Diabetes Volume 63, October 2014 3199

Kadiombo Bantubungi,1,2,3,4 Sarah-Anissa Hannou,1,2,3,4 Sandrine Caron-Houde,1,2,3,4 Emmanuelle Vallez,1,2,3,4 Morgane Baron,1,2,3,4 Anthony Lucas,1,2,3,4 Emmanuel Bouchaert,1,2,3,4 Réjane Paumelle,1,2,3,4 Anne Tailleux,1,2,3,4 and Bart Staels1,2,3,4

Cdkn2a/p16Ink4a Regulates Fasting-Induced Hepatic Gluconeogenesis Through the PKA-CREB-PGC1a Pathway

Diabetes 2014;63:3199–3209 | DOI: 10.2337/db13-1921

Type 2 diabetes (T2D) is hallmarked by insulin resis- required to identify new strategies for the prevention and tance, impaired insulin secretion, and increased hepatic treatment of T2D (3,4). Hence, recent - glucose production. The worldwide increasing preva- wide association studies (GWAS) have identified a poly- lence of T2D calls for efforts to understand its patho- morphism on 9p21 (rs10811661), located genesis in order to improve disease prevention and

~125 kb upstream of the CDKN2B and CDKN2A , METABOLISM management. Recent genome-wide association stud- that is strongly and reproducibly linked to T2D (5–7), ies have revealed strong associations between the establishing genes on the CDKN2A/B among the CDKN2A/B CDKN2A/B locus and T2D risk. The locus strongest candidates for conferring susceptibility to T2D contains genes encoding inhibitors, including Ink4a across different ethnicities (4). , which have not yet been implicated in the con- The products are the cyclin-dependent kinase trol of hepatic glucose homeostasis. Here, we show that Ink4a ARF Ink4a fi (CDK) inhibitors p16 and p14 for the CDKN2A p16 de ciency enhances fasting-induced hepatic Ink4b glucose production in vivo by increasing the expression locus and p15 for the CDKN2B locus, which are tumor Ink4a suppressors acting as cell cycle inhibitors (8,9). The of key gluconeogenic genes. p16 downregulation Ink4b Ink4a leads to an activation of PKA-CREB-PGC1a signaling p15 and p16 bind to either CDK4 or through increased phosphorylation of PKA regulatory CDK6, thus inhibiting the action of cyclin D and prevent- subunits. Taken together, these results provide evidence ing retinoblastoma phosphorylation and sub- that p16Ink4a controls fasting glucose homeostasis and sequent release of the E2F1 transcription factor. As a could as such be involved in T2D development. consequence, the transcription of genes required for cell cycle progression to the is restrained. However, how the CDKN2A/B gene products modulate Type 2 diabetes (T2D) is a complex metabolic disorder glucose metabolism is less clear. In murine models, involving a combination of insulin resistance, impaired increased expression of p15Ink4b in pancreatic islets is as- insulin secretion, and increased hepatic glucose produc- sociated with islet hypoplasia and impaired glucose-induced tion (1,2). The pathogenesis of T2D is multifactorial, in- insulin secretion (10). Moreover, p16Ink4a plays a crucial volving both genetic and environmental susceptibility role in senescence and aging. p16Ink4a expression increases factors (3). During these last few years, the search for with age in pancreatic b-cells and promotes an age- genetic determinants of T2D greatly progressed, identify- dependent decline in islet regenerative potential (11). Ad- ing new loci contributing to T2D. A better understand- ditionally, other cell cycle regulators, like CDK4, E2F1, and ing of the function of the gene products of these loci is cyclin D, also play roles in glucose homeostasis through

1Université Lille 2, Lille, France This article contains Supplementary Data online at http://diabetes 2INSERM, U1011, Lille, France .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1. 3 European Genomic Institute for Diabetes, Lille, France K.B. and S.-A.H. contributed equally to this study. R.P., A.T., and B.S. are senior 4 Institut Pasteur de Lille, Lille, France authors. Corresponding author: Bart Staels, [email protected]. © 2014 by the American Diabetes Association. Readers may use this article as Received 20 December 2013 and accepted 23 April 2014. long as the work is properly cited, the use is educational and not for profit, and the work is not altered. 3200 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014 actions in the pancreas, muscle, and/or adipose tissue (12– glucose control and the persistence of fasting hypergly- 16). However, whether the CDKN2A/B gene products mod- cemia are hallmarks of T2D (18). Increased rates of he- ulate hepatic glucose production is unknown (17). patic glucose production are a major cause of fasting Glucose homeostasis is determined by the balance hyperglycemia in T2D patients (1). In physiological con- of its production and utilization. Impaired postprandial ditions, during prolonged fasting, hepatic gluconeogenesis

Figure 1—p16Ink4a deficiency increases glucose production and gluconeogenic without modulating genes involved in glucose and lipid utilization in liver. A: 12-week-old p162/2 mice (n = 9) display higher blood glucose levels after 24-h fasting than wild-type p16+/+ mice (n = 9). Unpaired Student t test (*compared between the genotypes of the same treatment group; #compared between the treatment groups of the same genotype: * or #P < 0.05, ** or ##P < 0.01). Data are means 6 SEM. B: PTT shows increased glucose production in 12-week-old p162/2 compared with p16+/+ mice (n = 6). Two-way ANOVA and Newman-Keuls post hoc test (*compares genotypes: *P < 0.05, **P < 0.01). C: Area under the curve (iAUC) of PTT is higher in p162/2 vs. p16+/+ mice. Student t test (*P < 0.05). Data are means 6 SEM. D–F: The expression of gluconeogenic genes (G6pase, Fbp1, Pepck) is increased in p162/2 vs. p16+/+ mice (n = 10) after 24-h fasting. mRNA level of genes involved in glycolysis (Gk, Lpk, Pdk4)(G–I) and b-oxidation (Cpt1a, Lcad) (J and K) pathways is unchanged in livers of p16 2/2 vs. p16+/+ mice (n = 10). Two-way ANOVA and LSD Fisher post hoc test (*compared between the genotypes of the same treatment group; #compared between the treatment groups of the same genotype: * or #P < 0.05, ** or ##P < 0.01, ###P < 0.001, ####P < 0.0001). Data are means 6 SEM. diabetes.diabetesjournals.org Bantubungi and Associates 3201 is a major pathway for the maintenance of normal plasma firm texture. The soft liver was removed and cut into glucose levels (19) owing to the action of different hor- pieces and the homogenate filtered and centrifuged for mones, among which are glucagon and glucocorticoids, 2 min. The pellet was washed three times and resus- like cortisol. During starvation, low blood glucose levels in- pended in Williams medium supplemented with 0.1% duce pancreatic a-cell glucagon secretion and hypothalamic- BSA, 1% glutamine, 1% gentamycine, 100 nmol/L insulin, pituitary-adrenal axis activation. In the liver, glucagon and 100 nmol/L dexamethasone. Cell number and viability binds to its receptor, which then causes a GDP/GTP were assessed using trypan blue. Cells were plated on six-well exchange, hence stimulating adenylate-cyclase activity, plates during 2 h for hepatocyte selection and then incu- which converts ATP into cAMP (20). The rise in intra- bated in deprivation medium (1% penicillin-streptomycin, cellular cAMP levels stimulates the dissociation of the and 1% glutamine, with distinct concentrations of glucagon catalytic and regulatory subunits of protein kinase A [0, 1, 10, and 100 nmol/L]) for 6–8 h (for RNA measure- (PKA) (21). The catalytic PKA subunit then enters the ments) or 30 min (for protein analysis). 133 nucleus where it phosphorylates the CREB at Ser , Mouse Hepatocyte Cell Line Culture and Treatments converting it into its transcriptionally active form, which Alpha Mouse Liver 12 (AML12) (cat. no. CRL2254; induces gluconeogenic gene expression (22–24).Incon- American Type Culture Collection) cells were cultured in cert, glucocorticoids activate the glucocorticoid receptor, deprivation medium–Ham’s F-12 supplemented with 10% which binds to glucocorticoid-responsive elements in the FBS (Invitrogen), 5 g/mL insulin (Sigma), 5 g/mL trans- promoters of gluconeogenic genes (25,26). ferrin (Sigma), 5 ng/mL selenium (Sigma), 1% glutamine, Given the strong association of the CDKN2A/B locus and 1% penicillin-streptomycin and maintained at 37°C under with T2D risk, which in large population studies is mainly 5% CO . AML12 cells were transfected with small interfering established by the measurement of fasting hyperglycemia 2 RNA (siRNA) for CDKN2A (043107-00-005; Thermo Scien- (5), we set out to study whether p16Ink4a plays a role in tific [ON-TARGET plus SMART pool siRNA]), CDK4 (ON- hepatic glucose homeostasis using p16Ink4a-deficient mice 2 2 L-040106-00-0005; Thermo Scientific [ON-TARGET (p16 / ), mouse primary hepatocytes, and mouse hepatic Ink4a plus SMARTpool siRNA]), or control (D-001810-10-20; cell line. Our results identify p16 as a modulator of Thermo Scientific [ON-TARGET plus nontargeting pool the PKA-CREB–peroxisome proliferator–activated recep- tor g coactivator (PGC1a) signaling pathway and, hence, as a regulator of fasting hepatic glucose homeostasis, in- dependent of its function as cell cycle regulator.

RESEARCH DESIGN AND METHODS Animal Experiments 2 2 p16 / and littermate control (p16+/+) mice on a C57Bl6 background (.97%) were housed under standard condi- tions in conventional cages with free access to water and food unless indicated otherwise. Twelve-week-old male mice were killed by cervical dislocation at 9:00 A.M. after a 24-h fasting. Experimental procedures were conducted with the approval of the ethics committee for animal experimentation of the Nord Pas-de-Calais re- gion (CEEAA022008R). Pyruvate Test Overnight fasted mice (5:00 P.M. to 9:00 A.M.) were injected with sodium pyruvate (P4562; Sigma) (2 g/kg body wt i.p.). Blood glucose levels were measured from the tail vein at the indicated time points using an automatic glu- cose monitor (OneTouch; LifeScan).

Mouse Primary Hepatocyte Isolation, Culture, and Figure 2—p16Ink4a deficiency increases gluconeogenic gene ex- Treatments pression and glucose production in primary hepatocytes. The in- Mice were anesthetized with a mixture of ketamine (100 duction of gluconeogenic genes (G6pase [A], Fbp1 [B], Pepck [C]) by glucagon (8 h) is higher in primary hepatocytes isolated from mg/kg) and xylasine (20 mg/kg) administered intraperi- p162/2 vs. p16+/+ mice. Two-way ANOVA and LSD Fisher post toneally. Livers were perfused in situ through the inferior hoc test (*compared between the genotypes of the same treatment cava vein, with Hanks’ balanced salt solution (H9394; group; #compared between the treatment groups of the same ge- < < < Sigma) containing 0.5 mmol/L EGTA and 50 mmol/L notype: ** or ##P 0.01, *** or ###P 0.001, **** or ####P 0.0001). Data are means 6 SEM. Glucose production is higher in ’ 2 2 HEPES followed by Hanks balanced salt solution contain- primary hepatocytes isolated from p16 / vs. p16+/+ mice (D). Stu- ing 0.025% collagenase (C5138; Sigma) until loss of its dent t test (**P < 0.01). Data are means 6 SEM. 3202 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014 siRNA]) using the Dharmafect1 reagent (Thermo Scientific) Reverse transcribed cDNAs were quantified by Brilliant according to the manufacturer’s instructions. AML12 cells III Ultra-Fast SYBR green-based real-time PCR using spe- were treated for the indicated times points with 10 mmol/L cific oligonucleotides (Supplementary Table 1) on a Strata- forskolin. gene Mx3005P (Agilent Technologies) apparatus. mRNA Glucose Production Assay levels were normalized to Cyclophilin A expression as an internal control, and mRNA fold induction was calculated Primary hepatocytes were cultured in six-well plates in 2ΔΔ using the comparative Ct (2 Ct) method. Williams medium with 0.1% BSA, 100 nmol/L dexameth- asone, 1% penicillin-streptomycin, and 1% glutamine. Western Blot Analysis After 2 h, the medium was replaced with 1 mL glucose- AML12 cells and mouse primary hepatocytes were lysed production buffer consisting of glucose-free Krebs-ringer with cell lysis buffer (50 mmol/L Tris-HCl, pH 8; 137 buffer (115 mmol/L NaCl, 5.9 mmol/L KCI, 1.2 mmol/L mmol/L NaCl; 5 mmol/L Na EDTA; 2 mmol/L EGTA; 1% MgCl , 1.2 mmol/L NaH PO , and 2.5 mmol/L NaHCO 2 2 2 4 3 Triton; 20 mmol/L sodium pyrophosphate; 10 mmol/L pH 7.4) without phenol red, supplemented with 15 mmol/L b-glycerophosphate; 1mmol/L Na VO ;10mmol/L sodium lactate and 1 mmol/L sodium pyruvate. Glucose 3 4 leupeptin; and 5 mmol/L pepestatin A) (Sigma-Aldrich) on concentrations were measured at different time points ice. Cells were scraped and transferred to 1.5-mL Eppendorf with a colorimetric glucose assay kit (Sigma). The values tubes and rotated for 30 min at 4°C, followed by centri- were then normalized to total protein content determined fugation at 13,000g for 10 min at 4°C. The resulting on whole-cell lysates. supernatants were stored in aliquots at 280°C until Gene Expression Analysis they were required. Protein concentrationinthecell Liver total RNA was isolated using the guanidinium lysates was determined using a BCA protein assay kit isothiocyanate phenol/chloroform extraction method, (Pierce). The cell lysates were mixed with 4X-SDS sample and total RNA from cultured cells was extracted using buffer NOVEX (Life Technologies). Samples were heated the TRIzol reagent (Eurobio). One microgram of total at 100°C for 10 min before loading and being separated RNA was reverse transcribed to cDNA using the High- on precasted 4–12% or 3–8% SDS-PAGE (Invitrogen). Capacity cDNA Reverse Transcription kits (Applied Bio- Proteins were electrotransferred to a nitrocellulose systems) according to the manufacturer’s instructions. membrane (Millipore, Bedford, MA) in 1X transfer buffer

Figure 3—p16Ink4a downregulation increases gluconeogenic gene expression in AML12 cells. SiRNA CDKN2A treatment (which affects both p16Ink4a and p19ARF expression) in AML12 strongly decreases p16Ink4a mRNA level measured by RT–quantitative PCR (A) and p16Ink4a protein level measured by Western blot analysis (B). Student t test (***P < 0.001). Data are means 6 SEM. C: p16Ink4a protein level is comparable in liver, primary hepatocytes, and AML12 cells. D–F: p16Ink4a-silenced and p16Ink4a-expressing AML12 were treated with 10 mmol/L forskolin (FSK) for 16 h. The expression of G6pase and Fbp1 genes (D and E) was increased but that of Pepck was not (F)in p16Ink4a-silenced compared with p16Ink4a-expressing AML12 cells. Two-way ANOVA and LSD Fisher post hoc test (*compared between the genotypes of the same treatment group; #compared between the treatment groups of the same genotype: **P < 0.01, ***P < 0.001, **** or ####P < 0.0001). Data are means 6 SEM. diabetes.diabetesjournals.org Bantubungi and Associates 3203

(Invitrogen) using the Nupage Systeme for 1 h at 30 V. overnight with antibodies against p16ink4a (M-156, sc-1207; Nonspecific binding to the membrane was blocked for 1 h Santa Cruz Biotechnology) and phospho-PKAR2 (Ab-32390; at room temperature with 5% nonfat milk in Tween–Tris- Abcam) and subsequently incubated with a combination of buffered saline (TTBS) buffer (20 mmol/L Tris, 500 mmol/L Texas red–conjugated anti-rabbit IgG and FITC-conjugated sodium NaCl, and 0.1% Tween 20). Membranes were then anti-mouse IgG. A nuclear DAPI counterstain was also incubated overnight at 4°C with various primary antibodies performed. in blocking buffer containing 5% nonfat milk at the dilu- fi Statistics tion speci ed by the manufacturers. The following primary 6 133 Data are expressed as means SEM. Results were ana- antibodies were used: phospho-CREB (Ser )(9198;Cell lyzed by unpaired two-tailed Student t test or one-way Signaling Technology), CREB (9197; Cell Signaling Technol- ANOVA with least significant difference (LSD) Fisher ogy), phospho–(S/T)-PKA substrates (9621; Cell Signaling post hoc test or two-way ANOVA with LSD Fisher post Technology), phospho-pRb (3590; Cell Signaling Technol- hoc test as appropriate using GraphPad Prism software. A ogy), pRb (9313; Cell Signaling Technology), PGC1a (sc- P value of , 0.05 was considered statistically significant. 13067; Santa Cruz Biotechnology), GAPDH (sc-25778; Santa Cruz Biotechnology), p16ink4a (sc-1207; Santa Cruz RESULTS – Ink4a Biotechnology), phospho regulatory subunit 2 of PKA p16 Deficiency Results in Fasting Hyperglycemia (PKAR2) (ab32390; Abcam), and PKAR2 (ab-38949; and Increased Gluconeogenesis Abcam). Membranes were then incubated with the second- Since GWAS revealed an association between the ary antibody conjugated with the enzyme horseradish per- CDKN2A/B locus and T2D risk, primarily based on the oxidase. The visualization of immunoreactive bands was fasting plasma glucose trait, we first measured fed and 2 2 performed using the enhanced chemiluminescence plus fasted blood glucose levels in 12-week-old mice. p16 / Western blotting detection system (GE Healthcare). Quan- mice displayed a less pronounced hypoglycemia after 24 h tification of phospho-CREB level in mouse primary he- of fasting compared with p16+/+ mice (Fig. 1A). This effect patocytes and AML12 cells was performed by volume was not due to differences in plasma glucagon levels densitometry using the ImageJ 1.47t software (National Institutes of Health). Cyclic AMP and PKA Assay Intracellular cAMP concentrations were measured using a ready-to-use competitive enzyme immunoassay kit (R&D Systems). Briefly, cells were lysed according to the manufacturer’s protocol, and 100 mL sample was mixed with 50 mL cAMP conjugated and then added to cAMP- specific antibody precoated microplate. After 2 h of in- cubation at room temperature, substrate solution was added for 20 min. Color development was stopped, and the absorbance at 450 nm was measured using a Dynex MRX TC Revelation Microplate Reader. PKA activity was measured by the signaTECT cAMP-Dependent Protein Ki- nase Assay System by using the Kemptide (LRRASLG) as a peptide substrate.

Coimmunoprecipitation Assay Coimmunoprecipitation of CDK4 from whole AML12 cell extracts was performed using the Thermo Scientific Pierce Crosslink Magnetic IP/Co-IP kit. Briefly, 48 h after siRNA transfection, cells were lysed and 500 mg total protein extract was incubated with 3 mgCDK4antibody(sc-260; Figure 4—p16Ink4a downregulation increases Pgc1a gene expres- Santa Cruz Biotechnology) according to the manufactur- sion. Pgc1a mRNA levels are higher in livers of p162/2 compared ’ with p16+/+ mice (A), in primary hepatocytes isolated from p162/2 er s protocol. The eluate was then subjected to Western +/+ blot analysis using PKAR2 (ab-38949; Abcam) and CDK4 compared with p16 mice and treated with glucagon for8h(B), and in p16Ink4a-silenced compared with p16Ink4a-expressing AML12 (sc-260; Santa Cruz Biotechnology). cells treated with 10 mmol/L forskolin (FSK) for 16 h (C). D: Western blots show higher increase of PCG1a protein level in p16Ink4a- Immunofluorescence Assay in AML12 Cells silenced compared with p16Ink4a-expressing AML12 cells treated Cells were grown on cover slips. At 4 8h after siRNA with 10 mmol/L forskolin for 1 h. Two-way ANOVA and LSD Fisher transfection, cells were washed with PBS and fixed with post hoc test (*compared between the genotypes of the same treat- fi ment group; #compared between the treatment groups of the same 4% paraformaldehyde for 20 min. After xation and genotype: #P < 0.05, ** or ##P < 0.01, **** or ####P < 0.0001). permeabilization with 0.1% TRITON, cells were incubated Data are means 6 SEM. 3204 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014

2 2 between fasted p16+/+ and p16 / mice (Supplementary differences in liver weight compared with p16+/+ mice Fig. 1). For evaluation of whether gluconeogenesis was (Supplementary Fig. 2A and B). Moreover, immunohisto- influenced, a pyruvate tolerance test (PTT) was performed chemical Ki-67 staining of liver sections showed no differ- 2 2 2 2 2 2 in fasted p16 / and p16+/+ mice. Interestingly, p16 / ences between p16 / mice and their littermate controls mice produced higher blood glucose levels, upon pyruvate under fasting conditions (Supplementary Fig. 2C–H), in- administration, suggesting an increased hepatic glucose dicating that hepatocyte proliferation is not different. These production (Fig. 1B and C). Consistent with this, hepatic data indicate that p16Ink4a deficiency increases fasting- mRNA levels of gluconeogenic genes, such as G6pase and induced hepatic gluconeogenesis in vivo, independent of 2 2 Pepck,weresignificantly higher in livers of fasted p16 / any action on hepatocyte proliferation. +/+ versus p16 mice (Fig. 1D and F), while Fbp1 mRNA p16Ink4a Deficiency Increases Gluconeogenic Gene was not different between the genotypes (Fig. 1E). Con- Expression and Glucose Production In Vitro in versely,genesinvolvedinothermetabolicpathwaysreg- Hepatocytes ulated during fasting, such as glycolysis (Gk, Lpk)and For analysis of whether the altered regulation of hepatic 2 2 b-oxidation (Cpt1a, Lcad), were not differently expressed gluconeogenic gene expression in p16 / mice is a cell- between both genotypes upon fasting (Fig. 1G, H, J, and autonomous phenomenon, primary hepatocytes from 2 2 K), although mRNA levels of Pdk4, which block glycolysis p16 / mice and their littermate controls were isolated at the level of pyruvate dehydrogenase, tended to be and incubated with increasing concentrations of glucagon 2 2 higher in fasted p16 / livers (Fig. 1I). Altogether, these to mimic the fasting conditions. Basal levels of gluco- data indicate that among the different hepatic metabolic neogenic gene expression were 1.5-fold higher for pathways regulated by fasting, gluconeogenesis is the only G6Pase (60.15; P , 0.05), 4.4-fold higher for Pepck 2 2 one modulated in p16 / mice. (60.96; P , 0.01) and 2.3-fold higher for Fbp1 (0.17; 2 2 Since p16Ink4a is a tumor suppressor and a cell cycle P , 0.001) in p16 / compared with p16+/+ primary regulator and since hepatic proliferation and tumor hepatocytes (Fig. 2A–C). Moreover, glucagon, which growth may perturb glucose homeostasis, we investigated activates the PKA-CREB signaling pathway, more pro- whether p16Ink4a deficiency is associated with spontane- nouncedly induced G6pase, Pepck,andFbp1 (Fig. 2A–C) 2 2 ouslivertumorgrowthoralteredhepatocyteproliferationin mRNA levels in p16 / vs. p16+/+ primary hepatocytes. 2 2 our experimental conditions. At the age of 12 weeks, p16 / Further, hepatic glucose production was higher in pri- 2 2 mice did not display macroscopic liver abnormalities or mary hepatocytes of p16 / than of p16+/+ mice (Fig.

Figure 5—p16Ink4a downregulation increases the phosphorylation of CREB. Western blots show higher increase of p-CREB in primary hepatocytes isolated from p162/2 compared with p16+/+ mice and treated with glucagon for 1 h (A) and in p16Ink4a-silenced compared with p16Ink4a-expressing AML12 cells treated with 10 mmol/L forskolin (FSK) for 1 h (C). B and D: The bar graphs are the quantification of p-CREB Western blots in primary hepatocytes isolated from p162/2 and p16+/+ mice and treated with glucagon for 1 h and p16Ink4a- silenced and p16Ink4a-expressing AML12 cells treated with 10 mmol/L forskolin for 1 h. Two-way ANOVA and LSD Fisher post hoc test (*compared between the genotypes of the same treatment group; #compared between the treatment groups of the same genotype: * or #P < 0.05, ** or ##P < 0.01, *** or ###P < 0.001). Data are means 6 SEM. diabetes.diabetesjournals.org Bantubungi and Associates 3205

2D). Next, p16Ink4a was silenced using a CDKN2A compared with p16+/+ hepatocytes (Fig. 4B). p16Ink4a si- siRNA (which affects both p16Ink4a and p19ARF expres- lencing in AML12 cells significantly increased Pgc1a ex- sion) in AML12 cells (Fig. 3A and B), a mouse hepato- pression at both mRNA and protein levels upon forskolin cyte cell line that expresses very high levels of p16Ink4a treatment (Fig. 4C and D). compared with liver and primary hepatocytes (Fig. 3C). p16Ink4a Deficiency Increases the PKA-CREB Signaling Incubation with forskolin, to activate the PKA-CREB Pathway pathway, resulted in a more pronounced increase of Ink4a Ink4a To gain insight into how PGC1a is induced upon p16 - G6pase and Fbp1 gene expression when p16 was deficiency, we first analyzed the phosphorylation status of silenced, while no effect was observed on Pepck gene CREB,atranscriptionfactorinducingPGC1a expression. – 2 2 expression (Fig. 3D F). Moreover, although G6pase and p-Ser133-CREB was markedly higher in p16 / compared Fbp1 gene expression only marginally increased upon with p16+/+ hepatocytes at the basal level as well as after forskolin treatment in p16Ink4a-expressing AML12 Ink4a glucagon exposure (Fig. 5A and B). Similar results were cells, p16 silencing resulted in the restoration of obtained upon forskolin treatment (data not shown). a strong response (Fig. 3D and E). Altogether, these Likewise, p16Ink4a silencing in AML12 cells resulted in Ink4a fl results indicate that p16 expression levels in uence a stronger CREB phosphorylation both at the basal level the response to fasting-induced stimuli both in vivo and in and upon forskolin treatment (Fig. 5C and D). Al- vitro. together, these data demonstrate that p16Ink4a knock- p16Ink4a Levels Modulate PGC1a Expression in Vivo down increases CREB phosphorylation. It is well-known and In Vitro that the cAMP-PKA signaling pathway regulates fasting- For studying of the mechanism by which p16Ink4a regu- induced CREB phosphorylation (22,27). To test whether lates gluconeogenic gene expression, mRNA and protein alterations in PKA activity may explain the increased levels of PGC1a, a master regulator of the fasting adap- CREB phosphorylation upon p16Ink4a deficiency, p16Ink4a- tation process (24), were measured. The fasting response silenced AML12 cells were treated with H89, a specific of Pgc1a mRNA was significantly more pronounced in PKA inhibitor. H89 treatment prevented CREB phos- 2 2 livers of p16 / compared with p16+/+ mice (Fig. 4A). phorylation induced by p16Ink4a silencing (Fig. 6A). Ac- 2 2 In line, p16 / primary hepatocytes displayed a 3.4-fold cordingly, PKA activity in p16Ink4a-silenced AML12 cells increased Pgc1a mRNA level (60.83; P , 0.01, two-tailed was 1.5-fold higher compared with control (Fig. 6B). This Student t test) and a stronger induction by glucagon increase was substantiated by the increase in total PKA

Figure 6—p16Ink4a downregulation increases PKA activity. Western blots show a stronger decreased p-CREB in p16Ink4a-silenced com- pared with p16Ink4a-expressing AML12 cells treated with 20 mmol/L H89 (A) and an increased PKA activity upon p16Ink4a silencing in AML12 cells (B). Student t test (*P < 0.05). Data are means 6 SEM. Western blots show higher increase of global profile of PKA phosphorylated substrates in p16Ink4a-silenced and p16Ink4a-expressing AML12 under basal conditions (C) and in primary hepatocytes isolated from p162/2 and p16+/+ mice treated with glucagon for1h(D). 3206 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014 substrate phosphorylation profiles upon p16Ink4a silencing changes in intracellular cAMP levels. To understand the (Fig. 6C). Likewise, several PKA substrates were more underlying mechanism by which p16Ink4a increases phos- 2 2 phosphorylated in p16 / than in p16+/+ primary hepa- phorylation of PKAR2 and thereby the increase of gluco- tocytes both under basal conditions and after glucagon neogenic genes, we investigated the involvement of CDK4, stimulation (Fig. 6D). Since PKA activity is controlled at a well-known target of p16Ink4a. Silencing of CDK4 in least in part by the phosphorylation of its regulatory sub- p16Ink4a-silenced AML12 cells (Fig. 8A and B) abrogated units (PKAR2), the expression and phosphorylation of the induction of Ppc1a and Fbp1 mRNA levels by p16Ink4a PKAR2 were measured in p16Ink4a-silenced AML12 cells silencing (Fig. 8C and D). Moreover, coimmunoprecipita- 2 2 and in p16 / primary hepatocytes. p16Ink4a silencing or tion experiments in AML12 cells after p16Ink4a knockdown deficiency resulted in increased PKAR2 phosphorylation demonstrated a physical interaction between CDK4 and 2 2 in AML12 cells (Fig. 7A) and in p16 / primary hepato- PKAR2 (Fig. 8E). cytes both at the basal state and upon glucagon stimula- tion (Fig. 7B). This result was confirmed by the enhanced DISCUSSION p-PKAR2 immunostaining in p16Ink4a-silenced AML12 cells In recent years, a growing body of evidence supports the (Fig. 7C). Noteworthy, the increased PKA activity was not emerging notion that cell cycle regulatory proteins due to an increase in cAMP levels (Fig. 7D). Collectively, contribute to metabolic processes in addition to, or linked these data demonstrate that p16Ink4a-deficiency activates with, their role in cell growth (17,28). Today, these pro- the PKA-CREB-PGC1a signaling pathway independent of teins are perceived as sensors of external signals that

Figure 7—p16Ink4a downregulation increases PKAR2 phosphorylation without affecting intracellular cAMP levels. Western blots show higher p-PKAR2 in p16Ink4a-silenced than in p16Ink4a-expressing AML12 cells (A) and in primary hepatocytes from p162/2 than in p16+/+ mice treated or not with glucagon for 1 h (B). C: Immunofluorescent staining of p-PKAR2 in p16Ink4a-silenced and p16Ink4a-expressing AML12 cells. Original magnification 320. D: cAMP levels were measured in p16Ink4a-silenced and p16Ink4a-expressing AML12 under basal conditions. Student t test (not significant). Data are means 6 SEM. diabetes.diabetesjournals.org Bantubungi and Associates 3207

Figure 8—p16Ink4a downregulation increases gluconeogenic gene expression in AML12 cells in a CDK4-dependent manner. SiRNA CDK4 treatment in AML12 strongly decreases CDK4 mRNA level (A) measured by RT–quantitative PCR without affecting p16Ink4a mRNA level (B). The p16Ink4a downregulation–induced increase of Pgc1a and Fbp1 mRNA expression (C and D) was abrogated by siRNA CDK4 treatment. Two-way ANOVA and LSD Fisher post hoc test (*compared AML12 treated by SiRNA CDKN2A or not; #compared AML12 treated by siRNA CDK4:#P < 0.05, *** or ###P < 0.001, ****P < 0.0001). Data are means 6 SEM. E: Coimmunoprecipitation (IP) of CDK4 from whole AML12 cell extracts was performed. The eluate was then subjected to Western blot (WB) analysis against PKAR2. F: Proposed pathway for the control of hepatic gluconeogenesis through p16Ink4a and CDK4. When p16Ink4a is unable to bind CDK4 in the nucleus, CDK4 translocates to the cytoplasm where it interacts with the PKA complex through PKAR2. This interaction leads to an increase of PKA activity, independently of changes in intracellular cAMP levels. Increased PKA activity leads to the activation of the transcription factor CREB and expression of the PGC1a coactivator, which in turn drives the transcription of gluconeogenic enzymes such as PEPCK and G6Pase. AC, adenylate cyclase; C, catalytic subunit of PKA; HPG, hepatic glucose production; R, regulatory subunit of PKA. require a particular adapted metabolic response. The by controlling oxidative metabolism in adipose tissue CDK-Rb-E2F1 pathway, which is inhibited by p16Ink4a, (30). The CDK-Rb-E2F1 pathway is also a negative regulator has already been shown to control adipogenesis by mod- of energy expenditure through repression of mitochondrial ulating the expression of the nuclear receptor PPARg oxidative metabolism in muscle (16). Disruption of CDK (15,29), a master regulator of adipogenesis, as well as inhibitor genes in the mouse has not revealed profound 3208 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014 cell cycle abnormalities but does result in a specificmeta- described in tissues such as adipose tissue and the pan- bolic phenotype. Mice lacking p18Ink4c (31,32), p21cip1,or creas (28), this is the first study that demonstrates a role p27Kip1 display growth abnormalities and adipocyte hy- of a cell cycle regulator, p16Ink4a, in the liver, a master 2 2 2 2 perplasia (33). Double knockout mice (p21 / ;p27 / ) organ regulating glucose homeostasis in a manner inde- develop hypercholesterolemia, glucose intolerance, and pendent of its function in cell proliferation. Thus, altered insulin insensitivity (33). Surprisingly, the role of these p16Ink4a activity may contribute to the association be- cell cycle regulators in the liver, one of the main meta- tween the GWAS locus and the risk of developing T2D. bolic organs controlling glucose homeostasis, has not yet been demonstrated. Acknowledgments. The authors thank P. Krimpenfort for providing the It is well known that an increased rate of hepatic p16-deficient mice and J. Dumont for her assistance. gluconeogenesis contributes to fasting hyperglycemia Funding. This work was supported by grants from the European Genomic observed in T2D patients. Genetic analysis in GWAS Institute for Diabetes (ANR-10-LABX-46). The authors also thank Cost Action fi identi ed an association of the CDKN2A/B locus with (BM0602), Conseil régional Nord Pas-de-Calais, and Fonds Européens de T2D risk (5,34,35). The association is based on the Développement Régional (FEDER). K.B. was supported by a postdoctoral fellow- measurement of fasting glycemia and confers to the ship from Fondation pour la Recherche Médicale (FRM). S.-A.H. was supported CDKN2A/B locus a high susceptibility to T2D across by a doctoral fellowship from Université Lille 2/Conseil régional Nord Pas- different ethnicities. In this study, we tried to elucidate de-Calais and a FRM grant (FDT20130928340). the mechanism by which a product of CDKN2A/B, i.e., Duality of Interest. No potential conflicts of interest relevant to this article p16Ink4a, may influence the hepatic gluconeogenic pro- were reported. gram and thereby be implicated in T2D pathogenesis. Author Contributions. K.B. and S.-A.H. performed experiments, designed experiments, analyzed data, and wrote the manuscript. S.C.-H., E.V., We found that p16Ink4a deficiency raises PKAR2 phos- M.B., A.L., and E.B. performed experiments. R.P., A.T., and B.S. designed experi- phorylation leading to an increased PKA activity. The in- ments, analyzed data, and wrote the manuscript. B.S. is the guarantor of this creased PKA activity enhances CREB-PGC1a signaling, work and, as such, had full access to all the data in the study and takes regulating the gluconeogenic gene expression program. responsibility for the integrity of the data and the accuracy of the data analysis. Since the p16Ink4a protein shares several repeat domains, which are involved in protein-protein inter- References action, we assessed whether p16Ink4a may associate with 1. Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D. Relationships between the PKA complex. Immunoprecipitation of endogenous insulin secretion, insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent diabetic subjects. J Clin Invest 1984;74: p16Ink4a in AML12 cells failed to demonstrate an interac- Ink4a 1238–1246 tion of p16 with the PKA regulatory subunit or the 2. Rizza RA. 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