PKA-CREB-PGC1a Pathwayis a major pathway for the maintenance of normal plasma glucose levels (19) owing to the action of different hor-mones, among which are glucagon and glucocorticoids,

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 RegulatesFasting-Induced HepaticGluconeogenesis Through thePKA-CREB-PGC1a PathwayDiabetes 2014;63:3199–3209 | DOI: 10.2337/db13-1921

Type 2 diabetes (T2D) is hallmarked by insulin resis-tance, impaired insulin secretion, and increased hepaticglucose production. The worldwide increasing preva-lence of T2D calls for efforts to understand its patho-genesis in order to improve disease prevention andmanagement. Recent genome-wide association stud-ies have revealed strong associations between theCDKN2A/B locus and T2D risk. The CDKN2A/B locuscontains genes encoding cell cycle inhibitors, includingp16Ink4a, which have not yet been implicated in the con-trol of hepatic glucose homeostasis. Here, we show thatp16Ink4a deficiency enhances fasting-induced hepaticglucose production in vivo by increasing the expressionof key gluconeogenic genes. p16Ink4a downregulationleads to an activation of PKA-CREB-PGC1a signalingthrough increased phosphorylation of PKA regulatorysubunits. Taken together, these results provide evidencethat p16Ink4a controls fasting glucose homeostasis andcould as such be involved in T2D development.

Type 2 diabetes (T2D) is a complex metabolic disorderinvolving a combination of insulin resistance, impairedinsulin secretion, and increased hepatic glucose produc-tion (1,2). The pathogenesis of T2D is multifactorial, in-volving both genetic and environmental susceptibilityfactors (3). During these last few years, the search forgenetic determinants of T2D greatly progressed, identify-ing new loci contributing to T2D. A better understand-ing of the function of the gene products of these loci is

required to identify new strategies for the prevention andtreatment of T2D (3,4). Hence, recent human genome-wide association studies (GWAS) have identified a poly-morphism on chromosome 9p21 (rs10811661), located~125 kb upstream of the CDKN2B and CDKN2A genes,that is strongly and reproducibly linked to T2D (5–7),establishing genes on the CDKN2A/B locus among thestrongest candidates for conferring susceptibility to T2Dacross different ethnicities (4).

The gene products are the cyclin-dependent kinase(CDK) inhibitors p16Ink4a and p14ARF for the CDKN2Alocus and p15Ink4b for the CDKN2B locus, which are tumorsuppressors acting as cell cycle inhibitors (8,9). Thep15Ink4b and p16Ink4a proteins bind to either CDK4 orCDK6, thus inhibiting the action of cyclin D and prevent-ing retinoblastoma protein phosphorylation and sub-sequent release of the E2F1 transcription factor. As aconsequence, the transcription of genes required for cellcycle progression to the S phase is restrained.

However, how the CDKN2A/B gene products modulateglucose metabolism is less clear. In murine models,increased expression of p15Ink4b in pancreatic islets is as-sociated with islet hypoplasia and impaired glucose-inducedinsulin secretion (10). Moreover, p16Ink4a plays a crucialrole in senescence and aging. p16Ink4a expression increaseswith age in pancreatic b-cells and promotes an age-dependent decline in islet regenerative potential (11). Ad-ditionally, other cell cycle regulators, like CDK4, E2F1, andcyclin D, also play roles in glucose homeostasis through

1Université Lille 2, Lille, France2INSERM, U1011, Lille, France3European Genomic Institute for Diabetes, Lille, France4Institut Pasteur de Lille, Lille, France

Corresponding author: Bart Staels, [email protected].

Received 20 December 2013 and accepted 23 April 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1.

K.B. and S.-A.H. contributed equally to this study. R.P., A.T., and B.S. are seniorauthors.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 63, October 2014 3199

METABOLISM

http://crossmark.crossref.org/dialog/?doi=10.2337/db13-1921&domain=pdf&date_stamp=2014-09-02http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1

actions in the pancreas, muscle, and/or adipose tissue (12–16). However, whether the CDKN2A/B gene products mod-ulate hepatic glucose production is unknown (17).

Glucose homeostasis is determined by the balanceof its production and utilization. Impaired postprandial

glucose control and the persistence of fasting hypergly-cemia are hallmarks of T2D (18). Increased rates of he-patic glucose production are a major cause of fastinghyperglycemia in T2D patients (1). In physiological con-ditions, during prolonged fasting, hepatic gluconeogenesis

Figure 1—p16Ink4a deficiency increases glucose production and gluconeogenic gene expression without modulating genes involved inglucose 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-typep16+/+ mice (n = 9). Unpaired Student t test (*compared between the genotypes of the same treatment group; #compared between thetreatment groups of the same genotype: * or #P < 0.05, ** or ##P < 0.01). Data are means 6 SEM. B: PTT shows increased glucoseproduction in 12-week-old p162/2 compared with p16+/+ mice (n = 6). Two-way ANOVA and Newman-Keuls post hoc test (*comparesgenotypes: *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). Dataare 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 isunchanged in livers of p16 2/2 vs. p16+/+ mice (n = 10). Two-way ANOVA and LSD Fisher post hoc test (*compared between the genotypesof 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.

3200 p16Ink4a Controls Fasting-Induced Gluconeogenesis Diabetes Volume 63, October 2014

is a major pathway for the maintenance of normal plasmaglucose levels (19) owing to the action of different hor-mones, among which are glucagon and glucocorticoids,like cortisol. During starvation, low blood glucose levels in-duce pancreatic a-cell glucagon secretion and hypothalamic-pituitary-adrenal axis activation. In the liver, glucagonbinds to its receptor, which then causes a GDP/GTPexchange, hence stimulating adenylate-cyclase activity,which converts ATP into cAMP (20). The rise in intra-cellular cAMP levels stimulates the dissociation of thecatalytic and regulatory subunits of protein kinase A(PKA) (21). The catalytic PKA subunit then enters thenucleus where it phosphorylates the CREB at Ser133,converting it into its transcriptionally active form, whichinduces gluconeogenic gene expression (22–24). In con-cert, glucocorticoids activate the glucocorticoid receptor,which binds to glucocorticoid-responsive elements in thepromoters of gluconeogenic genes (25,26).

Given the strong association of the CDKN2A/B locuswith T2D risk, which in large population studies is mainlyestablished by the measurement of fasting hyperglycemia(5), we set out to study whether p16Ink4a plays a role inhepatic glucose homeostasis using p16Ink4a-deficient mice(p162/2), mouse primary hepatocytes, and mouse hepaticcell line. Our results identify p16Ink4a as a modulator ofthe 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 Experimentsp162/2 and littermate control (p16+/+) mice on a C57Bl6background (.97%) were housed under standard condi-tions in conventional cages with free access to water andfood unless indicated otherwise. Twelve-week-old malemice were killed by cervical dislocation at 9:00 A.M.after a 24-h fasting. Experimental procedures wereconducted with the approval of the ethics committeefor animal experimentation of the Nord Pas-de-Calais re-gion (CEEAA022008R).

Pyruvate TestOvernight fasted mice (5:00 P.M. to 9:00 A.M.) were injectedwith sodium pyruvate (P4562; Sigma) (2 g/kg body wti.p.). Blood glucose levels were measured from the tailvein at the indicated time points using an automatic glu-cose monitor (OneTouch; LifeScan).

Mouse Primary Hepatocyte Isolation, Culture, andTreatmentsMice were anesthetized with a mixture of ketamine (100mg/kg) and xylasine (20 mg/kg) administered intraperi-toneally. Livers were perfused in situ through the inferiorcava vein, with Hanks’ balanced salt solution (H9394;Sigma) containing 0.5 mmol/L EGTA and 50 mmol/LHEPES followed by Hanks’ balanced salt solution contain-ing 0.025% collagenase (C5138; Sigma) until loss of its

firm texture. The soft liver was removed and cut intopieces and the homogenate filtered and centrifuged for2 min. The pellet was washed three times and resus-pended in Williams medium supplemented with 0.1%BSA, 1% glutamine, 1% gentamycine, 100 nmol/L insulin,and 100 nmol/L dexamethasone. Cell number and viabilitywere assessed using trypan blue. Cells were plated on six-wellplates during 2 h for hepatocyte selection and then incu-bated in deprivation medium (1% penicillin-streptomycin,and 1% glutamine, with distinct concentrations of glucagon[0, 1, 10, and 100 nmol/L]) for 6–8 h (for RNA measure-ments) or 30 min (for protein analysis).

Mouse Hepatocyte Cell Line Culture and TreatmentsAlpha Mouse Liver 12 (AML12) (cat. no. CRL2254;American Type Culture Collection) cells were cultured indeprivation medium–Ham’s F-12 supplemented with 10%FBS (Invitrogen), 5 g/mL insulin (Sigma), 5 g/mL trans-ferrin (Sigma), 5 ng/mL selenium (Sigma), 1% glutamine,and 1% penicillin-streptomycin and maintained at 37°C under5% CO2. AML12 cells were transfected with small interferingRNA (siRNA) for CDKN2A (043107-00-005; Thermo Scien-tific [ON-TARGET plus SMART pool siRNA]), CDK4 (ON-L-040106-00-0005; Thermo Scientific [ON-TARGETplus SMARTpool siRNA]), or control (D-001810-10-20;Thermo Scientific [ON-TARGET plus nontargeting pool

Figure 2—p16Ink4a deficiency increases gluconeogenic gene ex-pression and glucose production in primary hepatocytes. The in-duction of gluconeogenic genes (G6pase [A], Fbp1 [B], Pepck [C])by glucagon (8 h) is higher in primary hepatocytes isolated fromp162/2 vs. p16+/+ mice. Two-way ANOVA and LSD Fisher posthoc test (*compared between the genotypes of the same treatmentgroup; #compared between the treatment groups of the same ge-notype: ** or ##P < 0.01, *** or ###P < 0.001, **** or ####P <0.0001). Data are means 6 SEM. Glucose production is higher inprimary hepatocytes isolated from p162/2 vs. p16+/+ mice (D). Stu-dent t test (**P < 0.01). Data are means 6 SEM.

diabetes.diabetesjournals.org Bantubungi and Associates 3201

siRNA]) using the Dharmafect1 reagent (Thermo Scientific)according to the manufacturer’s instructions. AML12 cellswere treated for the indicated times points with 10 mmol/Lforskolin.

Glucose Production AssayPrimary hepatocytes were cultured in six-well plates inWilliams medium with 0.1% BSA, 100 nmol/L dexameth-asone, 1% penicillin-streptomycin, and 1% glutamine.After 2 h, the medium was replaced with 1 mL glucose-production buffer consisting of glucose-free Krebs-ringerbuffer (115 mmol/L NaCl, 5.9 mmol/L KCI, 1.2 mmol/LMgCl2, 1.2 mmol/L NaH2PO4, and 2.5 mmol/L NaHCO3pH 7.4) without phenol red, supplemented with 15 mmol/Lsodium lactate and 1 mmol/L sodium pyruvate. Glucoseconcentrations were measured at different time pointswith a colorimetric glucose assay kit (Sigma). The valueswere then normalized to total protein content determinedon whole-cell lysates.

Gene Expression AnalysisLiver total RNA was isolated using the guanidiniumisothiocyanate phenol/chloroform extraction method,and total RNA from cultured cells was extracted usingthe TRIzol reagent (Eurobio). One microgram of totalRNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription kits (Applied Bio-systems) according to the manufacturer’s instructions.

Reverse transcribed cDNAs were quantified by BrilliantIII Ultra-Fast SYBR green-based real-time PCR using spe-cific oligonucleotides (Supplementary Table 1) on a Strata-gene Mx3005P (Agilent Technologies) apparatus. mRNAlevels were normalized to Cyclophilin A expression as aninternal control, and mRNA fold induction was calculatedusing the comparative Ct (22ΔΔCt) method.

Western Blot AnalysisAML12 cells and mouse primary hepatocytes were lysedwith cell lysis buffer (50 mmol/L Tris-HCl, pH 8; 137mmol/L NaCl; 5 mmol/L Na2EDTA; 2 mmol/L EGTA; 1%Triton; 20 mmol/L sodium pyrophosphate; 10 mmol/Lb-glycerophosphate; 1mmol/L Na3VO4; 10 mmol/Lleupeptin; and 5 mmol/L pepestatin A) (Sigma-Aldrich) onice. Cells were scraped and transferred to 1.5-mL Eppendorftubes and rotated for 30 min at 4°C, followed by centri-fugation at 13,000g for 10 min at 4°C. The resultingsupernatants were stored in aliquots at 280°C untilthey were required. Protein concentration in the celllysates was determined using a BCA protein assay kit(Pierce). The cell lysates were mixed with 4X-SDS samplebuffer NOVEX (Life Technologies). Samples were heatedat 100°C for 10 min before loading and being separatedon precasted 4–12% or 3–8% SDS-PAGE (Invitrogen).Proteins were electrotransferred to a nitrocellulosemembrane (Millipore, Bedford, MA) in 1X transfer buffer

Figure 3—p16Ink4a downregulation increases gluconeogenic gene expression in AML12 cells. SiRNA CDKN2A treatment (which affectsboth 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 iscomparable in liver, primary hepatocytes, and AML12 cells. D–F: p16Ink4a-silenced and p16Ink4a-expressing AML12 were treated with 10mmol/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 ) inp16Ink4a-silenced compared with p16Ink4a-expressing AML12 cells. Two-way ANOVA and LSD Fisher post hoc test (*compared between thegenotypes 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.


http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1

(Invitrogen) using the Nupage Systeme for 1 h at 30 V.Nonspecific binding to the membrane was blocked for 1 hat room temperature with 5% nonfat milk in Tween–Tris-buffered saline (TTBS) buffer (20 mmol/L Tris, 500 mmol/Lsodium NaCl, and 0.1% Tween 20). Membranes were thenincubated overnight at 4°C with various primary antibodiesin blocking buffer containing 5% nonfat milk at the dilu-tion specified by the manufacturers. The following primaryantibodies were used: phospho-CREB (Ser133) (9198; CellSignaling Technology), CREB (9197; Cell Signaling Technol-ogy), phospho–(S/T)-PKA substrates (9621; Cell SignalingTechnology), phospho-pRb (3590; Cell Signaling Technol-ogy), pRb (9313; Cell Signaling Technology), PGC1a (sc-13067; Santa Cruz Biotechnology), GAPDH (sc-25778;Santa Cruz Biotechnology), p16ink4a (sc-1207; Santa CruzBiotechnology), phospho–regulatory subunit 2 of PKA(PKAR2) (ab32390; Abcam), and PKAR2 (ab-38949;Abcam). Membranes were then incubated with the second-ary antibody conjugated with the enzyme horseradish per-oxidase. The visualization of immunoreactive bands wasperformed using the enhanced chemiluminescence plusWestern blotting detection system (GE Healthcare). Quan-tification of phospho-CREB level in mouse primary he-patocytes and AML12 cells was performed by volumedensitometry using the ImageJ 1.47t software (NationalInstitutes of Health).

Cyclic AMP and PKA AssayIntracellular cAMP concentrations were measured usinga ready-to-use competitive enzyme immunoassay kit(R&D Systems). Briefly, cells were lysed according to themanufacturer’s protocol, and 100 mL sample was mixedwith 50 mL cAMP conjugated and then added to cAMP-specific antibody precoated microplate. After 2 h of in-cubation at room temperature, substrate solution wasadded for 20 min. Color development was stopped, andthe absorbance at 450 nm was measured using a DynexMRX TC Revelation Microplate Reader. PKA activity wasmeasured by the signaTECT cAMP-Dependent Protein Ki-nase Assay System by using the Kemptide (LRRASLG) asa peptide substrate.

Coimmunoprecipitation AssayCoimmunoprecipitation of CDK4 from whole AML12 cellextracts was performed using the Thermo Scientific PierceCrosslink Magnetic IP/Co-IP kit. Briefly, 48 h after siRNAtransfection, cells were lysed and 500 mg total proteinextract was incubated with 3 mg CDK4 antibody (sc-260;Santa Cruz Biotechnology) according to the manufactur-er’s protocol. The eluate was then subjected to Westernblot analysis using PKAR2 (ab-38949; Abcam) and CDK4(sc-260; Santa Cruz Biotechnology).

Immunofluorescence Assay in AML12 CellsCells were grown on cover slips. At 4 8h after siRNAtransfection, cells were washed with PBS and fixed with4% paraformaldehyde for 20 min. After fixation andpermeabilization with 0.1% TRITON, cells were incubated

overnight with antibodies against p16ink4a (M-156, sc-1207;Santa Cruz Biotechnology) and phospho-PKAR2 (Ab-32390;Abcam) and subsequently incubated with a combination ofTexas red–conjugated anti-rabbit IgG and FITC-conjugatedanti-mouse IgG. A nuclear DAPI counterstain was alsoperformed.

StatisticsData are expressed as means 6 SEM. Results were ana-lyzed by unpaired two-tailed Student t test or one-wayANOVA with least significant difference (LSD) Fisherpost hoc test or two-way ANOVA with LSD Fisher posthoc test as appropriate using GraphPad Prism software. AP value of , 0.05 was considered statistically significant.

RESULTS

p16Ink4a Deficiency Results in Fasting Hyperglycemiaand Increased GluconeogenesisSince GWAS revealed an association between theCDKN2A/B locus and T2D risk, primarily based on thefasting plasma glucose trait, we first measured fed andfasted blood glucose levels in 12-week-old mice. p162/2

mice displayed a less pronounced hypoglycemia after 24 hof fasting compared with p16+/+ mice (Fig. 1A). This effectwas not due to differences in plasma glucagon levels

Figure 4—p16Ink4a downregulation increases Pgc1a gene expres-sion. Pgc1a mRNA levels are higher in livers of p162/2 comparedwith p16+/+ mice (A), in primary hepatocytes isolated from p162/2

compared with p16+/+ mice and treated with glucagon for 8 h (B),and in p16Ink4a-silenced compared with p16Ink4a-expressing AML12cells treated with 10 mmol/L forskolin (FSK) for 16 h (C). D: Westernblots show higher increase of PCG1a protein level in p16Ink4a-silenced compared with p16Ink4a-expressing AML12 cells treatedwith 10 mmol/L forskolin for 1 h. Two-way ANOVA and LSD Fisherpost hoc test (*compared between the genotypes of the same treat-ment group; #compared between the treatment groups of the samegenotype: #P < 0.05, ** or ##P < 0.01, **** or ####P < 0.0001).Data are means 6 SEM.


between fasted p16+/+ and p162/2 mice (SupplementaryFig. 1). For evaluation of whether gluconeogenesis wasinfluenced, a pyruvate tolerance test (PTT) was performedin fasted p162/2 and p16+/+ mice. Interestingly, p162/2

mice produced higher blood glucose levels, upon pyruvateadministration, suggesting an increased hepatic glucoseproduction (Fig. 1B and C). Consistent with this, hepaticmRNA levels of gluconeogenic genes, such as G6pase andPepck, were significantly higher in livers of fasted p162/2

versus p16+/+ mice (Fig. 1D and F), while Fbp1 mRNAwas not different between the genotypes (Fig. 1E). Con-versely, genes involved in other metabolic pathways reg-ulated during fasting, such as glycolysis (Gk, Lpk) andb-oxidation (Cpt1a, Lcad), were not differently expressedbetween both genotypes upon fasting (Fig. 1G, H, J, andK), although mRNA levels of Pdk4, which block glycolysisat the level of pyruvate dehydrogenase, tended to behigher in fasted p162/2 livers (Fig. 1I). Altogether, thesedata indicate that among the different hepatic metabolicpathways regulated by fasting, gluconeogenesis is the onlyone modulated in p162/2 mice.

Since p16Ink4a is a tumor suppressor and a cell cycleregulator and since hepatic proliferation and tumorgrowth may perturb glucose homeostasis, we investigatedwhether p16Ink4a deficiency is associated with spontane-ous liver tumor growth or altered hepatocyte proliferation inour experimental conditions. At the age of 12 weeks, p162/2

mice did not display macroscopic liver abnormalities or

differences in liver weight compared with p16+/+ mice(Supplementary Fig. 2A and B). Moreover, immunohisto-chemical Ki-67 staining of liver sections showed no differ-ences between p162/2 mice and their littermate controlsunder fasting conditions (Supplementary Fig. 2C–H), in-dicating that hepatocyte proliferation is not different. Thesedata indicate that p16Ink4a deficiency increases fasting-induced hepatic gluconeogenesis in vivo, independent ofany action on hepatocyte proliferation.

p16Ink4a Deficiency Increases Gluconeogenic GeneExpression and Glucose Production In Vitro inHepatocytesFor analysis of whether the altered regulation of hepaticgluconeogenic gene expression in p162/2 mice is a cell-autonomous phenomenon, primary hepatocytes fromp162/2 mice and their littermate controls were isolatedand incubated with increasing concentrations of glucagonto mimic the fasting conditions. Basal levels of gluco-neogenic gene expression were 1.5-fold higher forG6Pase (60.15; P , 0.05), 4.4-fold higher for Pepck(60.96; P , 0.01) and 2.3-fold higher for Fbp1 (0.17;P , 0.001) in p162/2 compared with p16+/+ primaryhepatocytes (Fig. 2A–C). Moreover, glucagon, whichactivates the PKA-CREB signaling pathway, more pro-nouncedly induced G6pase, Pepck, and Fbp1 (Fig. 2A–C)mRNA levels in p162/2 vs. p16+/+ primary hepatocytes.Further, hepatic glucose production was higher in pri-mary hepatocytes of p162/2 than of p16+/+ mice (Fig.

Figure 5—p16Ink4a downregulation increases the phosphorylation of CREB. Western blots show higher increase of p-CREB in primaryhepatocytes isolated from p162/2 compared with p16+/+ mice and treated with glucagon for 1 h (A) and in p16Ink4a-silenced compared withp16Ink4a-expressing AML12 cells treated with 10 mmol/L forskolin (FSK) for 1 h (C). B and D: The bar graphs are the quantificationof 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.


http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1921/-/DC1

2D). Next, p16Ink4a was silenced using a CDKN2AsiRNA (which affects both p16Ink4a and p19ARF expres-sion) in AML12 cells (Fig. 3A and B), a mouse hepato-cyte cell line that expresses very high levels of p16Ink4a

compared with liver and primary hepatocytes (Fig. 3C).Incubation with forskolin, to activate the PKA-CREBpathway, resulted in a more pronounced increase ofG6pase and Fbp1 gene expression when p16Ink4a wassilenced, while no effect was observed on Pepck geneexpression (Fig. 3D–F). Moreover, although G6pase andFbp1 gene expression only marginally increased uponforskolin treatment in p16Ink4a-expressing AML12cells, p16Ink4a silencing resulted in the restoration ofa strong response (Fig. 3D and E). Altogether, theseresults indicate that p16Ink4a expression levels influencethe response to fasting-induced stimuli both in vivo and invitro.

p16Ink4a Levels Modulate PGC1a Expression in Vivoand In VitroFor studying of the mechanism by which p16Ink4a regu-lates gluconeogenic gene expression, mRNA and proteinlevels of PGC1a, a master regulator of the fasting adap-tation process (24), were measured. The fasting responseof Pgc1a mRNA was significantly more pronounced inlivers of p162/2 compared with p16+/+ mice (Fig. 4A).In line, p162/2 primary hepatocytes displayed a 3.4-foldincreased Pgc1a mRNA level (60.83; P , 0.01, two-tailedStudent t test) and a stronger induction by glucagon

compared with p16+/+ hepatocytes (Fig. 4B). p16Ink4a si-lencing in AML12 cells significantly increased Pgc1a ex-pression at both mRNA and protein levels upon forskolintreatment (Fig. 4C and D).

p16Ink4a Deficiency Increases the PKA-CREB SignalingPathwayTo gain insight into how PGC1a is induced upon p16Ink4a-deficiency, we first analyzed the phosphorylation status ofCREB, a transcription factor inducing PGC1a expression.p-Ser133-CREB was markedly higher in p162/2 comparedwith p16+/+ hepatocytes at the basal level as well as afterglucagon exposure (Fig. 5A and B). Similar results wereobtained upon forskolin treatment (data not shown).Likewise, p16Ink4a silencing in AML12 cells resulted ina stronger CREB phosphorylation both at the basal leveland upon forskolin treatment (Fig. 5C and D). Al-together, these data demonstrate that p16Ink4a knock-down increases CREB phosphorylation. It is well-knownthat the cAMP-PKA signaling pathway regulates fasting-induced CREB phosphorylation (22,27). To test whetheralterations in PKA activity may explain the increasedCREB phosphorylation upon p16Ink4a deficiency, p16Ink4a-silenced AML12 cells were treated with H89, a specificPKA inhibitor. H89 treatment prevented CREB phos-phorylation induced by p16Ink4a silencing (Fig. 6A). Ac-cordingly, PKA activity in p16Ink4a-silenced AML12 cellswas 1.5-fold higher compared with control (Fig. 6B). Thisincrease 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 AML12cells (B). Student t test (*P < 0.05). Data are means 6 SEM. Western blots show higher increase of global profile of PKA phosphorylatedsubstrates 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 for 1 h (D).


substrate phosphorylation profiles upon p16Ink4a silencing(Fig. 6C). Likewise, several PKA substrates were morephosphorylated in p162/2 than in p16+/+ primary hepa-tocytes both under basal conditions and after glucagonstimulation (Fig. 6D). Since PKA activity is controlled atleast in part by the phosphorylation of its regulatory sub-units (PKAR2), the expression and phosphorylation ofPKAR2 were measured in p16Ink4a-silenced AML12 cellsand in p162/2 primary hepatocytes. p16Ink4a silencing ordeficiency resulted in increased PKAR2 phosphorylationin AML12 cells (Fig. 7A) and in p162/2 primary hepato-cytes both at the basal state and upon glucagon stimula-tion (Fig. 7B). This result was confirmed by the enhancedp-PKAR2 immunostaining in p16Ink4a-silenced AML12 cells(Fig. 7C). Noteworthy, the increased PKA activity was notdue to an increase in cAMP levels (Fig. 7D). Collectively,these data demonstrate that p16Ink4a-deficiency activatesthe PKA-CREB-PGC1a signaling pathway independent of

changes in intracellular cAMP levels. To understand theunderlying mechanism by which p16Ink4a increases phos-phorylation of PKAR2 and thereby the increase of gluco-neogenic genes, we investigated the involvement of CDK4,a well-known target of p16Ink4a. Silencing of CDK4 inp16Ink4a-silenced AML12 cells (Fig. 8A and B) abrogatedthe induction of Ppc1a and Fbp1 mRNA levels by p16Ink4a

silencing (Fig. 8C and D). Moreover, coimmunoprecipita-tion experiments in AML12 cells after p16Ink4a knockdowndemonstrated a physical interaction between CDK4 andPKAR2 (Fig. 8E).

DISCUSSION

In recent years, a growing body of evidence supports theemerging notion that cell cycle regulatory proteinscontribute to metabolic processes in addition to, or linkedwith, their role in cell growth (17,28). Today, these pro-teins are perceived as sensors of external signals that

Figure 7—p16Ink4a downregulation increases PKAR2 phosphorylation without affecting intracellular cAMP levels. Western blots showhigher 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-expressingAML12 cells. Original magnification 320. D: cAMP levels were measured in p16Ink4a-silenced and p16Ink4a-expressing AML12 under basalconditions. Student t test (not significant). Data are means 6 SEM.


require a particular adapted metabolic response. TheCDK-Rb-E2F1 pathway, which is inhibited by p16Ink4a,has already been shown to control adipogenesis by mod-ulating the expression of the nuclear receptor PPARg(15,29), a master regulator of adipogenesis, as well as

by controlling oxidative metabolism in adipose tissue(30). The CDK-Rb-E2F1 pathway is also a negative regulatorof energy expenditure through repression of mitochondrialoxidative metabolism in muscle (16). Disruption of CDKinhibitor genes in the mouse has not revealed profound

Figure 8—p16Ink4a downregulation increases gluconeogenic gene expression in AML12 cells in a CDK4-dependent manner. SiRNA CDK4treatment in AML12 strongly decreases CDK4mRNA 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 siRNACDK4: #P < 0.05, *** or ###P < 0.001, ****P< 0.0001). Data are means6 SEM. E: Coimmunoprecipitation (IP) of CDK4 from whole AML12cell extracts was performed. The eluate was then subjected to Western blot (WB) analysis against PKAR2. F: Proposed pathway for thecontrol of hepatic gluconeogenesis through p16Ink4a and CDK4. When p16Ink4a is unable to bind CDK4 in the nucleus, CDK4 translocates tothe cytoplasm where it interacts with the PKA complex through PKAR2. This interaction leads to an increase of PKA activity, independentlyof changes in intracellular cAMP levels. Increased PKA activity leads to the activation of the transcription factor CREB and expression of thePGC1a 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.


cell cycle abnormalities but does result in a specific meta-bolic phenotype. Mice lacking p18Ink4c (31,32), p21cip1, orp27Kip1 display growth abnormalities and adipocyte hy-perplasia (33). Double knockout mice (p212/2; p272/2)develop hypercholesterolemia, glucose intolerance, andinsulin insensitivity (33). Surprisingly, the role of thesecell cycle regulators in the liver, one of the main meta-bolic organs controlling glucose homeostasis, has not yetbeen demonstrated.

It is well known that an increased rate of hepaticgluconeogenesis contributes to fasting hyperglycemiaobserved in T2D patients. Genetic analysis in GWASidentified an association of the CDKN2A/B locus withT2D risk (5,34,35). The association is based on themeasurement of fasting glycemia and confers to theCDKN2A/B locus a high susceptibility to T2D acrossdifferent ethnicities. In this study, we tried to elucidatethe mechanism by which a product of CDKN2A/B, i.e.,p16Ink4a, may influence the hepatic gluconeogenic pro-gram and thereby be implicated in T2D pathogenesis.We found that p16Ink4a deficiency raises PKAR2 phos-phorylation leading to an increased PKA activity. The in-creased PKA activity enhances CREB-PGC1a signaling,regulating the gluconeogenic gene expression program.Since the p16Ink4a protein shares several ankyrin repeatdomains, which are involved in protein-protein inter-action, we assessed whether p16Ink4a may associate withthe PKA complex. Immunoprecipitation of endogenousp16Ink4a in AML12 cells failed to demonstrate an interac-tion of p16Ink4a with the PKA regulatory subunit or thePKA catalytic subunit (data not shown), suggesting theexistence of other proteins able to connect p16Ink4a toPKA complex. One good candidate bridging p16Ink4a tothe PKA complex was CDK4, a well-known interactionpartner of p16Ink4a. Indeed, siRNA knockdown of CDK4in AML12 cells abrogates the effect of p16Ink4a deficiencyon the expression of gluconeogenic genes, like Pgc1a andFbp1, suggesting a direct role of CDK4 in the regulationof PKA activity by p16Ink4a. Moreover, it has already beenshown that CDK4 can displace the interaction betweencyclin D and the PKAR2–A-kinase anchoring protein/AKAP95 complex when CDK4 is activated (36). Otherstudies have demonstrated that CDK1 also phophorylatesPKAR2 (37), suggesting that other CDKs than CDK4 canhave the same activity on PKAR2 (38). All of these datasupport the existence of a dynamic complex includingp16Ink4a-(CDK4/cyclin D)-PKA-AKAP95 involved in thecontrol of hepatic glucose production (Fig. 8F).

In summary, GWAS identified SNPs near CDKN2A/Bassociate with fasting glycemia and the risk of T2Ddevelopment. Our study establishes that the p16Ink4a geneproduct of this locus modulates hepatic glucose produc-tion by increasing hepatic gluconeogenic gene expression.Further, we provide evidence that p16Ink4a acts via thePKA-CREB-PGC1a signaling pathway. Although the func-tional role of several cell cycle regulators (CDK4, pRb, E2F,p21cip1, p27kip1, and p18Ink4c) in metabolic control has been

described in tissues such as adipose tissue and the pan-creas (28), this is the first study that demonstrates a roleof a cell cycle regulator, p16Ink4a, in the liver, a masterorgan regulating glucose homeostasis in a manner inde-pendent of its function in cell proliferation. Thus, alteredp16Ink4a activity may contribute to the association be-tween the GWAS locus and the risk of developing T2D.

Acknowledgments. The authors thank P. Krimpenfort for providing thep16-deficient mice and J. Dumont for her assistance.Funding. This work was supported by grants from the European GenomicInstitute for Diabetes (ANR-10-LABX-46). The authors also thank Cost Action(BM0602), Conseil régional Nord Pas-de-Calais, and Fonds Européens deDéveloppement Régional (FEDER). K.B. was supported by a postdoctoral fellow-ship from Fondation pour la Recherche Médicale (FRM). S.-A.H. was supportedby a doctoral fellowship from Université Lille 2/Conseil régional Nord Pas-de-Calais and a FRM grant (FDT20130928340).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. K.B. and S.-A.H. performed experiments,designed experiments, analyzed data, and wrote the manuscript. S.C.-H., E.V.,M.B., A.L., and E.B. performed experiments. R.P., A.T., and B.S. designed experi-ments, analyzed data, and wrote the manuscript. B.S. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

References1. Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D. Relationships betweeninsulin secretion, insulin action, and fasting plasma glucose concentration innondiabetic and noninsulin-dependent diabetic subjects. J Clin Invest 1984;74:1238–12462. Rizza RA. Pathogenesis of fasting and postprandial hyperglycemia in type 2diabetes: implications for therapy. Diabetes 2010;59:2697–27073. Pal A, McCarthy MI. The genetics of type 2 diabetes and its clinical rele-vance. Clin Genet 2013;83:297–3064. Billings LK, Florez JC. The genetics of type 2 diabetes: what have welearned from GWAS? Ann N Y Acad Sci 2010;1212:59–775. Saxena R, Voight BF, Lyssenko V, et al.; Diabetes Genetics Initiative of BroadInstitute of Harvard and MIT, Lund University, and Novartis Institutes of Bio-Medical Research. Genome-wide association analysis identifies loci for type 2diabetes and triglyceride levels. Science 2007;316:1331–13366. Bao XY, Xie C, Yang MS. Association between type 2 diabetes and CDKN2A/B:a meta-analysis study. Mol Biol Rep 2012;39:1609–16167. Duesing K, Fatemifar G, Charpentier G, et al. Strong association of commonvariants in the CDKN2A/CDKN2B region with type 2 diabetes in French Europids.Diabetologia 2008;51:821–8268. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cyclecontrol causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704–7079. Hannon GJ, Beach D. p15INK4B is a potential effector of TGF-b-induced cellcycle arrest. Nature 1994;371:257–26110. Moritani M, Yamasaki S, Kagami M, et al. Hypoplasia of endocrine andexocrine pancreas in homozygous transgenic TGF-beta1. Mol Cell Endocrinol2005;229:175–18411. Krishnamurthy J, Ramsey MR, Ligon KL, et al. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 2006;443:453–45712. Fajas L, Annicotte J-S, Miard S, Sarruf D, Watanabe M, Auwerx J. Impairedpancreatic growth, beta cell mass, and beta cell function in E2F1 (-/- )mice. J ClinInvest 2004;113:1288–129513. Fajas L, Blanchet E, Annicotte J-S. CDK4, pRB and E2F1: connected toinsulin. Cell Div 2010;5:6


14. Annicotte J-S, Blanchet E, Chavey C, et al. The CDK4-pRB-E2F1 pathwaycontrols insulin secretion. Nat Cell Biol 2009;11:1017–102315. Fajas L, Landsberg RL, Huss-Garcia Y, Sardet C, Lees JA, Auwerx J. E2Fsregulate adipocyte differentiation. Dev Cell 2002;3:39–4916. Blanchet E, Annicotte J-S, Lagarrigue S, et al. E2F transcription factor-1regulates oxidative metabolism. Nat Cell Biol 2011;13:1146–115217. Aguilar V, Fajas L. Cycling through metabolism. EMBO Mol Med 2010;2:338–34818. Monnier L, Colette C, Dunseath GJ, Owens DR. The loss of postprandialglycemic control precedes stepwise deterioration of fasting with worsening di-abetes. Diabetes Care 2007;30:263–26919. Exton JH, Park CR. Control of gluconeogenesis in liver. I. General featuresof gluconeogenesis in the perfused livers of rats. J Biol Chem 1967;242:2622–263620. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am JPhysiol Endocrinol Metab 2003;284:E671–E67821. Taylor SS, Buechler JA, Yonemoto W. cAMP-dependent protein kinase:framework for a diverse family of regulatory enzymes. Annu Rev Biochem 1990;59:971–100522. Gonzalez GA, Montminy MR. Cyclic AMP stimulates somatostatin genetranscription by phosphorylation of CREB at serine 133. Cell 1989;59:675–68023. Quinn PG, Granner DK. Cyclic AMP-dependent protein kinase regulatestranscription of the phosphoenolpyruvate carboxykinase gene but not binding ofnuclear factors to the cyclic AMP regulatory element. Mol Cell Biol 1990;10:3357–336424. Herzig S, Long F, Jhala US, et al. CREB regulates hepatic gluconeogenesisthrough the coactivator PGC-1. Nature 2001;413:179–18325. Imai E, Stromstedt PE, Quinn PG, Carlstedt-Duke J, Gustafsson JA, GrannerDK. Characterization of a complex glucocorticoid response unit in the phos-phoenolpyruvate carboxykinase gene. Mol Cell Biol 1990;10:4712–471926. Vander Kooi BT, Onuma H, Oeser JK, et al. The glucose-6-phosphatasecatalytic subunit gene promoter contains both positive and negative glucocorti-coid response elements. Mol Endocrinol 2005;19:3001–302227. Hagiwara M, Brindle P, Harootunian A, et al. Coupling of hormonalstimulation and transcription via the cyclic AMP-responsive factor CREB is

rate limited by nuclear entry of protein kinase A. Mol Cell Biol 1993;13:4852–485928. Fajas L. Re-thinking cell cycle regulators: the cross-talk with metabolism.Front Oncol 2013;3:429. Fajas L, Egler V, Reiter R, et al. The retinoblastoma-histone deacetylase 3complex inhibits PPARgamma and adipocyte differentiation. Dev Cell 2002;3:903–91030. Dali-Youcef N, Mataki C, Coste A, et al. Adipose tissue-specific inactivationof the retinoblastoma protein protects against diabesity because of increasedenergy expenditure. Proc Natl Acad Sci U S A 2007;104:10703–1070831. Franklin DS, Godfrey VL, Lee H, et al. CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tu-morigenesis. Genes Dev 1998;12:2899–291132. Latres E, Malumbres M, Sotillo R, et al. Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis.EMBO J 2000;19:3496–350633. Naaz A, Holsberger DR, Iwamoto GA, Nelson A, Kiyokawa H, Cooke PS. Lossof cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obe-sity. FASEB J 2004;18:1925–192734. Scott LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide associationstudy of type 2 diabetes in Finns detects multiple susceptibility variants. Science2007;316:1341–134535. Zeggini E, Weedon MN, Lindgren CM, et al.; Wellcome Trust CaseControl Consortium (WTCCC). Replication of genome-wide association signalsin UK samples reveals risk loci for type 2 diabetes. Science 2007;316:1336–134136. Arsenijevic T, Degraef C, Dumont JE, Roger PP, Pirson I. G1/S Cyclins in-teract with regulatory subunit of PKA via A-kinase anchoring protein, AKAP95.Cell Cycle 2006;5:1217–122237. Carlson CR, Witczak O, Vossebein L, et al. CDK1-mediated phosphorylationof the RIIalpha regulatory subunit of PKA works as a molecular switch thatpromotes dissociation of RIIalpha from centrosomes at mitosis. J Cell Sci 2001;114:3243–325438. Gupte RS, Traganos F, Darzynkiewicz Z, Lee MYWT. Phosphorylation ofRIalpha by cyclin-dependent kinase CDK 2/cyclin E modulates the dissociation ofthe RIalpha-RFC40 complex. Cell Cycle 2006;5:653–660


Documents

PKA-CREB-PGC1a Pathwayis a major pathway for the maintenance of normal plasma glucose levels (19) owing to the action of different hor-mones, among which are glucagon and glucocorticoids,