/enhancer-binding protein α (25
), and BETA2/NeuroD (26
). SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan nuclear receptors SF-1, LRH-1, ER-related receptor γ, and the bile acid receptor FXR (16
). SHP plays a crucial role in regulating glucose metabolism (13
, 27
). Mutations in the SHP gene are associated with mild obesity in Japanese subjects (28
). Metformin, hepatocyte growth factor, and sodium arsenite increase SHP gene expression and inhibit the PEPCK and G6Pase gene expression; fenofibrate inhibits PAI-1 expression through induction of SHP (13
, 14
, 29
, 30
).The basic leucine zipper (bZIP) protein CREB binds to CREs that contain the 5′-TGACGTCA-3′ consensus motif and activate the transcription of CRE-bearing genes, such as G6Pase and PEPCK (
31
). CREB coactivator CRTC2 significantly contributes to the CRE-dependent transcriptional activation of hepatic gluconeogenesis (11
). Under feeding or in the presence of insulin, CRTC2 is located in the cytoplasm via its phosphorylation at Ser171 by members of the AMPK family of Ser/Thr kinase, including AMPK and SIK1 (salt-inducible kinase 1) (11
, 32
). Fasting triggers activation of cAMP-dependent protein kinase (PKA) to promote dephosphorylation and nuclear entry of CRTC2, which results in the increased occupancy of CRTC2 over promoters of PEPCK, G6Pase, or PGC-1α and activation of the entire gluconeogenic program in mouse liver or in rat primary hepatocytes (33
, 34
). Indeed, knock-out of CRTC2 decreases circulating glucose concentrations during fasting, due to the attenuation of the gluconeogenic gene expression (35
).In this study, we have demonstrated that AMPK inhibits phosphorylation-defective mutant CRTC2 (S171A)-dependent hepatic gluconeogenesis. SHP inhibits CRE promoter activity by direct interaction with CREB, thus inhibiting the recruitment of CRTC2 on the chromatin. Metformin or constitutively active AMPK inhibits not only WT CRTC2 but also S171A-dependent activation of hepatic gluconeogenic genes, whereas knockdown of SHP negated these effects, suggesting that AMPK could regulate CREB·CRTC2-dependent gluconeogenesis via an alternative mechanism by activating transcription of SHP. Taken together, our result suggests that SHP is an important contributor of AMPK-dependent suppression of CREB·CRTC2-mediated hepatic gluconeogenic gene expression.
DISCUSSION
In this study, we found that the inhibitory effect of chronic metformin treatment on CREB-mediated hepatic gluconeogenic enzyme gene expression and hepatic glucose production was mediated by induction of SHP. SHP decreases CREB-dependent induction of gluconeogenic gene expression and hepatic glucose production via direct interaction with CREB. We suggest that the acute response of metformin-mediated activation of AMPK inhibits CREB-dependent hepatic gluconeogenesis through CRCT2 phosphorylation, whereas chronic treatment of metformin inhibited CREB-dependent hepatic gluconeogenesis through induction of SHP gene expression.
AMPK suppresses CREB-mediated hepatic gluconeogenesis by regulation of CREB-binding protein (
10
) and CRTC2 (11
). Moreover, it has been previously reported that PXR represses glucagon-activated transcription of the G6Pase gene by directly binding to CREB (39
), and direct inactivation of GSK3β also inhibits transcriptional activity of CREB through AMPK-induced phosphorylation of GSK3β (12
). In this study, we demonstrated a novel mechanism by which the AMPK signaling pathway inhibits CREB-mediated hepatic gluconeogenic gene expression. Our results demonstrated that SHP directly interacted with CREB (Fig. 4) and inhibited CRTC2-mediated CREB transcriptional activity (Fig. 2). These results suggest that SHP might inhibit the activities of both CREB and CRTC2.In obese and diabetic db/db mouse liver, a higher expression level of CRTC2 protein was observed due to the defects in refeeding-dependent mechanisms for CRTC2 phosphorylation and degradation (
33
). CRTC2 null mice displayed low circulating blood glucose concentrations and improved insulin sensitivity in the context of diet-induced obesity, suggesting that regulation of CREB·CRTC2 activity is critical in the maintenance of glucose homeostasis (35
). Therefore, we suggest that SHP may inhibit CRTC2-induced hepatic gluconeogenic gene expression and blood glucose concentrations in insulin-resistant conditions and may improve CRTC2-mediated insulin resistance via increasing insulin sensitivity.CBP/p300 is required for the recruitment of CRTC2 following exposure to cAMP, and CRTC2 promotes CBP recruitment to the CREB target gene, indicating that the CRTC2·CBP complex has reciprocal effects on the recruitment of both proteins to CREB target gene promoter (
40
). Previous studies reported that AMPK regulated hepatic gluconeogenesis through phosphorylation of CBP (10
), and SHP repressed the expression of gluconeogenic enzyme genes through the dissociation of FOXO1 or HNF4 from CBP (41
). In this study, we demonstrated that SHP competed with CREB transcriptional coactivator CRTC2 to inhibit the CREB·CRTC2 complex (FIGURE 5, FIGURE 6). These results suggest that SHP plays an additional role in suppression of CREB-dependent hepatic gluconeogenesis via co-factor competition. However, we do not rule out the possibility that SHP competed with CBP. We have found that recruitment of CRTC2 onto the PEPCK promoter was significantly inhibited by SHP without affecting CREB recruitment (Fig. 7). This phenomenon is similar to the previous observation that CRTC2 recruitment does not appear to modulate CREB DNA binding activity but rather enhances the interaction of CREB with the TAF-(II)-130 component of TFIID following its recruitment to the promoter (42
). Therefore, our study suggests the possibility that SHP may inhibit the CREB·CBP·CRTC2 complex via inhibition of stable CBP occupancy over the promoter and prevent recruitment of other transcription components via a cofactor competition mechanism.CRTC2 was shown to be phosphorylated at Ser171 by AMPK agonist AICAR and relocated from the nucleus to the cytoplasm even in the presence of FSK (
11
). Our previous studies have demonstrated that AMPK elevated SHP gene expression via USF-1 (upstream stimulatory factor) (29
). AMPK-mediated suppression of hepatic gluconeogenic genes is likely to be mediated quickly by CRTC2 phosphorylation. Here, we explored the role of the delayed effect of metformin-induced AMPK to inhibit hepatic gluconeogenesis via SHP. In this study, chronic treatment of AMPK activator, metformin, and overexpression of AMPK suppress phosphorylation-defective CRTC2 (S171A)-mediated hepatic gluconeogenic enzyme gene expression and hepatic glucose production by induction of SHP gene expression, and this effect was totally abolished by depletion of SHP using Ad-siRNA. Consistent with these results, prolonged provision of AICAR also decreased PEPCK protein levels through AMPK-mediated SHP gene expression. These results suggest that metformin- and AICAR-mediated chronic activation of AMPK disrupts CREB-dependent hepatic gluconeogenesis via induction of SHP gene expression. Mouse models of hepatic insulin resistance display increased dephosphorylation and nuclear accumulation of CRTC2, but metformin is still effective in treating the hyperglycemia in these conditions. Therefore, we suggest that SHP can be a potential regulator of metformin-mediated anti-diabetic effects. However, a more detailed study and elucidation of SHP is required in animal models, like SHP knock-out mice as well as insulin-resistant rodent models.In conclusion, we suggest that metformin regulates the CREB-mediated hepatic gluconeogenic process by both CRTC2 phosphorylation and direct inhibition of CREB via modulation of SHP expression. Metformin-dependent activation of AMPK could acutely phosphorylate CRTC2 at Ser171 and leads to its nuclear exclusion. Chronic treatment of metformin antagonizes the stimulatory effects of CREB·CRTC2 complex-mediated hepatic gluconeogenesis through induction of SHP expression, suggesting that SHP has an important role in delayed metformin action (Fig. 9). Our study provides a novel insight into the dual molecular mechanisms by which AMPK affects CREB-mediated glucose metabolism. Further studies are required to elucidate the process by which other metabolic regulators and inducers of SHP gene may improve CREB-mediated hyperglycemia and provide therapeutic benefit for treatment of insulin resistance conditions prevalent in diabetes.
FIGURE 9Schematic diagram of SHP-mediated inhibition of CREB·CRTC2 complex-dependent gluconeogenesis. Early response (left) of metformin phosphorylates CRTC2 at Ser171 and leads to its nuclear exclusion through AMPK activation. On the other hand, delayed response (right) of metformin antagonizes the stimulatory effects of CREB·CRTC2 complex-mediated gluconeogenesis through induction of SHP expression. SHP directly interacts with CREB, which subsequently inhibits CREB-dependent gluconeogenic enzyme gene expression via competition with CRTC2.
Supplementary Material
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Article Info
Publication History
Published online: August 05, 2010
Received in revised form:
August 4,
2010
Received:
April 15,
2010
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© 2010 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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