Control of hepatic gluconeogenesis by the promyelocytic leukemia zinc finger protein.

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RESEARCH

Control of Hepatic Gluconeogenesis by the Promyelocytic Leukemia Zinc Finger Protein Siyu Chen,* Jinchun Qian,* Xiaoli Shi, Tingting Gao, Tingming Liang, and Chang Liu Jiangsu Key Laboratory for Molecular and Medical Biotechnology and College of Life Sciences (S.C., J.Q., X.S., T.G., T.L., C.L.), Nanjing Normal University, Nanjing, Jiangsu 210023, China; and State Key Laboratory of Natural Medicines (C.L.), China Pharmaceutical University, Nanjing, Jiangsu 210009, China

The promyelocytic leukemia zinc finger (PLZF) protein is involved in major biological processes including energy metabolism, although its role remains unknown. In this study, we demonstrated that hepatic PLZF expression was induced in fasted or diabetic mice. PLZF promoted gluconeogenic gene expression and hepatic glucose output, leading to hyperglycemia. In contrast, hepatic PLZF knockdown improved glucose homeostasis in db/db mice. Mechanistically, peroxisome proliferator-activated receptor ␥ coactivator 1␣ and the glucocorticoid receptor synergistically activated PLZF expression. We conclude that PLZF is a critical regulator of hepatic gluconeogenesis. PLZF manipulation may benefit the treatment of metabolic diseases associated with gluconeogenesis. (Molecular Endocrinology 28: 1987–1998, 2014)

epatic gluconeogenesis plays an important role in the maintenance of blood glucose homeostasis in mammals. Under fasting conditions, pancreatic ␣-cells secrete glucagon, which stimulates glycogen breakdown (glycogenolysis) and generates glucose from the liver. Prolonged fasting or starvation induces de novo glucose synthesis in the liver, termed hepatic gluconeogenesis. In addition to glucagons, glucocorticoids are also important hormonal signals for initializing gluconeogenesis. The response to glucocorticoids is mediated by the glucocorticoid receptor (GR) that binds to glucocorticoid responsive elements in the promoters of gluconeogenic genes (1). As is known, several key regulatory enzymes are required to correctly trigger the process of gluconeogenesis, including glucose 6-phosphatase (G6Pc), fructose 1,6-bisphosphatase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase (Pck1) (2, 3). In contrast, under feeding conditions, insulin is secreted from pancreatic ␤-cells in response to the elevated levels of plasma glucose. In the liver, insulin inhibits glycogenolysis and gluconeogenesis while stimulating glycolysis and glycogenesis, thus reducing the plasma glucose levels in the body (4). Hepatic gluconeogenesis is tightly controlled in healthy individuals; however, it is constitutively activated due to insulin

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ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received May 28, 2014. Accepted October 14, 2014. First Published Online October 21, 2014

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resistance in certain pathophysiological conditions, such as type 2 diabetes, and leads to excess glucose secretion and aggravated hyperglycemia (5, 6). To date, numerous transcriptional regulators of hepatic gluconeogenesis have been identified. Among these, the transcriptional coactivator peroxisome proliferatoractivated receptor ␥ coactivator 1␣ (PGC-1␣) has gained a lot of attention because of its robust roles in the regulation of various metabolic pathways including hepatic gluconeogenesis (7). In the liver, expression of PGC-1␣ is markedly induced upon fasting via a cAMP response element binding protein/cAMP response element binding protein-regulated transcription coactivator 2-dependent transcriptional mechanism (8). Overexpression of PGC-1␣ in the mouse liver or primary hepatocytes increases the expression levels of gluconeogenic genes by enhancing the transactivating potential of various transcriptional factors such as GR, hepatocyte nuclear factor 4␣ (9), and forkhead box class O1 (FoxO1) (10). Conversely, either acute knock* S.C. and J.Q. contributed equally to the study. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; Br, bromo; ChIP, chromatin immunoprecipitation; CoIP, coimmunoprecipitation; Dex, dexamethasone; FoxO1, forkhead box class O1; FSK, forskolin; GFP, green fluorescent protein; G6Pc, glucose 6-phosphatase; GTT, glucose tolerance test; GR, glucocorticoid receptor; H3K4me3, trimethylation of lysine 4 of histone 3; HCS, hydrocortisone; HFD, high-fat diet; IFN, interferon; IRS1, insulin receptor substrate 1; ITT, insulin tolerance test; Pck1, phosphoenolpyruvate carboxykinase; PGC-1␣, peroxisome proliferator–activated receptor ␥ coactivator 1␣; PLZF, promyelocytic leukemia zinc finger; PTT, pyruvate tolerance test; RNAi, RNA interference; Scrb, scrambled; siRNA, small interfering RNA; STZ, Streptozotocin.

Mol Endocrinol, December 2014, 28(12):1987–1998

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PLZF Activates Hepatic Gluconeogenesis

down or knockout of PGC-1␣ in mice results in the lower blood glucose levels that are associated with the reduced expression of gluconeogenic genes (11). These findings indicate that PGC-1␣ is a critical factor in the regulation of gluconeogenesis and glucose homeostasis. The promyelocytic leukemia zinc finger (PLZF) protein, also known as Zbtb 16 or Zfp 145, was first identified in 1993 in a patient with acute promyelocytic leukemia, in whom a reciprocal chromosomal translocation t(11;17) (q23;q21) resulted in a fusion with the RARA gene encoding retinoic acid receptor ␣ (12). PLZF has been shown to be involved in major developmental and biological processes, such as spermatogenesis (13), hind limb formation (13), hematopoiesis (14, 15), and immune regulation (16). Interestingly, recent studies have indicated that PLZF may potentially play a role in the pathogenesis of metabolic diseases. For example, a single nucleotide polymorphism leading to a threonine to serine substitution at amino acid position 208 (T208S) in the rat plzf gene not only results in the development of polydactyly-luxate syndrome but also affects total body weight, adiposity, the lipid profile, and the insulin sensitivity of skeletal muscle (17). In addition, PLZF expression in visceral adipose tissue of obese diabetic women is significantly lower than that in healthy control women (18). In contrast, PLZF overexpression in brown adipocytes leads to the induction of genes involved in fatty acid oxidation, glycolysis, and mitochondrial function (18). Although these findings clearly demonstrate that PLZF is a robust metabolic regulator, they are generally descriptive and the detailed mechanism through which PLZF regulates energy metabolism remains unknown. Based on the findings mentioned above, we aimed to explore whether PLZF regulates hepatic gluconeogenesis and whether this process requires PGC-1␣ participation. To answer this question, we used the gain- and loss-offunction strategy to manipulate the PLZF expression levels both in vivo and in vitro using adenoviral transduction and small interfering RNA (siRNA) delivery. Our studies revealed a positive role for PLZF in the regulation of gluconeogenesis and suggested that PLZF may be a therapeutic target for the treatment of various disorders associated with gluconeogenesis dysregulation.

Materials and Methods Animal experiments Eight-week-old male C57BL/6 mice and diabetic db/db mice on a C57BKS background were purchased from the Model Animal Research Center of Nanjing University. All animal procedures in this investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996)


and the approved regulations set by the Laboratory Animal Care Committee at Nanjing Normal University (permit number 2090658, date issued April 20, 2008). For the fasting time course experiment, mice were fed ad libitum or fasted during the dark period for 2, 5, and 16 hours. For the streptozotocin (STZ) (Sigma-Aldrich) diabetic mouse experiments, 6- to 8-week-old male mice were injected intraperitoneally for 2 days consecutively with either sodium citrate solution or STZ (125 mg/kg body mass). Animals were killed after 10 days, at which point the mean blood glucose in the STZ-treated group rose to ⬎19.4 mM. To induce type 2 diabetes, 4-week-old mice were fed a high-fat diet (HFD) (fat content 60%; Research Diets) for 1 month and then challenged with STZ for 5 days at a dose of 25 mg/kg body mass through intraperitoneal injection.

Adenovirus and plasmid information Adenoviruses (ad-green fluorescent protein [GFP], ad-PGC1␣, ad-scrambled (Scrb), and ad-PGC-1␣ RNA interference [RNAi]) and plasmids encoding PGC-1␣ and GR were kindly provided by Dr Jiandie Lin (Life Sciences Institute, University of Michigan, Ann Arbor, MI). PLZF adenoviruses (ad-PLZF) were designed, validated, and synthesized by Yingrun Biotechnologies Inc. All of the adenoviruses were concentrated and purified before use. For in vivo PLZF knockdown, 3 sets of stealth siRNAs for each gene were designed, validated, and synthesized by Invitrogen. To improve gene silencing efficiency, an siRNA cocktail comprising these 3 sets of siRNA oligonucleotides (an equal molar mixture) was used. The sequences of these siRNA oligonucleotides are listed in Supplemental Table 1. Mouse plzf promoter-luciferase (mplzf-luc) reporter plasmids (⫺1121 to ⫺630) were amplified from mouse genomic DNA using the primers listed in Supplemental Table 2, and the conserved half GR binding sequence AGAACA was mutated to ACAGCA. These sequences were validated by sequencing and cloned into a pGL3-basic vector. Commercial siRNA oligonucleotides against GR were purchased from Santa Cruz Biotechnology.

In vivo adenovirus transduction We transduced adenoviruses encoding PLZF or PGC-1␣ as well as their negative control GFP into mice at a dose of 1.0 ⫻ 109 plaque-forming units per mouse through tail vein injection. Four days after the injection, all mice were killed, and the livers were collected.

Glucose tolerance tests (GTTs), insulin tolerance tests (ITTs), and pyruvate tolerance tests (PTTs) For GTTs, mice were fasted overnight and intraperitoneally injected with glucose (2 g/kg per normal mouse, 1 g/kg per db/db mouse). For ITTs, mice were fasted for 6 hours and intraperitoneally injected with insulin (0.75 U/kg per normal mouse, 2.5 U/kg per db/db mouse). For PTTs, mice were fasted overnight and then intraperitoneally injected with pyruvate (2 g/kg per normal mouse, 1 g/kg per db/db mouse). Blood glucose levels were measured at 0, 20, 40, 60, and 90 minutes after injection using a glucose monitor (Roche).

Serological analysis Blood was obtained from the vena cava, and the serum was isolated and stored at ⫺80°C until analysis. Serum insulin, alanine aminotransferase (ALT), and aspartate aminotransferase


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(AST) levels were measured with the use of commercial kits (Crystal Chem [insulin] and Jiancheng Institute of Biotechnology [ALT and AST]) according to the manufacturer’s protocols.

Cell culture Mouse AML-12 hepatocytes were cultured with DMEM/ F-12 medium (Gibco) supplemented with insulin, transferrin, selenium (Gibco), and 40 ng/mL dexamethasone (Dex) (SigmaAldrich) in a humidified atmosphere containing 5% CO2 at 37°C. To demonstrate the effects of gluconeogenic hormones on PLZF expression, AML-12 mouse hepatocytes were treated with 1 ␮M Dex, 1 ␮M hydrocortisone (HCS), 10 ␮M forskolin (FSK), or 1 mM 8-bromo (Br)-cAMP, respectively, for 6 hours. For mplzf-luc activity, 200 ng of reporter plasmids were transfected into AML-12 cells. At 36 hours later, cells were treated with 1 ␮M Dex or 1 ␮M HCS for another 6 hours. Mouse primary hepatocytes were prepared from C57BL/6 mice via the collagenase IV (Gibco) perfusion method and subjected to treatments similar to those performed in AML-12 cells.


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Western blots For protein analysis, tissue samples were homogenized, and cells were lysed in radioimmunoprecipitation assay buffer. The protein concentration was quantified with a Dc protein assay reagent (Bio-Rad). Equal amounts of protein were loaded and separated by 10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore). The membranes were incubated overnight with the appropriate primary antibodies. Bound antibodies were then visualized using horseradish peroxidase-conjugated secondary antibodies. Quantitative analysis was performed by NIH ImageJ 1.32j software. For antibody information, anti-G6Pc, anti-Pck1, anti-GR antibodies were purchased from Santa Cruz Biotechnology, and anti-PGC-1␣ antibody was obtained from Calbiochem. Anti-glyceraldehyde-3-phosphate dehydrogenase, anti-phospho-insulin receptor substrate (IRS) 1 (Ser 307), anti-total IRS1, anti-phospho-Akt (Ser 473), anti-total Akt, anti-phospho-FoxO1 (Ser 319), and anti-total FoxO1 antibodies were purchased from Cell Signaling Technology. Anti-PLZF antibody was from Abcam.

Chromatin immunoprecipitation (ChIP) assay Glucose output assays Mouse primary hepatocytes were isolated and seeded into 6-well plates (1⫻106 cells/well). For the glucose output assay, cells were washed twice with PBS and incubated with glucosefree DMEM medium (pH 7.4, phenol red free, supplemented with 20 mM sodium lactate and 2 mM pyruvate). Six hours later, the medium was assayed for glucose production with a colorimetric kit (Sigma-Aldrich). Values were normalized to the protein content of the whole-cell lysates. Dex (1 ␮M, treatment time: 6 hours) was used as a positive control.

Chromatin lysates were prepared from AML-12 cells, precleared with protein-A/G agarose beads (Roche), and immunoprecipitated with antibodies against PGC-1␣, trimethylation of lysine 4 of histone 3 (H3K4me3) (Abcam), and acetylated histone 3 (Millipore), or normal mouse IgG (Santa Cruz Biotechnology) in the presence of BSA and salmon sperm DNA. Beads were extensively washed before reverse cross-linking. DNA was purified using a PCR purification kit (Qiagen) and subsequently analyzed by PCR using primers flanking the proximal binding sites for GR on the mouse plzf promoter, as detailed in Supplemental Table 3.

Transient transfection and luciferase reporter assays

Coimmunoprecipitation (CoIP) assay

Transient transfections were conducted using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For luciferase reporter assays, 200 ng of reporter plasmids were mixed with 200 ng of GR expression construct in the presence or absence of 500 ng of PGC-1␣ expression construct. Equal amounts of DNA were used for all transfection combinations by addition of appropriate vector DNA. Relative luciferase activities were determined 48 hours after transfection using the DualLuciferase System (Promega). To identify the essential roles of PGC-1␣ and GR in regulating PLZF expression, cells were transfected with Scrb or GR siRNA oligonucleotides together with plasmid vector DNA or plasmids encoding PGC-1␣ for 48 hours. To verify that PLZF functions downstream in PGC-1␣controlled gluconeogenesis, cells were transfected with Scrb or PLZF siRNA oligonucleotides together with plasmid vector DNA or plasmids encoding PGC-1␣ for 48 hours. All transfection experiments were performed in triplicate.

RT-quantitative PCR (qPCR) Total RNA was isolated using TRIzol reagents (Invitrogen), reverse transcribed, and analyzed by qPCR using SYBR Green and the Mastercycler ep realplex2 system (Eppendorf, Hamburg, Germany). Primers for mouse 36B4 were included for normalization. A complete list of PCR primers is shown in Supplemental Table 3.

AML-12 cells were lysed in radioimmunoprecipitation assay buffer. After centrifugation, 50 ␮g of protein lysate was incubated with 20 ␮l of protein-A/G agarose beads and 2 ␮g of normal mouse IgG or anti-PGC-1␣. After 24 hours of incubation, the immune complexes were centrifuged and washed 4 times with ice-cold lysis buffer. The immunoprecipitated protein was further analyzed using Western blotting.

Statistical analysis The groups of data are presented as the means ⫾ SD. The data were analyzed using a one-way ANOVA followed by the Fisher least significant difference post hoc test. The calculations were performed using the SPSS for Windows version 12.5S statistical package (SPSS). A value of P ⬍ .05 was considered statistically significant.

Results Gluconeogenic signals induce hepatic PLZF expression To explore whether nutritional signals regulate PLZF expression, we examined the hepatic PLZF expression levels in mice by performing a time course of fasting. As expected, induction of 2 gluconeogenic genes (G6Pc and


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ingly, the hepatic expression levels of plzf mRNA were significantly induced by short-term fasting (2 and 5 hours) and decreased upon refeeding. The protein expression levels of PLZF in the livers showed a similar pattern (Figure 1A, bottom panel). Thus, the induction of PLZF in liver is a relatively early event, consistent with a possible role in control of gluconeogenic genes. We also examined PLZF expression levels in other metabolic tissues under the fasting state and found that both PLZF mRNA and protein expression levels were induced in brown adipose tissues and skeletal muscles in response to fasting. However, PLZF was inhibited in white adipose tissues (Supplemental Figure 1). The dominant regulator of gluconeogenesis in vivo is insulin. We first examined the expression levels of PLZF in the livers of mice treated with STZ, which is the most commonly used experimental model of insulin-deficient type 1 diabetes. As shown in Figure 1B, both the mRNA and protein expression levels of PLZF were consistently increased in the livers of these mice. In addition, diabetic mice induced by leptin receptor deficiency (db/db) or HFD feeding plus STZ injection develop insulin resistance and show overactiFigure 1. Gluconeogenic signals induce hepatic PLZF expression. A, RT-qPCR and Western blot vated gluconeogenesis. We found analysis of PLZF and gluconeogenic gene expression in the liver from mice subjected to fasting and ## that PLZF expression levels were refeeding. *, P ⬍ .05 and **, P ⬍ .01 vs Fed group; , P ⬍ .01 vs Fasted 2 h group. n ⫽ 6. B–D, mRNA and protein analysis in the livers of STZ-induced type 1 diabetic mice (B), db/db mice (C), HFD/ also increased in the livers of these STZ-induced type 2 diabetic mice (D), and their corresponding controls under fed condition. n ⫽ 5. mice (Figure 1, C and D). **, P ⬍ .01 vs control groups. E, Regulation of PLZF expression by gluconeogenic hormones in AMLGlucocorticoids have long been 12 cells. plzf mRNA expression was quantified by RT-qPCR. **, P ⬍ .01 vs control group. F, Reporter gene analysis of transcriptional activity of mouse plzf promoter in response to Dex and HCS in AMLknown to stimulate hepatic gluco12 cells. **, P ⬍ .01 vs mplzf-luc alone. G, Dex induces PLZF expression in AML-12 cells in a doseneogenesis by binding to GR and inand time-dependent manner. **, P ⬍ .01 vs control group. H, Western blot analysis of PLZF and ducing transcription of G6Pc and gluconeogenic gene expression in AML-12 cells treated with 1 ␮M Dex for 6 or 12 hours, respectively. I and J, RT-qPCR (I) and Western blot (J) analyses of PLZF expression in mouse primary Pck1 genes. On the other hand, hepatocytes treated with indicated gluconeogenic hormones. **, P ⬍ .01 vs control group. K, RTcAMP rises in the liver during a fast. qPCR analysis of PLZF and gluconeogenic gene expression in primary hepatocytes treated with 1 ␮M Therefore, we investigated whether Dex for 6 or 12 hours, respectively. *, P ⬍ .05 and **, P ⬍ .01 vs control group. CTL, control WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. PLZF is induced by glucocorticoid hormone signals in AML-12 liver Pck1) was first detected at 2 hours, persisting during the cells and mouse primary hepatocytes. Cells were treated entire starvation period (Figure 1A, top panel). An in- with glucocorticoids (Dex and HCS), FSK, an activator of crease in pgc-1␣ mRNA was also observed after 2 hours adenylate cyclase that is used to increase intracellular and peaked at 5 hours after food deprivation. Interest- cAMP levels, or 8-Br-cAMP (a cAMP analog). Whereas


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plzf promoter (Figure 1F). The induction of plzf mRNA expression levels by Dex showed a dose- and time-dependent manner (Figure 1G). Similarly, PLZF protein expression was up-regulated by Dex (Figure 1H). In mouse primary hepatocytes, Dex and HCS also promoted PLZF mRNA and protein expression levels, whereas FSK and 8-Br cAMP only increased PLZF expression at the translational level (Figure 1, I and J). Specifically, the maximum induction of Dex occurred at 6 hours and then gradually declined (Figure 1K). PLZF activates hepatic gluconeogenesis To address the possibility that PLZF may regulate aspects of hepatic glucose metabolism, PLZF was overexpressed in cultured liver cells with an adenovirus-based vector. As shown in Figure 2A, forced expression of exogenous PLZF in AML-12 cells by adenovirus infection induced the mRNA and protein expression levels of gluconeogenic genes in a dose-dependent manner. Overexpression of PLZF in mouse primary hepatocytes had similar effects (Figure 2B) and functionally increased the glucose production (Figure 2C). In vivo studies indicated that the hepatic expression levels of gluconeogenic genes in normal mice were increased in response to PLZF overFigure 2. PLZF activates hepatic gluconeogenesis. A, Regulation of gluconeogenic gene expression by PLZF in vitro. RT-qPCR and Western blot analysis of PLZF and gluconeogenic gene expression, and such inductions expression in AML-12 cells transduced with adenoviruses encoding GFP (ad-GFP, as control) or came more robustly under the fastPLZF (ad-PLZF) for 48 hours. B, Gene expression assessments in mouse primary hepatocytes ing condition (Figure 2D). Coincitreated as for A. C, Glucose output assays in mouse primary hepatocytes. For A–C, **, P ⬍ .01 dent with these findings, the basal vs ad-GFP. D, Gene expression analysis in the liver of mice transduced with ad-GFP or ad-PLZF (0.1 absorbance unit per mouse). *, P ⬍ .05 and **, P ⬍ .01 vs ad-GFP/Fed group; #, P ⬍ .05 and fasted blood glucose levels were and ##, P ⬍ .01 group vs ad-GFP/Fasted group. n ⫽ 5. E, Basal blood glucose levels under fed or higher (Figure 2E). Insulin is an imovernight fasted condition. F, Serum insulin levels under fed condition. G–I, GTTs, PTTs, and ITTs. portant hormone in regulation of For E–I, *, P ⬍ .05 and **, P ⬍ .01 vs ad-GFP. n ⫽ 5. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. blood glucose homeostasis; thus, we next quantified the serum insulin levels and found they were higher either FSK or 8-Br-cAMP produced little effect on plzf mRNA, treatment with Dex or HCS caused a large increase under the fed condition when PLZF was overexpressed in in the plzf transcription (Figure 1E) in AML-12 cells, which liver (Figure 2F). The hyperinsulinemia in these mice may be due to the enhanced transcriptional activity of the might be due to a compensatory response to the elevated


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blood glucose levels. In addition, these mice also showed impaired glucose and pyruvate tolerance and insulin sensitivity (Figure 2, G–I). Collectively, our results strongly suggest that PLZF activates hepatic gluconeogenesis and impairs insulin sensitivity. Knockdown of PLZF suppresses hepatic gluconeogenesis We next used a loss-of-function strategy to confirm the above findings. The liver-specific knockdown of PLZF in normal mice did not affect body weight, but significantly decreased blood glucose levels (Supplemental Figure 2A). Accordingly, the mRNA and protein expression levels of gluconeogenic genes were decreased under both feeding and fasting conditions (Supplemental Figure 2B). Knockdown of PLZF also caused improved glucose and pyruvate tolerance and insulin sensitivity (Supplemental Figure 2, C–E). Coincident with the in vivo results, transfection of plasmids encoding PLZF siRNA oligonucleotides in AML-12 cells down-regulated the basal expression levels of gluconeogenic genes and antagonized their induction by Dex (Supplemental Figure 2F). Finally, PLZF knockdown functionally decreased the glucose production rate in mouse primary hepatocytes (Supplemental Figure 2G). Knockdown of PLZF ameliorates hyperglycemia in db/db mice The importance of PLZF in the regulation of hepatic gluconeogenesis under normal physiological conditions has now been shown. However, whether it affects glucose metabolism in pathological settings remains unknown. Therefore, we transduced diabetic db/db mice with scramble or PLZF siRNA oligonucleotides for 3 days and examined the effects of PLZF knockdown on the gluconeogenic pathway and glucose intolerance. Gene expression analysis revealed that RNAi knockdown of PLZF resulted in an approximately 54% reduction in plzf mRNA levels in the liver (Figure 3A). The expression of G6Pc and Pck1 was decreased in response to PLZF knockdown (Figure 3A). Compared with those in controls, blood glucose levels were significantly lower in mice with liver-specific PLZF knockdown when measured under fed and fasted conditions (Figure 3B). Serum insulin levels were also decreased (Figure 3C). Physiologically, these mice showed reduced food intake, water drinking, and body weight (Figure 3D). Serological analysis indicated that serum levels of ALT and AST did not alter in these mice (Figure 3E), suggesting that the reduction in food intake is not due to the possible liver toxicity caused by PLZF knockdown. More importantly, GTTs, ITTs, and PTTs in these mice revealed that they had improved glucose and pyruvate tol-


erance and insulin sensitivity (Figure 3, F–H). These results demonstrate that depletion of PLZF in the liver improves glucose homeostasis in an insulin-resistant state. PLZF negatively regulates the insulin signaling pathway The insulin signaling pathway plays a critical role in the regulation of gluconeogenesis. It was interesting to investigate whether PLZF regulates key components in this signaling cascade. As shown in Figure 4A, overexpression of PLZF reduced the phosphorylation levels of IRS1, Akt, and FoxO1 in normal mouse liver under the fed state. Similarly, infection of adenoviruses encoding PLZF in AML-12 cells antagonized insulin-induced phosphorylation of these factors (Figure 4B). Conversely, the liver-specific knockdown of PLZF in db/db mice resulted in a marked increase in the phosphorylation of IRS1, Akt, and FoxO1 (Figure 4C). In addition, synergistic effects of PLZF siRNA and insulin on the phosphorylation of these factors were observed in vitro (Figure 4D). These results suggest that PLZF negatively regulates key components involved in the insulin signaling pathway, which may contribute to the positive control of PLZF in gluconeogenesis. On the other hand, PLZF has forkhead response elements (A/G TAAA T/C A) on its promoter and is activated by Foxo3a, a FoxO family member (19). Given the importance of FoxO1 in gluconeogenesis, we questioned whether PLZF expression is conversely controlled by FoxO1. As shown in Supplemental Figure 3, forced overexpression of FoxO1 by plasmid transfection in AML-12 cells did not alter PLZF mRNA and protein expression levels, thus excluding such a possibility. PGC-1␣ and GR synergistically activate PLZF expression PGC-1␣ is a key nuclear coactivator of gluconeogenesis and may serve as an upstream regulator for PLZF expression. Indeed, overexpression of PGC-1␣ increased the mRNA and protein expression levels of PLZF both in vivo (Figure 5A) and in vitro (Supplemental Figure 4A). In contrast, PGC-1␣ knockdown inhibited PLZF expression in the liver (Figure 5B) and in cultured AML-12 cells (Supplemental Figure 4B). ChIP analysis indicated that PGC-1␣ was present near a glucocorticoid responsive element on the proximal plzf promoter (Figure 5C). Epigenetically, histone hyperacetylation is associated with transcriptional activation. Similarly, H3K4me3 is also found in activated genes. We found that PGC-1␣ overexpression led to a robust increase in acetylated histone 3 and H3K4me3 levels on the plzf promoter. The knockdown of PGC-1␣ caused opposite results (Figure 5D).


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Figure 3. Liver-specific knockdown of PLZF relieves hyperglycemia in db/db mice. A, Hepatic PLZF and gluconeogenic gene expression. **, P ⬍ .01 vs Scrb. n ⫽ 6. B, Basal blood glucose levels. *, P ⬍ .05 vs Scrb/Fed group; **, P ⬍ .01 vs Scrb/Fasted group. n ⫽ 6. C, Serum insulin levels under the fed condition. **, P ⬍ .01 vs Scrb group. n ⫽ 6. D, Food intake, water drinking, and body weight. *, P ⬍ .05 vs Scrb. n ⫽ 6. E, Serum levels of ALT and AST. n.s., no significant difference. n ⫽ 6. F–H, GTTs, PTTs, and ITTs. *, P ⬍ .05 and **P ⬍ .01 vs PLZF siRNA (siPLZF). n ⫽ 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

These results indicate that PGC-1␣ stimulates plzf transcription by altering the local chromatin environment from a repressive to an active state. We next attempted to identify the transcriptional factors that mediate the activation of plzf transcription by PGC-1␣. A bioinformatics analysis indicated that the plzf promoter (⫺1121 to ⫺630) possesses a conserved half GR binding sequence AGAACA (20). Importantly, when this GR binding domain was mutated, Dex-induced activation of plzf-luc was blocked (Supplemental Figure 5A). Consistent with this finding, induction of PLZF expression by Dex was attenuated in AML-12 cells with GR knockdown (Supplemental Figure 5B). Furthermore, a reporter gene assay indicated that PGC-1␣ augmented the transcriptional activity of GR on a plzf promoter reporter (Figure 5E). However, when the GR binding site was mutated, such synergistic effects disappeared. To confirm that PGC-1␣ requires GR to activate PLZF, we knocked down GR in AML-12 cells and found that PGC-1␣-induced PLZF expression was repressed by the siRNA against GR (Figure 5F). Collectively, these results

suggest that PGC-1␣ and GR synergistically activate PLZF expression. PLZF is a downstream effector for PGC-1␣-controlled gluconeogenesis To explore whether PLZF is a downstream effector for PGC-1␣-controlled gluconeogenesis, we knocked down PLZF in AML-12 cells overexpressing PGC-1␣ and observed that PLZF knockdown inhibited PGC-1␣-induced gluconeogenic gene expression (Figure 6A). Moreover, glucose output from mouse primary hepatocytes was decreased by PLZF knockdown (Figure 6B). At the molecular level, PGC-1␣ reduced Akt phosphorylation in response to insulin treatment, and knockdown of PLZF antagonized the effects of PGC-1␣ (Figure 6C). Physically, CoIP analysis demonstrated that PGC-1␣ and PLZF formed a complex in vitro (Figure 6D). These data demonstrate that PLZF is an important factor acting downstream of PGC-1␣ in the regulation of the hepatic gluconeogenic program.


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tool. These mice display major musculoskeletal limb defects with homeotic transformation of vertebral segments, physically disabled cartilage and skeleton patterning, and alterations in digit formation (21). PLZF⫺/⫺ male mice also show partial sterility caused by markedly impaired spermatogenesis (22). More importantly, PLZF expression is lost in many cancer cells (23), which is believed to transform cells to malignancy (24). Indeed, we found that PLZF expression was lower in human hepatoma HepG2 cells than that in the normal liver cell line LO2 (Qian, J., Zhang, W., Wang, D., unpublished data). Therefore, although the exact mechanism has yet to be elucidated, PLZF is considered to be a tumor suppressor protein (25, 26). At the molecular level, PLZF functions as a versatile transcriptional regulator, either repressing or activating certain physiological programs by selectively interacting with various chaperones. For example, it recruits nuclear corepressors to form repressive complexes via the N-terminal BTB/POZ domain and then silences the activation of target gene promoters (27). Conversely, when acting as an activator, PLZF interacts with GATA1 and stimulates megakaryocyte differentiation (28). In R3T3 cells, PLZF is required for PI3K-P85␣ gene activation, which is a crucial process in cardiac hypertrophy (29). Figure 4. PLZF negatively regulates the insulin signaling pathway. A, Phosphorylation levels of Thus, the roles of PLZF are variable IRS1, Akt, and FoxO1 in the livers of mice with liver-specific overexpression of PLZF. **, P ⬍ .01 and are dependent on specific pathovs ad-GFP. n ⫽ 5. B, Phosphorylation levels of IRS1, Akt, and FoxO1 in AML-12 cells. The cells were infected by ad-GFP or ad-PLZF for 48 hours and then treated with 100 nM insulin for the physiological circumstances. Furtherindicated time period. *, P ⬍ .05 and **, P ⬍ .01 vs ad-GFP/insulin 0 minutes; #, P ⬍ .05 and ##, P ⬍ more, the active effects of PLZF on .01 vs ad-GFP/insulin, the same time period. C, Phosphorylation levels of IRS1, Akt, and FoxO1 in the some cellular processes can also be livers of db/db mice with liver-specific knockdown of PLZF. **, P ⬍ .01 vs Scrb. n ⫽ 6. D, Synergistic achieved by inhibiting the expression effects of PLZF knockdown and insulin on IRS1, Akt, and FoxO1 phosphorylation levels in AML-12 cells. *, P ⬍ .05 and **, P ⬍ .01 vs Scrb/insulin 0 minutes; #, P ⬍ .05 and ##, P ⬍ .01 vs Scrb/insulin, of microRNAs, such as microRNAthe same time period. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 146a and microRNA-221/222. In these cases, although PLZF essentially Discussion functions as a repressor, the overall regulation by PLZF PLZF was first identified as a zinc finger family member that leads to the activation of megakaryopoiesis (30) and melaexhibited a pronounced effect on promyelocytic leukemia nogenesis (31), respectively. pathology. Recent studies have revealed the versatile biologOn the other hand, the metabolic properties of cancer ical functions of PLZF by using PLZF-deficient mice as a cells diverge significantly from those of normal cells. The


doi: 10.1210/me.2014-1164


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Figure 5. PGC-1␣ and GR activate PLZF expression. A, RT-qPCR (left panel) and Western blot (right panel) analyses of hepatic PLZF expression in liver-specific PGC-1␣ overexpression mice. **, P ⬍ .01 vs ad-GFP. n ⫽ 7. B, RT-qPCR (left) and Western blot (right) analyses of hepatic PLZF expression in liver-specific PGC-1␣ knockdown mice. **, P ⬍ .01 vs ad-Scrb. n ⫽ 7. C, ChIP assays with IgG or anti-PGC-1␣ antibody in AML-12 cells. D, ChIP assays (left panel) with indicated antibodies using AML-12 cells infected with ad-GFP or ad-PGC-1␣. The enrichment was quantified by qPCR analysis. **, P ⬍ .01 vs ad-GFP. ChIP assays (right panel) in AML-12 cells infected with ad-Scrb or ad-siPGC-1␣. *, P ⬍ .05 and **, P ⬍ .01 vs ad-Scrb. E, Reporter gene assays in AML-12 cells transfected with the indicated plasmids. **, P ⬍ .01 vs basal levels; ##, P ⬍ .01 vs PGC-1␣ and GR cotransfection group. F, RT-qPCR (left panel) and Western blot (right panel) analyses of PGC-1␣, GR, and PLZF expression in AML-12 cells. **, P ⬍ .01 vs Vector⫹Scrb, #, P ⬍ .05 vs PGC-1␣⫹Scrb. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

best studied metabolic phenotype of cancer is aerobic glycolysis, known as the Warburg effect, which is characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen (32). A recent metabolomic analysis indicated that the serum metabolite composition from acute myeloid leukemia patients differs a lot from that of the controls, highlighted by system differences in multiple metabolic pathways including gluconeogenesis (33). In the liver, gluconeogenesis is a fundamental feature of hepatocytes but is not present in mouse or human malignant hepatocytes (34). This is particularly interesting when the positive relationship between PLZF and gluconeogenesis is considered. First, both PLZF expression and gluconeogenesis are impaired during the development of liver cancer. Second, Dex is reported to restore gluconeogenesis in malignant liver cells, leading to therapeutic efficacy against hepatocarcinoma (34). In our system, Dex increases PLZF expression in

AML-12 cells. Therefore, PLZF may serve as a node integrating metabolism switch and tumorigenesis. In addition to its role in glucose homeostasis, PLZF also affects lipid metabolism. A comparative analysis of genome-wide chromatin state maps discovered that PLZF possesses antiadipogenic activity (35). In the heart, PLZF is robustly induced by dietary fatty acids in a PPAR␣independent manner (36). Considering that fatty acids are important energy donors for the healthy heart, PLZF may link dietary lipids to heart function. In our study, hepatic PLZF expression is induced in db/db and diet-induced obese mice, which exhibited severe hepatic steatosis. PLZF is also induced by fasting, which leads to physiological hepatic steatosis. These findings suggest that PLZF may be involved in the pathogenesis of lipid dysregulation and accumulation. Further studies are required to elucidate the role of PLZF in the regulation of lipid metabo-


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terferon-stimulated genes but also triggers gluconeogenesis to provide sufficient energy for the immune defense. PLZF robustly responds to glucocorticoids, which are synthesized and secreted by the adrenal cortex. Through binding to GR, glucocorticoids regulate many essential biological functions, such as lipolysis (38), gluconeogenesis (39), and cardiovascular (40) and immunological processes (41). The induction of PLZF by glucocorticoids has been shown in various cells, including the T47D/A1-2 breast cancer cell line (42), primary human endometrial stromal cells, and myometrial smooth muscle cells (43). Importantly, such induction is believed to be, in part, the cause of breast cancer cell death Figure 6. PLZF is a downstream effector for PGC-1␣-controlled gluconeogenesis. A, RT-qPCR (left panel) and Western blot (right panel) analyses of gluconeogenic gene expression in AML-12 after Dex treatment (42). In agreecells were transfected with Scrb or PLZF siRNA oligonucleotides, together with plasmid vector ment with previous studies, we also DNA or plasmids encoding PGC-1␣, for 48 hours. **, P ⬍ .01 vs Vector⫹Scrb; ##, P ⬍ .01 vs found that PLZF is induced by Dex PGC-1␣⫹Scrb. B, Glucose output assays in mouse primary hepatocytes. **, P ⬍ .01 vs Vector⫹Scrb; ##, P ⬍ .01 vs PGC-1␣⫹Scrb. C, Western blot analysis for Akt phosphorylation and HCS in liver cells. In contrast, levels. AML-12 cells were treated as described for A. Before harvest, the cells were challenged PLZF expression is not altered in the with 100 nM insulin for 10 minutes. **, P ⬍ .01 vs Vector⫹Scrb; ##, P ⬍ .01 vs PGC-1␣⫹Scrb. glucocorticoid-treated KG-1 cell D, CoIP analysis in the AML-12 cells. E, A model illustrating the role of PLZF in the regulation of line (human Caucasian bone marhepatic gluconeogenesis. Gluconeogenic signals activate the PGC-1␣/GR complex, which in turn induces PLZF expression. As a downstream effector, PLZF triggers hepatic gluconeogenesis and row myelogenous leukemia), alincreases the glucose output. Moreover, PLZF negatively regulates the insulin signaling pathway. though these cells have endogenous GR (43). This finding indicates that induction of PLZF by glucocorticolism, including fatty acid oxidation, lipid synthesis, and ids is a cell type–specific event caused by the interaction of the mobilization and/or conversion of triglycerides, fatty GR with cell-specific cofactors. acids, and cholesterol. Hepatic insulin signaling ranks as the most important Another important function of PLZF is to regulate inpathway in regulation of glucose homeostasis. When in⫺/⫺ nate immunity (16). PLZF mice are more prone to sulin is secreted, it will bind to the insulin receptors loviral infections and are not protected by interferon (IFN). cated in the cell membrane, which in turn phosphorylate This is due to the failure to induce a subset of interferonand activate IRS1 and IRS2. Both substrates in active stimulated genes and the impairment of IFN-induced actiforms trigger the PI3K/Akt cascade to inhibit FoxO1 tran⫺/⫺ vation of natural killer cells in PLZF mice (16). Thus, scriptional activity, thus turning off the gluconeogenesis PLZF is required in the IFN-mediated antiviral innate imby repressing key regulators, such as PGC-1␣ and glucomune response in vivo. On the other hand, the immune neogenic genes (44). In this view, the IRS/Akt/FoxO1 sigresponse is an energy-demanding biological process accom- naling axis contributes importantly to insulin actions. In panied by the consequential reduction in food intake (37); our study, we found that PLZF negatively regulates the therefore, the host’s metabolic activities should be coordi- insulin signaling pathway by decreasing the phosphorynately regulated to increase energy availability. Hepatic glu- lation of IRS1, Akt, and FoxO1 in normal mice. This may coneogenesis, as well as muscle catabolism, usually occurs contribute to the impaired insulin sensitivity observed in during this process. The requirement for PLZF in both the vivo. Therefore, the elevated blood glucose levels induced immune response and gluconeogenesis makes it a very by PLZF not only are due to its positive regulation of promising molecule finely orchestrating these 2 processes. gluconeogenesis but also are dependent on its negative Activation of PLZF not only drives the transcription of in- effects on the insulin signaling pathway. Our results col-


doi: 10.1210/me.2014-1164

lectively demonstrate that PLZF regulates glucose homeostasis in a systemic manner. More interestingly, PLZF is induced in energy-consuming tissues (brown adipose tissues and skeletal muscles) but is inhibited in energystoring tissues (white adipose tissues), under the fasting state. Further studies are required to elucidate how nutritional signals coordinately regulate PLZF expression in various metabolic tissues to achieve the balance of energy metabolism. Taken together, our results reveal a positive role for PLZF in the regulation of hepatic gluconeogenesis (Figure 6E), which expands our knowledge for understanding the important metabolic coordination necessary for proper blood glucose control. Because gluconeogenesis is tightly linked to the pathogenesis of various diseases, such as tumor, infections, and metabolic diseases, manipulation of PLZF expression levels may offer a useful strategy to treat these diseases in the view of metabolic control.

Acknowledgments We thank Dr Jiandie Lin (Life Sciences Institute, University of Michigan, Ann Arbor, Michigan) for providing us adenoviruses (ad-GFP, ad-PGC-1␣, ad-scrambled, and ad-PGC-1␣ RNAi) and plasmids encoding PGC-1␣ and GR. Address all correspondence and requests for reprints to: Chang Liu, PhD, Jiangsu Key Laboratory for Molecular and Medical Biotechnology and College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, China. E-mail: [email protected]. This work was supported by grants from the National Basic Research Program of China (973 Program) (2012CB947600 and 2013CB911600), the National Natural Science Foundation of China (31422028, 31171137, 31271261, and 31401009), the Program for the Top Young Talents by the Organization Department of the CPC Central Committee, the Science Fund for Distinguished Young Scholars of Jiangsu Province (BK20140041), the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine (Nanjing Medical University), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The innovative Projects of Scientific Research for Graduate Students of Jiangsu Province. Disclosure Summary: The authors have nothing to disclose.

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Analysis of the interaction between Zinc finger protein 179 (Znf179) and promyelocytic leukemia zinc finger (Plzf).

Interleukin-32α modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase Cɛ-dependent sumoylation.

Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20.

Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia.

The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization.

Zinc finger protein 382 is downregulated by promoter hypermethylation in pediatric acute myeloid leukemia patients.

Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt.

CDKN1A) gene repression.

Ca2(+)-fatty acid interaction in the control of hepatic gluconeogenesis.

Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure.

CDKN1A) and tumor protein p53 (TP53) genes.

Inhibition of hepatic gluconeogenesis by dichloroacetate.

Inhibition of hepatic gluconeogenesis by phenethylhydrazine (phenelzine).

Investigation of zinc-binding affinities of Moloney murine leukemia virus nucleocapsid protein and its related zinc finger and modified peptides.

C2H2 zinc finger protein.

The B-box 1 dimer of human promyelocytic leukemia protein.

Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

Fluorescent protein-based molecular beacons by zinc finger protein-guided assembly.

Promyelocytic Leukemia Protein (PML) Controls Listeria monocytogenes Infection.

Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation.

Hormonal control of gluconeogenesis.

In-depth study of DNA binding of Cys2His2 finger domains in testis zinc-finger protein.

Regulation of hepatic energy metabolism and gluconeogenesis by BAD.

Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein.