CDK4 is an essential insulin effector in adipocytes.
Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.The first suggestion of a role of CDK4 in adipose tissue biology came from the finding that CDK4 and 2 D-type cyclins (CCND2 and CCND3) are highly expressed in epididymal WAT (eWAT) compared with the other tissues analyzed (Figure 1A). The high levels of expression of CCND3 in eWAT (Figure 1A and Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI81480DS1) are consistent with previous findings showing increased CCND3 expression during adipogenesis (15). Protein expression analysis in visceral adipose tissue (VAT) cellular fractions showed that CDK4 was better expressed in mature adipocytes compared with the stromal vascular fraction (SVF) (Figure 1B and Supplemental Figure 1C). Furthermore, CDK4 expression was also higher in differentiated 3T3-L1 adipocytes compared with nondifferentiated 3T3-L1 preadipocytes (Supplemental Figure 1C). Interestingly, the subcellular localization of CDK4 and CCND3 as well as of the other D-type cyclins revealed that these proteins are not only found in the nucleus; rather, they are mainly localized in the cytoplasm of adipocytes (Figure 1C and Supplemental Figure 1D), suggesting a role for CDK4 that is independent of the RB/E2F pathway in these cells. Moreover, since the duplication rate in mature adipocytes is low (16), these results suggested a novel cell-cycle independent role for CDK4. In order to analyze the participation of CDK4 in adipose tissue biology, we set to determine the phenotype of CDK4 mutant mice. The previously generated Cdk4neo/neo mice are diabetic and have impaired pancreatic β cell development and decreased insulin levels (11). Analysis of adipose tissue function in these mice would be confusing, since any observed effect could be secondary to insulin deficiency. We therefore used Cdk4neo/neo Rip-Cre (Cdk4neo/neo;cre/cre; herein referred to as Cdk4nc) mice that reexpress Cdk4 in β cells and thus have normal insulin levels (13). We also used a mouse model of CDK4 hyperactivation, the R24C model. Cdk4R24C/R24C mice express a mutant CDK4 protein that is not sensitive to INK4a inhibitors (11) and is consequently more active. A first analysis showed that Cdk4nc mice had decreased body weight, whereas Cdk4R24C/R24C mice exhibited increased body weight compared with Cdk4+/+ mice (Figure 1, D and E). Significant changes in WAT mass accounted for body weight variation. Cdk4nc and Cdk4R24C/R24C mice had decreased and increased WAT mass, respectively, as measured by EchoMRI (Figure 1, D and E, and Supplemental Figure 1, E and F). Changes in fat mass were consistent with variation in adipocyte size (Figure 1F and Supplemental Figure 1G). Overall, severe atrophy could be observed in fat pads from Cdk4nc mice, whereas Cdk4R24C/R24C mice developed adipose tissue hypertrophy (Supplemental Figure 1H).To demonstrate that the effects of Cdk4 deletion in adipose tissue were cell autonomous, we used an approach involving systemic administration of adeno-associated viral vectors of serotype 8 (AAV8), which has been previously reported as leading to genetic engineering of white and brown adipocytes in adult mice and has very poor tropism for macrophages (17). We infected Cdk4flox/flox mice (Supplemental Figure 1I) with AAV8 vectors expressing the Cre recombinase under the control of the mini/aP2 adipose tissue–specific promoter (AAV8-mini/aP2-cre) or with the control vector (AAV8-mini/aP2-null). First of all, we determined the tissues that were infected by assessing the presence of viral genome (vg) using Cre PCR. The vg was only present in brown adipose tissue (BAT), eWAT, s.c. WAT, and liver, whereas we could not detect it in pancreas and muscle (Supplemental Figure 1J). Quantitative reverse-transcription–PCR (RT-qPCR) analysis showed a significant decrease of Cdk4 mRNA in eWAT and s.c. WAT, whereas no changes were observed in liver and BAT (Supplemental Figure 1K). After 3 weeks, the systemic administration of AAV8-mini/aP2-cre triggered a decrease in fat mass gain; indeed, AAV8-mini/aP2-cre–infected mice gained significantly less fat mass (Figure 1G) and experienced a reduction in adipocyte size (Figure 1H). However, no differences were found in body weight and lean mass in Cdk4flox/flox mice infected with AAV8-mini/aP2-cre vector (Figure 1G and Supplemental Figure 1L). The use of this adipose tissue–specific Cdk4 depletion model supports a cell-autonomous contribution for CDK4 in adipose tissue. Overall, these 3 models (Cdk4nc, Cdk4R24C/R24C, and Cdk4flox/flox mice infected with AAV8 mini/aP2-cre) clearly demonstrate a positive correlation between CDK4 activity and WAT mass/size.E2F1, a known proproliferative downstream effector of CDK4, was previously shown to promote adipogenesis (16). Therefore, in order to determine whether adipocyte proliferation was not affected with the modulation of CDK4 activity, we generated Cdk4R24C/R24C E2f1–/– mice. No significant changes were observed in adiposity, adipocyte size, lean mass, or adipocyte proliferation as measured by Ki67 expression in Cdk4R24C/R24C E2f1–/– compared with Cdk4R24C/R24C E2f1+/+ mice (Figure 1, I and J, and Supplemental Figure 1, M and N). These results demonstrate that when CDK4 is hyperactive, the deletion of E2f1 does not affect fat mass, mature adipocyte size, and proliferation. Because Cdk4R24C/R24C mice develop a wide spectrum of tumors (18, 19), we investigated to determine whether the WAT phenotype observed in these mice could be secondary to tumor development. We could not find any correlation between fat mass and tumor development. Indeed, all mice used in this study were tumor free (Supplemental Figure 1O). Moreover, tumor development was negatively correlated with fat mass in 60-week-old Cdk4R24C/R24C mice, proving that the increased WAT mass in these mice was not secondary to tumor formation (Supplemental Figure 1P).All experiments with the CDK4 inhibitor (PD0332991, Azasynth Co.) were done using 1 μM of PD0332991 in mature 3T3-L1 adipocytes. All chemicals, unless stated otherwise, were purchased from Sigma-Aldrich. Actrapid human recombinant insulin was purchased from Novo Nordisk Pharma SA. AKT inhibitor (catalog 124017) was purchased from Calbiochem and used at 10 μM for 30 minutes. 14C‑acetate, and γ‑33P-ATP were purchased from PerkinElmer.Members of the Fajas laboratory are acknowledged for support and discussions. We thank M. Barbacid for providing Cdk4R24C/R24C and Cdk4nc mice. We thank A.-C. Thomas and F. Thévenaz for technical support. We thank J.-C. Stehle from the Mouse Pathology Facility. F. Bosch is the recipient of an award from the ICREA Academia, Generalitat de Catalunya, Spain. Vector generation and production were funded by Ministerio de Economía y Competitividad (SAF 2014-54866-R), Spain. This work was supported by grants from the Swiss Ligue Contre le Cancer, the French Ligue Contre le Cancer, and the Swiss National Science Foundation. S. Lagarrigue was supported by a grant from the French Ligue Contre le Cancer and the Swiss National Science Foundation.Conflict of interest: The authors have declared that no conflict of interest exists.Reference information:J Clin Invest. 2016;126(1):335–348. doi:10.1172/JCI81480.