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0Cell Reports
Article
PCAF Improves Glucose Homeostasis by Suppressing the Gluconeogenic Activity of PGC-1 a
Graphical Abstract
Authors
Cheng Sun, Meihong Wang.....Fei Ding,
Xiaosong Gu
Correspondence suncheng1975@ntu.edu.cn (C.S.), nervegu@ntu.edu.cn (X.G.)
In Brief
The ectopic gluconeogenic activity of PGC-1 a is a causative factor for hyperglycemia. Sun et al. show that PCAF acetylates PGC-1 a and thus triggers its proteasomal degradation. By downregulating PGC-1 a activity, PCAF improves glucose homeostasis and insulin sensitivity in the obese mice.
Highlights
• PCAF inversely correlates with PGC-1 a in fasted and diabetic states
• PCAF acetylates PGC-1 a at K328 and K450 and triggers proteasomal degradation
• PCAF overexpression improves glucose homeostasis and insulin sensitivity
• PCAF knockdown stimulates hepatic gluconeogenesis
Sun et al., 2014, Cell Reports 9, 2250-2262 ciossMark December 24, 2014 ©2014 The Authors
http://dx.d0i.0rg/l 0.1016/j.celrep.2014.11.029
CelPress
Cell Reports
Article
PCAF Improves Glucose Homeostasis by Suppressing the Gluconeogenic Activity of PGC-1a
Cheng Sun,12 * Meihong Wang,12 Xiaoyu Liu,12 Lan Luo,3 Kaixuan Li,3 Shuqiang Zhang,12 Yongjun Wang,12 Yumin Yang,12 Fei Ding,12 and Xiaosong Gu12 *
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PRC 2Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu 226001, PRC 3Department of Geratology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, PRC 'Correspondence: suncheng1975@ntu.edu.cn (C.S.), nervegu@ntu.edu.cn (X.G.) http://dx.doi.org/10.10167j.celrep.2014.11.029
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd73.07).
SUMMARY
PGC-1 a plays a central role in hepatic gluconeogen-esis and has been implicated in the onset of type 2 diabetes. Acetylation is an important posttransla-tional modification for regulating the transcriptional activity of PGC-1 a. Here, we show that PCAF is a pivotal acetyltransferase for acetylating PGC-1 a in both fasted and diabetic states. PCAF acetylates two lysine residues K328 and K450 in PGC-1 a, which subsequently triggers its proteasomal degradation and suppresses its transcriptional activity. Adenoviral-mediated expression of PCAF in the obese mouse liver greatly represses gluconeogenic enzyme activation and glucose production and improves glucose homeostasis and insulin sensitivity. Moreover, liver-specific knockdown of PCAF stimulates PGC-1a activity, resulting in an increase in blood glucose and hepatic glucose output. Our results suggest that PCAF might be a potential pharmacological target for developing agents against metabolic disorders associated with hyperglycemia, such as obesity and diabetes.
INTRODUCTION
Obesity and the associated disorder type 2 diabetes have become leading causes of adult morbidity and mortality worldwide (Saltiel, 2001; Schenk et al., 2008). Hyperglycemia is a hallmark of severe obesity and type 2 diabetes (Bornfeldt and Tabas, 2011; Zimmet et al., 2001). Excessive hepatic glucose production (HGP) is a major contributor for fasting hyperglyce-mia in diabetes (Saltiel, 2001; Shulman, 2000). Hepatic gluco-neogenesis mainly accounts for HGP in diabetes, which is largely controlled by a set of transcriptional factors including CREB, FoxO1, C/EBPs, HNF4a, and GR (Herzig et al., 2001; Lin et al., 2004; Puigserver et al., 2003; Yoon et al., 2001). PGC-1a was initially identified as a cold-inducible coactivator for PPARg in brown fat (Puigserver et al., 1998). Subsequently, accumulating evidence reveals that PGC-1a regulates several metabolic processes including mitochondrial biogenesis and respiration, muscle-fiber-type switching, and fatty acid synthesis (Finck
et al., 2006; Lehman et al., 2000; Lin et al., 2002; Puigserver et al., 2003; Wu et al., 1999). Additionally, adenoviral-mediated expression of PGC-1a in the hepatocytes strongly activates the entire program of gluconeogenesis and increased glucose output (Yoon et al., 2001), leading to a conclusion that PGC-1a is a key transcriptional coactivator in hepatic gluconeogenesis. Notably, PGC-1 a modulates the gluconeogenic pathway in fasting and diabetic states. For example, PGC-1 a mRNA level in the liver is rapidly induced following short-term fasting (Yoon et al., 2001). Likewise, hepatic PGC-1 a gene expression is robustly increased in the diabetic mice (Yoon et al., 2001). Furthermore, specific knockout of PGC-1 a in mouse liver results in abnormal HGP and eventually leads to a reduction in blood glucose level (Handschin et al., 2005). Liver-specific knockdown of PGC-1 a in the diabetic mice is sufficient to normalize blood glucose levels and to improve systemic glucose homeostasis (Koo et al., 2004). Therefore, manipulating the hepatic PGC-1 a transcriptional activity is an effective strategy for treating metabolic disorders in which hepatic glucose output is dysregulated.
Acetylation, an important posttranslational modification (PTM), is involved in multiple cellular events including growth, proliferation, differentiation, survival, and apoptosis (Sadoul et al., 2008). Histone acetyltransferases (HATs) are the enzymes catalyzing protein acetylation, whereas histone deacetylases (HDACs) are responsible for protein deacetylation (Roth et al., 2001). HATs and HDACs govern gene silence or activation by catalyzing the acetylation or deacetylation of histones, respectively. In addition to posttranslationally modifying histones, HATs and HDACs can also modify nonhistone proteins, such as p27, Skp2, and FoxO1 (Inuzuka et al., 2012; Motta et al., 2004; Perez-Luna et al., 2012). PGC-1 a was recently found to be acetylated and deacetylated, respectively, byGCN5andSIRT1 for regulating its transcriptional activity (Lerin et al., 2006; Rodgers et al., 2005). In particular, PGC-1 a acetylation by GCN5 attenuates its activity, whereas its deacetylation by SIRT1 enhances its activity. Multiple lysine residues in PGC-1 a were identified as the deacetylation sites by SIRT1 (Rodgers et al., 2005). In contrast, the potential acetylation sites in PGC-1 a by GCN5 or other HATs remain yet to be characterized (Lerin et al., 2006). Moreover, whether and which HAT is the key acetyltransferase responsible for the upregulation of PGC-1 a gluconeogenic activity in fasting and diabetic states are still not understood well.
Herein, our results show that PCAF is a pivotal HAT that acetylates PGC-1 a in both fasting and diabetic states. PCAF
Figure 1. PCAF Reversely Correlates and Physically Interacts with PGC-1 a
(A) Protein levels of acetylated PGC-1 a, PCAF, GCN5, CBP, and p300 In the lean and diet-Induced obese (DIO) mouse livers.
(B) Densitometrie quantification of the immunoblot data in (A).
(C) Protein levels of acetylated PGC-1 a, PCAF, GCN5, CBP, and p300 in the wild-type (WT) and ob/ob mouse livers.
(D) Densitometric quantification of the immunoblot data in (C).
(E) Physical interaction between PCAF and PGC-1 a. The HEK293 cells were transfected with the plasmids expressing PCAF (left) or PGC-1 a (right). IP was performed using antibodies indicated.
(F) Physical interaction between CBP and PGC-1 a. The HEK293 cells were transfected with the plasmids expressing CBP (left) or PGC-1 a (right). IP was performed using antibodies indicated.
IgG, immunoglobulin G; IP, immunoprecipitation; WCL, whole cell lysate. *p < 0.05; **p < 0.01 versus the lean or WT mice. All values represent mean ± SEM.
acetylates K328 and K450 residues in PGC-1 a, leading to its proteasomal degradation. Adenoviral-mediated expression of PCAF improves glucose homeostasis in the obese mice by attenuating PGC-1 a-driven hepatic gluconeogenesis. Liver-specific knockdown of PCAF enhances the transcriptional activity of PGC-1 a and stimulates hepatic gluconeogenesis. Together, our data suggest that PCAF might be a pharmacological target for developing agents against hyperglycemia-associ-ated disorders such as obesity, insulin resistance, and diabetes.
RESULTS
PCAF Reversely Correlates and Physically Interacts with PGC-1 a
It has been reported that PGC-1 a acetylation suppresses its gluconeogenic activity (Lerin et al., 2006). To elucidate whether
PGC-1 a acetylation contributes to hyperglycemia in vivo, we compared hepatic acetylated PGC-1 a levels between the lean and obese mice. As shown in Figure 1A, PGC-1 a acetylation levels (Ac-K) were mildly decreased in the diet-induced obese (DIO) mouse livers, whereas total PGC-1 a protein levels were markedly increased. As a consequence, Ac-K/PGC-1a is significantly reduced in the obese mice (Figure 1B). To identify which acetyltransferase is responsible for this reduction, several acetyltransferase protein levels were measured. The results show that PCAF and CBP protein levels are decreased in the DIO mice, whereas GCN5 and p300 are not altered (Figures 1A and 1B). Furthermore, we also measured these proteins levels in the ob/ob mice. Similarly, the Ac-K/PGC-1a is significantly decreased in the ob/ob mice (Figures 1C and 1D). PCAF and CBP were dramatically reduced in the ob/ob mice, whereas no significant changes in GCN5 and p300 were observed (Figures
Figure 2. PCAF Acetylates PGC-1 a and Triggers Its Proteasomal Degradation
(A) PCAF acetylates PGC-1 a in a histone acetyltransferase (HAT) activity-dependent manner.
(B and C) Adenovirus-mediated knockdown of PCAF. MEFs were infected with adenovirus expressing shRNAs against Pcaf. PCAF mRNA (B) and protein levels
(C) were measured.
(D) PCAF knockdown decreases PGC-1 a acetylation in the MEFs.
(E) The decrease in PGC-1 a protein level induced by PCAF in the HEK293 cells requires PCAF acetyltransferase activity.
(F) PGC-1 a mRNA levels were not altered by PCAF in the HEK293 cells.
(G) PCAF promotes PGC-1 a protein degradation in the HEK293 cells. CHX, cycloheximide.
(H) Densitometric quantification of the immunoblot data as shown in (G).
(I) PCAF-mediated degradation in PGC-1 a via proteasome pathway. HEK293 cells were transfected with plasmids as indicated, and the cells were treated with 10 mM MG132.
*p < 0.05; **p < 0.01. All values represent mean ± SEM. See also Figure S1.
1C and 1D). These data imply that the decrease in Ac-K/PGC-1 a in DIO or ob/ob mouse liver might result from the reductions in PCAF or CBP. Coimmunoprecipitation (colP) experiments show that PGC-1 a interacts with PCAF, but not CBP (Figures 1E and 1F). These data strongly indicate that PCAF acetyltrans-ferase is responsible for the reduction of PGC-1 a acetylation in the obese mouse livers.
PCAF Acetylates PGC-1 a and Promotes Its Degradation
We next investigated whether PCAF directly acetylates PGC-1 a. As shown in Figure 2A, PCAF dramatically enhances PGC-1 a acetylation whereas inactive PCAF (PCAF DHAT) does not. We then investigated whether PCAF downregulation affects PGC-1 a acetylation. mRNA levels of PCAF were significantly reduced by Ad-PCAF small hairpin RNA (shRNA) no. 2 and
no. 3 in the mouse embryonic fibroblasts (MEFs) (Figure 2B). Consistent with the changes in mRNA levels, PCAF protein levels were noticeably reduced by Ad-PCAF shRNA no. 2 and no. 3 (Figure 2C). Of these adenoviruses, Ad-PCAF shRNA no. 2 exhibits the best effect; hence, we chose Ad-PCAF shRNA no. 2 for the subsequent experiments. PGC-1 a acetylation was gradually reduced along with the decrease in PCAF (Figure 2D). These data clearly show that PCAF directly acetylates PGC-1 a.
Previous studies show that acetylation affects the transcrip-tional activity of PGC-1 a (Lerin et al., 2006; Rodgers et al., 2005). However, the underlying molecular mechanism is unclear. Here, we noticed that PGC-1 a protein levels were significantly reduced or enhanced by PCAF overexpression or knockdown, respectively (Figures 2A and 2D). To confirm these effects, we ectopically expressed PGC-1 a together with PCAF or inactive
PCAF in the human embryonic kidney 293 (HEK293) cells. PGC-1 a protein levels were reduced continuously as PCAF increases, whereas no significant alterations were occurred by inactive PCAF (Figure 2E). Next, we aim to explore the molecular mechanism responsible for PGC-1 a reduction by PCAF. We first measured the effect of PCAF on PGC-1 a mRNA level but found that PCAF does not affect its mRNA level (Figure 2F). We then examined whether PCAF decreases PGC-1 a expression at protein level. Pulse-chase experiments show that PGC-1 a protein was gradually degraded with cycloheximide (CHX) treatment. Interestingly, PCAF further accelerates the degradation of PGC-1a protein (Figures 2G and 2H).The decrease in PGC-1 a induced by PCAF was almost completely restored by MG132 (Figure 2I). Immunostaining and colP experiments showed that PCAF and PGC-1 a colocalize in the nucleus, the primary site for acetylation (Figures S1A and S1B). These data indicate that PGC-1 a acetylation by PCAF triggers its proteasomal degradation.
PCAF Acetylates PGC-1 a at Lysine Residues K328 and K450
To identify the site(s) in PGC-1 a targeted by PCAF acetyltrans-ferase for acetylation, we cotransfected the HEK293 cells with the plasmids expressing PGC-1 a and PCAF. PGC-1 a was purified by immunoprecipitation for subsequent tandem mass spectrometry analysis. The results showed that two lysine residues K328 and K450 in PGC-1 a were acetylated (Figures 3A and 3B). These two sites are highly conserved among different species (Figure 3C). We next examined whether these site mutations affect PCAF-mediated acetylation on PGC-1 a. The HEK293 cells were transfected with PCAF along with wild-type or mutated PGC-1 a. The cells were treated with MG132 to inhibit PGC-1 a degradation, which will facilitate IP experiments. PGC-1 a with either K328R or K450R single-site mutation shows decreased acetylation level by PCAF compared with the wild-type PGC-1 a (Figure 3D). Double mutations (K328R/ K450R) almost completely abolish the acetylation of PGC-1 a by PCAF. PCAF reduces the protein levels of PGC-1 a, PGC-1 aK328R, and PGC-1 aK450R but shows no reduction effect on PGC-1 aK328R/K450R (Figure 3E). Furthermore, the degradation
of PGC-1 aK328R/K450R induced by PCAF was significantly attenuated (Figures 3F and 3G). These data clearly show that K328 and K450 in PGC-1 a are the target acetylation sites by PCAF and replacing both lysines with arginines would abolish the degradation of PGC-1 a induced by PCAF.
Acetylation by PCAF Attenuates PGC-1 a Gluconeogenic Activity
We next investigated whether PGC-1 a acetylation by PCAF affects its hepatic gluconeogenic activity. We transfected the primary mouse hepatocytes with a luciferase reporter driven by PEPCK or G6Pase promoter together with PGC-1 a and PCAF. PEPCK transcription was robustly stimulated by PGC-1 a, and this stimulation was markedly attenuated by PCAF (Figure 4A). However, PCAF has no effect on PEPCK transcription activation stimulated by PGC-1 aK328R/K450R. Similar results were also observed using a luciferase reporter driven by G6Pase promoter (Figure 4A). Consistent with the luciferase assay, PCAF blocks the increases in PEPCK and G6Pase induced by PGC-1 a.
However, PCAF fails to downregulate PEPCK and G6Pase induced by PGC-1 aK328R/K450R (Figure 4B). Furthermore, we investigated the effects of PCAF on PEPCK and G6Pase mRNA levels. Pck1 expression was significantly enhanced by PGC-1 a, and this enhancement was blocked by PCAF, whereas inactive PCAF has no such an effect (Figure 4C). Similar results in G6pc expression were observed. The protein levels of PEPCK and G6Pase were altered accordingly by PCAF and inactive PCAF (Figure 4D). PCAF reduces the glucose production stimulated by PGC-1 a, and this effect requires acetyltransferase activity of PCAF (Figure 4E).
To ascertain whether PCAF affects the recruitment of PGC-1 a to gluconeogenic gene promoters, we ectopically expressed PCAF in the H4IIE cells and the cells were treated with MG132 to inhibit PGC-1 a degradation. Forskolin/3-isobutyl-1-methyl-xanthine (FSK/IMX) was used to stimulate hepatic gluconeogen-esis. Chromatin immunoprecipitation (ChIP) analysis showed that the recruitment of PGC-1 a to Pck1 and G6pc promoters was increased by FSK/IMX, whereas PCAF overexpression has no effect on this recruitment (Figures S2A and S2B). It has been reported that GCN5 could lead to PGC-1 a redistribution in a nuclear punctuate pattern (Lerin et al., 2006), raising a prediction that GCN5, as well as PCAF, might directly affect the coactivity of PGC-1 a on gluconeogenic gene expression. To test this hypothesis, we transfected the H4IIE cells with GCN5 or PCAF and ChIP experiments were performed. Indeed, the recruitment of GCN5 to Pck1 and G6pc promoters was significantly impaired in the FSK/IMX-treated cells (Figure S2C). However, no obvious alterations were observed with regard to PCAF (Figure S2D). These data indicate that PGC-1 a acetylation by PCAF attenuates its hepatic gluconeogenic activity without affecting the recruitment of PGC-1 a to Pck1 and G6pc promoters.
PCAF Improves Glucose Homeostasis by Repressing PGC-1 a Activity
It has been shown that PGC-1 a activity was regulated by nutritional status (Rodgers et al., 2005; Yoon et al., 2001). To elucidate whether PCAF is involved in this process, mice were placed under control, fasted, and refed conditions. The mRNA levels of PGC-1 a, PEPCK, and G6Pase in the lean mouse liver were dramatically stimulated by fasting and restored to nearly control levels upon refeeding, whereas no significant changes in PCAF mRNA levels were observed (Figure 5A). For the obese mice, the mRNA levels of PGC-1 a and PEPCK were not altered by fasting and G6pc expression was even decreased (Figure 5B). Pcaf expression was not altered as well. Similar to the mRNA levels, the protein levels of PGC-1 a, as well as its downstream targets PEPCK and G6Pase, were stimulated upon fasting. These protein levels were restored to the control levels under refed state (Figures 5C and 5D). Interestingly, the PCAF level decreased upon fasting and recovered upon refeeding. In the obese mice, no significant changes in PCAF, PGC-1 a, PEPCK, and G6Pase were observed (Figures 5E and 5F). These data indicate that PCAF might act as a negative regulator of PGC-1 a in response to different nutritional status.
Therefore, PCAF likely improves glucose homeostasis by repressing PGC-1 a activity. To verify this possibility, we increased
Figure 3. PCAF Acetylates PGC-1 a at K328 and K450
(A and B) Identification of acetylation sites in PGC-1 a by tandem mass spectrometry.
(C) Sequence alignment of the putative acetylation sites of K328 and K450 in PGC-1 a from different species.
(D) Mutations of K328 and K450 blocks PCAF-mediated acetylation modification on PGC-1 a.
(E) Effects of PCAF on wild-type and mutated PGC-1 a protein levels.
(F) Protein stability analysis of wild-type and mutated PGC-1 a.
(G) Densitometric quantification of the immunoblot data as shown in (F). Results are expressed as percentage relative to levels observed at time 0. **p < 0.01; ***p < 0.001 versus the cells transfected with PGC-1 a. All values represent mean ± SEM.
PCAF expression in the obese mouse liver by tail vein injection with Ad-PCAF. Blood glucose level was decreased by PCAF (Figure 5G), and plasma insulin was reduced as well (Figure 5H).
Almost 8-fold increase in Pcaf expression was observed in the Ad-PCAF-infected mouse liver (Figure 5I). Pck1 and G6pc were decreased significantly by PCAF, whereas PGC-1 a mRNA level
Figure 4. PCAF Represses PGC-1 a Gluconeogenic Activity
(A) Acetylation site mutation diminishes PCAF-mediated repression on PGC-1 a activity. The HEK293 cells were transfected with the indicated plasmlds together with PEPCK or G6Pase reporter luciferase construct. Luciferase activity was measured 24 hr after transfection.
(B) Mutations of K328 and K450 blocks PCAF-mediated repression of PGC-1 a transcriptional activity on PEPCK and G6Pase. The primary mouse hepatocytes were infected with adenovirus as indicated. Thirty-six hours postinfection, total cell lysates were subjected to western blot analysis.
(C and D) Repression of PGC-1 a transcriptional activity through PCAF acetyltransferase activity. The mRNA levels of Pck1 and G6pc (C) and the protein levels of PEPCK, G6Pase, PGC-1 a, and PCAF (D) in the primary mouse hepatocytes were analyzed.
(E) Repression of PGC-1 a-stimulated glucose production in the primary mouse hepatocytes requires PCAF acetyltransferase activity. *p < 0.05; **p < 0.01 versus the control cells. All values represent mean ± SEM. See also Figure S2.
was not affected (Figure 5I). The increased PCAF results in declines in PGC-1 a, PEPCK, and G6Pase (Figures 5J and 5K). Glucose tolerance was improved by PCAF, and glucose production from pyruvate was attenuated (Figures 5L and 5M). In addition, PCAF accelerates glucose clearance rate based on the insulin tolerance test (Figure 5N). We also analyzed the insulin signal transduction, and the results showed that phosphorylated Akt and GSK3P were significantly increased by PCAF (Figures 5O and 5P). To investigate whether PGC-1 a plays a major role in PCAF-mediated enhancement of glucose homeostasis, we reconstituted PGC-1 a expression in the PCAF-infected mice and analyzed glucose metabolism. The ameliorations induced by PCAF in blood glucose levels, Pck1 and G6pc expression, and pyruvate tolerance were restored to the control levels (Figures S3A-S3C). The decreases in protein levels of PGC-1 a, PEPCK, and G6Pase were increased to the levels comparable to that observed in the control mice (Figure S3D). Furthermore, we measured liver mitochondria function because PGC-1 a is a pivotal factor for mitochondrial biogenesis (Cui et al., 2006;
Wu et al., 1999). mtDNA copy number, as well as oxygen flux in mitochondria and mitochondrial gene expression, was not altered by PCAF (Figures S4A-S4C). The expression levels of three fatty-acid-oxidation-related genes, LCAD, MCAD, and SCAD, were analyzed. Of the three genes, LCAD expression level was decreased whereas MCAD and SCAD were not affected (Figure S4D). These results strongly indicate that PCAF acetyltransferase improves glucose homeostasis and insulin sensitivity in the obese mice by repressing the gluconeo-genic activity of PGC-1 a.
PCAF Knockdown Stimulates the Gluconeogenic Activity of PGC-1 a
The observation that PCAF represses the gluconeogenic activity of PGC-1 a led us to wonder whether PCAF knockdown stimulates PGC-1 a activity. To address this issue, PCAF knockdown in liver was achieved by tail vein injection with Ad-PCAF shRNA. The mRNA level of PCAF was significantly reduced in the Ad-PCAF-shRNA-no. 2-infected mice (Figure 6A). Pck1
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Figure 6. PCAF Knockdown Potentiates Hepatic Gluconeogenesis
(A) Adenovirus-mediated PCAF knockdown in the mouse liver. Mice were injected with Ad-PCAF shRNA no. 2 or Ad-LacZ shRNA via tail vein. Seven days postinfection, PCAF mRNA levels were measured.
(B) Pck1 and G6pc expression in liver.
(C) Protein levels of acetylated PGC-1 a, PCAF, PEPCK, G6Pase, and GCN5 in liver.
(D) Densitometric quantification of the immunoblot data shown in (C).
(E) Increased blood glucose levels by PCAF knockdown.
(F) Stimulation of glucose production from pyruvate by PCAF knockdown.
Values are presented as mean ± SEM with n = 5 from three independent experiments; *p < 0.05; **p < 0.01 versus the Ad-LacZ-shRNA-infected mice. See also Figure S5.
expression was markedly stimulated by PCAF knockdown, whereas G6pc was not affected (Figure 6B). PCAF protein level was dramatically reduced by Ad-PCAF shRNA no. 2, which in turn stimulates PGC-1 a and PEPCK (Figures 6C and 6D). The acetylated PGC-1 a was decreased by the reduced expression of PCAF. G6Pase protein level was not altered by PCAF reduction. The blood glucose level was enhanced by PCAF knockdown (Figure 6E), and glucose de novo synthesis from pyruvate was also upregulated (Figure 6F). Next, we wished to determine whether PCAF knockdown mediates gluconeogenesis via PGC-
1a. To address this issue, we generated adenovirus-bearing shRNAs against PGC-1 a (Ad-PGC-1a shRNA) to reduce PGC-1a expression. Both the mRNA and protein levels of PGC-1 a were decreased dramatically in the adenovirus-infected Hepa 1-6 cells, and Ad-PGC-1 a shRNA no. 1 showed the best knockdown efficiency (Figures S5A and S5B). Thus, Ad-PGC-1 a shRNA no. 1 was chosen for the following experiments. The mice were injected with Ad-PCAF shRNA no. 2 or Ad-PCAF shRNA no. 2 in combination with Ad-PGC-1 a shRNA no. 1 via tail vein. The increases in blood glucose, Pck1 expression, and
Figure 5. PCAF Improves Glucose Homeostasis and Insulin Sensitivity in Obese Mice
(A and B) Ppargcla, Pck1, G6pc, and Pcaf gene expressions in the lean (A) or DIO mouse (B) livers during fasting.
(C-F) Immunoblot analysis of PGC-1 a, PCAF, PEPCK, and G6Pase in the livers of lean (C) or DIO mice (E) under control, fasted, or refed conditions. Densitometric quantification of the immunoblot data in (C) and (E) are shown in (D) and (F), respectively.
(G and H) DIO mice were injected with Ad-PCAF via tail vein. Seven days later, after a 6 hr fast, blood glucose (G) and plasma insulin levels (H) were measured. (I) mRNA levels of PCAF, PGC-1 a, PEPCK, and G6Pase in liver.
(J and K) Protein levels of acetylated PGC-1 a, PCAF, GCN5, PEPCK, and G6Pase in liver. Densitometric quantification of the immunoblot data in (J) is shown in (K).
(L-N) Glucose tolerance test (L), pyruvate tolerance test (M), and insulin tolerance test (N) were performed.
(O) Insulin signaling transduction was analyzed by western blot.
(P) Densitometric quantification of the immunoblot data as shown in (O).
Values are presented as mean ± SEM with n = 8 from three independent experiments; *p < 0.05; **p < 0.01; ***p < 0.001 versus the control mice or the Ad-LacZ-infected mice. See also Figures S3 and S4.
glucose production from pyruvate induced by PCAF knockdown were reduced to the control levels (Figures S5C-S5E). The PCAF knockdown-mediated upregulations of PGC-1a and PEPCK protein levels were decreased as well (Figure S5F). These data clearly show that PGC-1a-mediated gluconeogenesis was stimulated by PCAF knockdown.
DISCUSSION
Our current work shows that PCAF is a key acetyltransferase that regulates PGC-1 a activity under diabetic and fasting conditions. As a transcriptional coactivator for hepatic gluconeogenesis, the expression level of PGC-1 a is finely controlled by nutritional state indicators such as insulin and glucagon (Herzig et al., 2001, 2003; Yoon et al., 2001). However, this control mechanism is impaired in the obese mice. For example, PGC-1 a is dramatically increased in the obese mouse liver, even in the presence of high insulin levels. Furthermore, PGC-1 a fails to respond to the fasting and refeeding regimen in the obese mice. These data suggest that PGC-1 a-driven hepatic gluconeogenesis is dysre-gulated in the obese mice. In addition to the fine control by insulin and glucagon at the transcriptional level, protein acetylation is also an effective approach for posttranslationally regulating PGC-1 a activity. It has been reported that GCN5, another his-tone acetyltransferase, acetylates PGC-1 a to repress its activity (Lerin etal., 2006). However, it is still unclear whether this repression effect of GCN5 on PGC-1 a is correlated with the hepatic gluconeogenesis in diabetic and fasting states. In the present work, we observed that PCAF reversely correlates PGC-1 a both in diabetic and fasting conditions. These observations led us to conclude that PCAF is a key negative regulator for hepatic gluconeogenesis by affecting PGC-1 a transcriptional activity.
It is worthy to note that the protein levels of both CBP and PCAF are decreased in the obese mouse liver whereas the protein level of p300 remains unchanged. It has been shown that both insulin and metformin trigger the Ser436 phosphorylation in the CH1 domain of CBP, which causes the disassembly of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme expression (He et al., 2009; Zanger et al., 2001; Zhou et al., 2004). Such insulin- or metformin-medi-ated response is generally thought unique of CBP, as p300 lacks an equivalent serine residue in CH1. However, some published results suggest that CBP and p300 are both involved in the inhibitory effects of insulin upon hepatic gluconeogenic gene expression (Bedford et al., 2011; Liu et al., 2008). Thus, in obese mice, the decreased CBP protein level may lead to a decline in hepatic gluconeogenesis, which is inconsistent with the enhanced Pck1 and G6pc expression observed in obese animals (Yoon et al., 2001). Whereas it is plausible that p300 may play a compensatory role to enhance the expression of Pck1 and G6pc, its protein level is only comparable to the lean mice. Therefore, p300 may not be a causative factor for the unconstrained hepatic glu-coneogenesis in obese mice. Considering our findings in the present work, an alternative explanation for the ectopic expressions in Pck1 and G6pc in obese mice is that the decreased PCAF protein level results in enhanced PGC-1 a activity, which eventually stimulates the expression of its downstream targets, such as Pck1 and G6pc.
Whereas PCAF stimulates proteasomal degradation of PGC-1a, PCAF knockdown significantly enhances the protein level of PGC-1 a. Our experiments revealed that both lysine residues (K328 and K450) in PGC-1 a are acetylated by PCAF. Double mutation (K328R/K450R) in PGC-1 a almost completely abolishes its degradation induced by PCAF. The protein stability of PGC-1 a is vital for the control of PGC-1 a activity because of its relatively short half-life (2 or 3 hr; Sano et al., 2007). It is well established that PTMs play an important role in regulating PGC-1 a stability. For example, p38 mitogen-activated protein kinase phosphorylates PGC-1 a at T262, S265, and T298 in response to cytokine stimulation to enhance its stability in muscle cells (Puigserver et al., 2001). Upon O-GlcNAcylation by O-GlcNAc transferase/host cell factor C1 complex, PGC-1 a shows enhanced protein stability and hepatic gluconeogenic transcriptional activity (Ruan et al., 2012). In our current work, PCAF acetylates PGC-1 a at K328 and K450, which subsequently accelerates its proteasomal degradation. Both acetyla-tion sites were conserved among different species, and K450 was previously identified as a substrate of SIRT1 (Rodgers et al., 2005). Our results are also consistent with the previous observation that p300/CBP-mediated acetylation stimulates transcriptional factor E2F1 ubiquitination after DNA damage to cause its degradation (Galbiati et al., 2005). Acetylation of HIF-1 at K532 by ARD1 acetyltransferase most likely enhances its interaction with pVHL and its subsequent ubiquitination and degradation (Jeong et al., 2002). Recently, acetylation of cyclin-dependent kinase inhibitor p27 by PCAF was shown to trigger the proteasomal degradation of p27 (Perez-Luna et al., 2012). However, acetylation may improve the stability for some proteins. For example, Skp2 protein stability was enhanced after acetylation at K68 and K71 by p300 (Inuzuka et al., 2012). p300/CBP-mediated acetylation in p53 enhances its stability by preventing MDM2-dependent ubiquitination, whereas deace-tylation by HDAC1 accelerates the degradation of p53 (Ito et al., 2002; Jin et al., 2002). Lysine ubiquitination is an important hallmark for ubiquitin-mediated protein degradation (Pickart, 2001). It is highly likely that acetylation and ubiquitination compete for the same lysine substrate in some proteins, and as a result, lysine acetylation can enhance the stability of a protein by preventing ubiquitination at the same residue from taking place. However, it is unclear how acetylation can cause reduction in the stability of some proteins.
SIRT1-mediated deacetylation improves PGC-1 a activity (Rodgers et al., 2005), whereas acetylation of PGC-1 a by GCN5 exhibits the opposite effect (Lerin et al., 2006). The mechanism underlying acetylation-mediated repression of PGC-1 a activity is still not well understood. Others have shown that acetylation of PGC-1 a by GCN5 causes a repression of PGC-1 a activity by impairing its binding capacity to the promoter sequence of its target genes (Lerin et al., 2006). In the present study, we observed that PCAF, an acetyltransferase, also acetylates PGC-1 a and results in a reduction in its transcriptional activity. However, PCAF has no effects on the recruitment of PGC-1 a to Pck1 and G6pc promoters. It is also reasonable to predict that GCN5 or PCAF itself may directly bind to gluco-neogenic gene promoter sites and thus impairs downstream gene expression. The results showed that GCN5 recruitment
was decreased in the FSK/IMX-treated cells, whereas PCAF recruitment was not affected. These results indicate that PCAF inhibits PGC-1a activity via a different mechanism whereby acetylation of PGC-1a by PCAF triggers its degradation. It is possible that GCN5 and PCAF on PGC-1a may use different lysine residues for acetylation. In the present work, we identified two lysine residues in PGC-1a, K328 and K450, are both targets for acetylation by PCAF, and double mutation K328R/K450R completely abolishes the effect of PCAF. It remains to be determined which lysine residues are targeted by GCN5 for the acetylation of PGC-1a (Lerin et al., 2006).
Upon acetylation by PCAF, PGC-1 a is prone to proteasomal degradation, resulting in a decrease in protein stability in the HEK293 cells. Consistent with this notion, we observed a reverse correlation between PCAF and PGC-1 a in the lean and obese mouse livers. We also observed similar phenomena in the lean mice under control and fasted states. These in vitro and in vivo data suggest that PCAF is likely a negative regulator of PGC-1a. In this case, increased expression of PCAF in liver should lead to a decrease in the protein level of PGC-1 a and its transcriptional activity and eventually cause euglycemic effect in diabetic mice. Indeed, forced ectopic expression of PCAF in the obese mouse liver ameliorates hyperglycemia and hyperin-sulinemia. Moreover, glucose tolerance, insulin tolerance, and insulin signal transduction were improved as well. When we specifically reduced PCAF expression in the lean mouse liver, we noticed that the entire hepatic gluconeogenesis program was stimulated by PCAF knockdown. Collectively, we propose that PCAF is a key element for regulating PGC-1 a activity that is required for maintaining normal physiological blood glucose level.
Our results provide strong evidence that PCAF acetyltransfer-ase is potentially a novel target for treating disorders associated with dysregulated hepatic glucose output, such as hyperglycemia, insulin resistance, obesity, and diabetes. Specific activators of PCAF may be potential candidates for treating these diseases. The two lysine residues K328 and K450 in PGC-1 a are important for the maintenance of its stability, and we demonstrated in this study that PCAF is a key acetyltransferase for the acetylation of PGC-1 a via these two lysine residues. Future experiments need to be conducted to identify the endogenous deacetylase for these two sites. The reverse correlation between PCAF and PGC-1 a we observed here also exists in diseases other than obesity and type 2 diabetes. For example, knockdown of PCAF in colon cancer cells markedly inhibits cell migration and represses xenograft tumorigenesis and tumor growth in nude mice (Ge et al., 2009). Whereas PCAF inhibitors are potential antitumor drugs, our study cautions that application of PCAF inhibitors may stimulate PGC-1 a activity and cause hypergly-cemia. Therapeutic strategies using PCAF inhibitors for the treatment of tumors should take into account the potential side effects on PCG-1 a and its consequence.
EXPERIMENTAL PROCEDURES Cell Culture
293A cells were from Invitrogen. HEK293, MEF, and Hepa 1-6 cells were from American Type Tissue Collection. H4IIE cells were kindly provided by Prof.
Wei-Fen Xie (Department of Gastroenterology of Changzheng Hospital, the Second Military Medical University). These cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 10 U/ml penicillin, and 1 mg/ml streptomycin at 37°C and 5% CO2. Cultures of primary hepatocytes were prepared from C57BL/6J as described previously (Li et al., 2010). See Supplemental Experimental Procedures for a more-detailed description.
PCAF and PGC-1 a Knockdown with Adenoviral shRNAs
Adenovirus-mediated PCAF knockdown was prepared by using BLOCK-iTTM Adenoviral RNAi Expression System (Invitrogen). Briefly, four pairs of shRNAs against PCAF and three pairs of small hairpin RNAs against PGC-1 a were designed and synthesized at Genscript. PCAF shRNAs were then ligated into U6 Entry vector according to the manual instruction (BLOCK-iT U6 RNAi Entry Vector Kit; Invitrogen). By using LR Clonase, PCAF shRNA was transferred from U6 Entry vector to pAd/BLOCK-iTTM-DEST vector (Invitrogen) for producing adenovirus bearing PCAF shRNA. The shRNA sequences were listed in Table S2.
Adenovirus Production
Adenovirus expressing PCAF, PCAF shRNAs, PGC-1 a shRNAs, PGC-1 a, PGC-1 a K328R, PGC-1 a K450R, or PGC-1 a K328R/K450R was produced with ViraPower Adenoviral Expression System (Invitrogen) according to manufacturer's instruction.
Mass Spectrometric Analysis by LC-MS/MS
To identify PGC-1 a acetylation status in vivo, the HEK293 were transfected with PGC-1 a and PCAF using Lipofectamine. Thirty-six hours posttrans-fection, HEK293 cells were treated with 1 mM trichostatin A (TSA) and 5 mM nicotinamide (NAM) for 12 hr to inhibit the HDACs activity. HEK293 cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 2 mM EGTA, 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, 100 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 10 mg/ml leupeptin, 10 mg/ml aproptonin, 2 mM phenylmethanesulfonylfluoride [PMSF], and 20 nM okadaic acid) containing 2 mM TSA and 10 mM NAM. The whole-cell lysates were incubated with anti-PGC-1a antibody overnight at 4°C in the presence of 2 mM TSA and 10 mM NAM. Protein G Sepharose beads were added for additional 2 hr. Immunoprecipitates were washed three times with RIPA buffer containing 150 mM NaCl, 2 mM TSA, and 10 mM NAM and boiled for 5 min in 2x Laemmli buffer for elution of immunoprecipitated PGC-1 a. Samples were resolved in SDS-PAGE and stained with Coomassie blue. In gel digestion and reversed-phase microcapillary/tandem mass spectrometry (LC-MS/MS) were performed. MS/MS spectra were assigned by searching them against the PGC-1 a protein sequence using the SEQUEST algorithm.
Total Protein Extraction from Cells and Tissues
Cells were lysed in lysis buffer (25 mM Tris-HCl [pH 7.4], 10 mM NaF, 10 mM Na4P2O7, 2 mM Na3VO4,1 mM EGTA, 1 mM EDTA, 1% Nonidet P-40 [NP-40], 10 mg/ml Leupeptin, 10 mg/ml Aproptonin, 1 mM PMSF, and 20 nM Okadaic acid). After 20 min rotation at 4°C, cell lysates were centrifuged at 13,200 rpm for 20 min at 4°C. Tissues were homogenized with a dounce ho-mogenizer in ice-cold tissue lysis buffer (25 mM Tris-HCl [pH 7.4], 100 mM NaF, 50 mM Na4P2O7, 10 mM Na3VO4, 10 mM EGTA, 10 mM EDTA, 1% NP-40, 10 mg/ml Leupeptin, 10 mg/ml Aproptonin, 2 mM PMSF, and 20 nM Okadaic acid). After homogenization, lysates were rotated for 1 hr at 4°C and then subjected to centrifugation at 13,200 rpm for 20 min at 4°C. Super-natants were collected, and protein concentration was quantified by using Protein Assay Kit (Bio-Rad). The concentrations of protein were normalized with lysis buffer to have equivalent amounts of protein and volume. Protein was denatured by boiling at 100°C for 5 min in Laemmli buffer. The lysates were cooled to room temperature before loading for western blot analysis.
Protein Stability Analysis
HEK293 cells were transfected with PGC-1 a together with mock or PCAF using Lipofectamine 2000. For determining mutated PGC-1 a stability, HEK293 cells were transfected with wild-type PGC-1 a or PGC-1 aK328R/K450R together
with PCAF. After 36 hr posttransfection, the cells were treated with cyclohex-¡mide (10 mg/ml) to inhibit protein translation initiation. At various time points, the cells were snap frozen in liquid nitrogen. Protein levels were determined via western blot. The intensity of PGC-1 a bands were quantified, normalized for tubulin, and plotted. Results are expressed as percentage relative to levels observed at time 0.
Transcriptional Activity Assay
The primary mouse hepatocytes were infected with adenovirus expressing wild-type or mutated PGC-1 a together with or without adenovirus bearing PCAF. Six hours postinfection, the culture medium was changed with fresh medium and the cells were transfected with luciferase reporters and p-galac-tosidase using Lipofectamine. The cells were cultured for additional 24 hr, and luciferase and p-galactosidase enzyme activities were measured by the kits. Relative luciferase activity was determined by normalizing to p-galactosidase activity.
Hepatic Glucose Output
The primary mouse hepatocytes were grown in 6-well plates and infected with indicated adenovirus. Thirty-six hours postinfection, culture medium was replaced with 1 ml of glucose-free DMEM supplemented with 0.5% BSA, 20 mM sodium lactate, and 2 mM sodium pyruvate. After 6 hr incubation, medium was collected and the glucose concentration was measured using glucose oxidation kit. The readings were then normalized to the total protein content.
Overexpression or Knockdown of PCAF in Mouse Liver
To increase expression of PCAF in the liver, 5.0 x 106 plaque-forming units (pfu) Ad-PCAF or Ad-LacZ were injected into mice via tail vein. Four days postinjection, blood glucose and plasma insulin were assayed in the fed animals. Six days and eight days post-adenovirus injection, glucose tolerance test and pyruvate tolerance test were performed, respectively. Ten days post-adenovirus injection, insulin tolerance test was carried out. Twelve days post-adenovirus injection, the mice were sacrificed after 6 hr of fasting for experimental analysis. To knockdown PCAF expression in the liver, Ad-PCAF shRNA no. 2 was injected via tail vein at the dosage of 1.5 x 108 pfu. Ad-LacZ shRNA was used as a control virus. Twelve days post-adenovirus injection, blood glucose level was measured in the fed animals. Fourteen days post-adenovirus injection, pyruvate tolerance test was performed. Sixteen days post-adenovirus injection, the mice were sacrificed after 6 hr of fasting for experimental analysis.
Animal Experiments
Four-week-old male C57BL/6 mice were fed with standard diet or high-fat diet (45% fat content) for 8 weeks. After 6 hr starvation, mice were sacrificed and the livers were removed and snap frozen in liquid nitrogen. As for analyzing hepatic PCAF and gluconeogenic gene expression under different nutritional states, 7-week-old C57BL/6 mice were fed ad libitum, fasted for 24 hr, and refed for 24 hr (Rodgers et al., 2005). The study protocol was approved by the Institutional Animal Care and Use Committee, Model Animal Research Center of Nantong University.
Blood Glucose and Insulin Measurements
Mice were fasted for 6 hr, after which their blood was analyzed for glucose measurement with a glucose meter (Bayer). For insulin analysis, mice were fasted for 12 hrand serum insulin level was measured with an Ultra-Sensitive Mouse Insulin ELISA kit.
GTT, PTT, and ITT Assays
Glucose tolerance test (GTT) and pyruvate tolerance test (PTT) were performed after an overnight fast (Sun et al., 2007; Zhou et al., 2011). Mice were intraperitoneally injected with D-glucose (1 g/kg body weight) for GTT or sodium pyruvate (2 g/kg body weight) for PTT. For insulin tolerance test (ITT) analysis, mice were fasted for 6 hr (from 8 a.m. to 2 p.m.) and intraperitoneally injected with recombinant human insulin (2 IU/kg). Tail vein blood was collected at 0, 15, 30, 60, and 120 min following glucose or sodium py-
ruvate or insulin injection, and blood glucose was measured with a glucose meter.
Analysis of In Vivo Insulin Signaling
For in vivo insulin signaling analysis, mice were anaesthetized with xylazine/ ketamine after 6 hr of fasting. Insulin (0.75 IU/kg) or saline was infused into liver via portal vein. Five minutes after infusion, livers were excised quickly, frozen in liquid nitrogen immediately, and stored at —80°C freezer until use.
Immunoprecipitation and Western Blot Analysis
Western blot analysis and immunoprecipitation procedure were performed as previously described (Lee et al., 2011). Briefly, 1 mg lysates were incubated with the appropriate antibody (1 or 2 mg) for 6-8 hr at 4°C followed by 2 hr incubation with Protein G Sepharose beads (Roche). Immunocomplexes were washed five times with the cell lysis buffer before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
Real-Time PCR
Total RNA was extracted from animal tissues using Trizol reagent and transcribed into cDNA using cDNA synthesis kit. The gene expression analysis was performed with StepOne Real-Time PCR Detection System (Applied Biosystems) with SYBR Green Supermix. The mRNA level was normalized to 18S as a housekeeping gene. Sequences of the primers used for real-time PCR assay were listed in Table S1.
Statistics and Data Analyses
Data are presented as a mean ± SEM. The comparisons between two groups were performed using unpaired two-tailed Student's t test. For multiple-group comparisons, one-way ANOVA was applied to test for no differences among the group means. Post hoc comparisons were adjusted using Bonferroni corrections. Significance was accepted at *p < 0.05, **p < 0.01, or ***p < 0.001.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, five figures, and two tables and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2014.11.029.
AUTHOR CONTRIBUTIONS
C.S. designed and performed the experiments, analyzed the data, and wrote the manuscript. M.W., X.L., K.L., S.Z., and L.L. carried out experiments. Y.W., Y.Y., and F.D. analyzed the data and provided helpful comments and advice throughout the project. X.G. designed the experiments, analyzed the data, and wrote the manuscript.
ACKNOWLEDGMENTS
We thank Dr. Weihong Qiu (Department of Physics, Oregon State University) for his critical reading of the manuscript. We thank Prof. Wei-Fen Xie for providing H4IIE cells. This work was supported by grants from the National Natural Science Foundation of China (31271260 and 81471037), the Natural Science Foundation of Jiangsu Province (BK2011132), the Basic Research of Jiangsu Education Department (14KJA180006), the Project of "Six Kinds of Talents Summit'' of Jiangsu Province (2013-WSN-071), the Project of Social Scientific and Technological Innovation and Demonstration of Nantong City (HS2013024), the Neural Regeneration Co-innovation Center of Jiangsu Province, and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.
Received: June 8, 2014 Revised: October 28, 2014 Accepted: November 19, 2014 Published: December 11, 2014
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