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Loss of cyclin-dependent kinase inhibitors produces adipocyte hyperplasia and obesity

AFIA NAAZ^*, DENISE R. HOLSBERGER^*, GARY A. IWAMOTO^*, AMANDA NELSON, HIROAKI KIYOKAWA and PAUL S. COOKE^*,,1

Departments of
^* Veterinary Biosciences and
Kinesiology, and
Division of Nutritional Sciences, University of Illinois-Urbana, Urbana, Illinois, USA; and
Department of Biochemistry and Molecular Genetics, University of Illinois-Chicago, Chicago, Illinois, USA

¹Correspondence: Dept. of Veterinary Biosciences, Univ. of Illinois, 2001 S. Lincoln Ave., Urbana, IL 61802, USA. Email: p-cooke{at}uiuc.edu

SPECIFIC AIM

The cell cycle inhibitors p27 and p21 play a role in adipocyte differentiation in vitro and might be important factors in the determination of adipocyte number in vivo. The aim of this research was to determine the effect of these cell cycle inhibitors on adipose tissue by using animals lacking one or both of these proteins. To examine this, we compared adipose development and metabolic parameters in p27 and p21 knockouts (p27KO and p21KO, respectively), p27/p21 double knockout (DBKO) and wild-type (WT) mice.

PRINCIPAL FINDINGS

1. Mice lacking p27 and/or p21 show temporal differences in body weight and adipose depots
Body weights of single KO mice were not initially different from WT, but DBKO weights were heavier than WT by 45 days of age. From days 60–120, both p27KOs and p21KOs were heavier than WT mice and DBKO mice were heavier than all other groups. By day 120, DBKO mice weighed 100% more than WT. At 120 days of age, DEXA analysis indicated that adipose mass in DBKO mice was increased 670% compared with WT, while lean mass was increased only 24%. Thus, the augmentation in body weight in DBKO mice is predominantly due to increased adipose mass. Increased adipose mass was apparent in DBKO mice by 60 days, and became more pronounced with advancing age. DEXA analysis was corroborated by direct measurement of adipose and other tissues at 125–130 days of age. Parametrial fat pads showed 80% and 90% increases in wet weight in p27KO and p21KO mice, respectively, compared with WT. In contrast, parametrial fat pad weights in DBKO mice were 500% that of WT controls (Fig. 1 a, b). Inguinal fat pads showed changes similar to those seen in parametrial fat pads in all groups. Brown fat depots were increased by 25%, 38%, and 142% in p27KO, p21KO, and DBKO mice, respectively, compared with WT. Kidney weight was increased 37%, 18%, and 105% in p27KO, p21KO and DBKO mice, respectively, compared with WT. Other organs typically increased in weight, though the increase was variable by organ. For example, brain weights in DBKO mice were increased less than 25%.

View larger version (36K): [in this window] [in a new window]   Figure 1. Adiposity is increased by loss of p27 and/or p21 in female mice. A) Parametrial fat pad weight in 120-day-old WT, p27KO, p21KO, and DBKO mice (n7 for all genotypes). *Significantly different from WT. B) Whole mounts of parametrial fat pads from 120-day-old WT and DBKO mice showing increased fat pad size in mice lacking p27 and p21. C) Cell number in parametrial fat pads of WT, p27KO, p21KO, and DBKO mice (n5 for all genotypes). Loss of either p27 or p21 caused adipocyte hyperplasia while loss of both CDKIs further increased these parameters.

2. Increase in adipose depots due to loss of p27 and/or p21 is due to adipocyte hyperplasia
Adipocyte numbers in parametrial fat pads of p27KO, p21KO, and DBKO mice were 1.9-, 1.7-, and 6.1-fold those in WT mice, respectively (Fig. 1c ). Hyperplasia was seen in the small (<30 µm in diameter), medium (30–130 µm) and large (>130 µm) adipocytes in p27KO, p21KO, and DBKO mice compared with WT mice. The numbers of small adipocytes in p27KO, p21KO, and DBKO mice were markedly increased (3.0-, 1.7-, and 9.5-fold that of WT, respectively), and were consistent with greater generation of adipocytes in all of the knockout mice. There were similar increases in large adipocytes. Medium adipocytes in p27KO, p21KO, and DBKO mice showed less pronounced increases than the small and large adipocytes and two categories of medium adipocytes (50–70 µm and 70–90 µm) were not elevated in p27KO mice. Despite disproportionate increases in small and large adipocytes, average adipocyte diameter did not differ in any of the knockout groups compared with WT controls.

3. Increase in adipose depots due to hyperplasia in mice lacking p27 and/or p21 leads to metabolic changes
Lack of both p27 and p21 caused glucose intolerance (Fig. 2 a) and insulin insensitivity (Fig. 2b ). Loss of only p27 or p21 produced fewer pronounced changes in insulin sensitivity, and glucose tolerance was not significantly different than controls in single knockout mice. Loss of p27 and/or p21 induced hypercholesterolemia (total serum cholesterol=103±12, 124±15, 159±10, 180±14 mg/dL in WT, p27KO, p21KO, and DBKO, respectively; P<0.05 for all groups vs. WT).

View larger version (22K): [in this window] [in a new window]   Figure 2. Metabolic changes result from loss of p27 and/or p21. A) Glucose tolerance in knockout and WT mice. Loss of both p27 and p21 led to impaired glucose tolerance, while loss of either p27 or p21 alone did not produce a significant impairment in glucose tolerance, although there was a trend toward impaired glucose tolerance in p27KO. B) Insulin sensitivity in knockout and WT mice. Loss of either p27 or p21 led to impaired insulin sensitivity, while loss of both of these CDKIs led to further decreases in sensitivity. *Significantly different from WT. Asterisks over the time points in panel C indicate that the values for all 3 genotypes (p27KO, p21KO, and DBKO) were different from WT (n5 for all points).

Feed consumption from 30 to 120 days of age in DBKO mice was increased 40% compared with WT (total feed consumption=468±42 g and 656±46 g in WT and DBKO mice, respectively, from 30 to 120 days of age; P<0.05). When feed consumption was expressed relative to lean body mass, DBKO mice did not show increased feed consumption per g of lean body weight, indicating that their obesity was not simply due to hyperphagia.

CONCLUSIONS AND SIGNIFICANCE

Our results indicate that both p27 and p21 are important regulators of adipogenesis, and loss of both of these CDKIs results in pronounced adipocyte hyperplasia, obesity, and associated metabolic changes. The loss of either of these CDKIs has a marked stimulatory effect on adipogenesis and causes adipose changes that are not allometric but far exceed the modest gains in body weight in either the p27KO or p21KO. The mice lacking both p27 and p21 have large increases in adipocyte number, fat pad weights and obesity that far exceed those induced by loss of a single CDKI. Although both p27 and p21 are important in establishment of adult adipocyte number, their roles are not totally redundant. Deletion of one induces critical effects that are not compensated for by the other, and absence of both causes greater adipose changes than those seen in either single knockout. Our results show that DBKO adipocytes differentiate, indicating that neither p27 nor p21 are obligatory for this process, although there could be a redundancy or compensatory change where some other factor substitutes for the absence of these CDKIs in the DBKO, allowing adipocyte differentiation to occur.

Increases in adipose depots can involve both hypertrophy and hyperplasia. Hypertrophy of adipocytes is considered the main cause of adult obesity, and hyperplasia of adipocytes in adult obesity sometimes occurs secondarily to adipocyte hypertrophy, possibly due to a critical increase in the size of adipocytes leading to production of growth factors that induce adipocyte hyperplasia. Adipocyte number is established early in life, and childhood obesity results in adipocyte hyperplasia. This increased adipocyte number during early life may strongly predispose for later obesity. Lack of p27 and/or p21 produces adipocyte hyperplasia, and provides a model to study factors regulating the establishment of adipocyte number and specific consequences of adipocyte hyperplasia (Fig. 3 ).

View larger version (131K): [in this window] [in a new window]   Figure 3. Schematic diagram of adipose development in WT vs. DBKO mice. In wild-type mice, cells committed to the adipogenic lineage and preadipocytes (adipocyte precursor cells) proliferate and differentiate to establish adipocyte number in young animals; this initial establishment of adipocyte number determines the adult adipose phenotype in wild-type mice. In DBKO mice, loss of p27 and p21 causes an increase in proliferation and adipocyte hyperplasia. The presence of increased number of adipocytes induces increased lipogenesis and increases in adipose depots. An obese phenotype with consequent metabolic changes results.

Insulin responsiveness decreases in larger adipocytes, and this change is believed to be critical for the well-known impaired glucose tolerance and insulin sensitivity in obesity. The metabolic changes in mice lacking both p27 and p21 also emphasize the importance of overall adipose mass in these changes, since the metabolic sequelae common to obesity (hypercholesterolemia, glucose intolerance, and insulin insensitivity) are seen in DBKO animals lacking significant adipocyte hypertrophy. Although both the p27KO and p21KO show decreased insulin sensitivity relative to the WT control, neither single knockout had significant impairments in glucose tolerance, in contrast to the clear glucose intolerance seen in the DBKO.

The present results emphasize the importance of adipocyte number and how changes in adipose cellularity can be a major factor regulating fat pad size and obesity, a topic that heretofore has not received extensive attention but appears to be critical in the global regulation of adipose stores. This adipocyte hyperplasia may explain the overwhelming persistence of obesity in human conditions such as childhood obesity, obesity in infants born to mothers with gestational diabetes, and morbid obesity. In the latter, the adipocyte hyperplasia has been considered to be secondary to adipocyte hypertrophy. Our results showing that adipocyte hyperplasia can induce obesity indicate that the adipocyte hyperplasia in morbid obesity may be important for the subsequent clinical course of the disease. The enlarged adipocyte population may alter the adipose tissue to favor continued obesity. In addition, if an increased adipocyte population induces obesity, the present epidemic of childhood obesity becomes of greater concern, as this suggests that this type of obesity may be essentially a permanent consequence of the adipocyte hyperplasia. This may account for the high proportion of obesity seen even 40 years later in adults who were overweight children (in some cases despite adult food intake that was consistent with recommended norms). Definitively establishing how increased adipocyte populations lead to obesity will be critical for explaining the obese phenotype in knockout animals as well as gaining insights into human conditions involving adipocyte hyperplasia.

Proliferation and differentiation of the adipocyte lineage is regulated by various endocrine and paracrine factors, although how these various factors regulate adipocyte number is not completely understood. Our study indicating the centrality of p27 and p21 for establishment of adipocyte number suggests that these CDKIs may be targets for factors regulating establishment of adipocyte number. This could occur through direct effects on the CDKIs themselves, or more likely through effects on proteins that regulate the CDKIs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2631fje;

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