High Expression Level of PPARγ in CD24 Knockout Mice and Gender-Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity

Abstract

Background: The heat-stable HSA/CD24 gene encodes a protein that shows high expression levels in adipocyte precursor cells but low levels in terminally differentiated adipocytes. Its high expression in many types of human cancer suggests an association between cancer, diabetes, and obesity, which is currently unclear. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) is a regulator of adipogenesis that plays a role in insulin sensitivity, lipid metabolism, and adipokine expression in adipocytes.

Aim: To assess gender-dependent changes in CD24 KO and its association with PPARγ expression.

Experimental approach: WT and CD24 KO mice were monitored from birth up to 12 months, and various physiological and molecular characteristics were analysed. Mean body weight and adipose mass were higher in KO mice than in WT mice. Male, but not female, KO mice showed increased insulin sensitivity, glucose uptake, adipocyte size, and PPARγ expression than WT mice. In addition, enteric bacterial populations, assessed through high-throughput sequencing of stool 16S rRNA genes, were significantly different between male KO and WT mice.

Conclusions: CD24 may negatively regulate PPARγ expression in male mice. Furthermore, the association between the CD24 and insulin sensitivity suggests a possible mechanism for diabetes as a cancer risk factor. Finally, CD24 KO male mice may serve as a model of obesity and insulin hyper-sensitivity.

Keywords: CD24; PPARγ; cancer; diabetes; insulin sensitivity.

Figure 1

Genotype verification by FACS analysis. Heparinized blood samples from KO (A) and WT (B) mice were collected and analyzed for CD24 expression by FACS analysis. The red curves represent the negative control (secondary antibody only), and the black curves represent the binding of M1/69 anti-CD24 antibody. (C) Blood samples were taken from KO and WT mice and peripheral blood leukocytes were isolated. Protein extracts (20 µg) were subjected to SDS-PAGE (odium dodecyl sulphate–polyacrylamide gel electrophoresis) and Western blotting using anti-CD24 M1/69 monoclonal antibodies. The membrane was re-probed with anti-tubulin to confirm uniform loading of the samples.

Figure 2

Whole body and organ weights. Representative pictures of male, 15-week-old KO (A) and WT (B) mice demonstrate the differences in body weight. Both male (C) and female (D) KO and WT mice were monitored, and their body weights were recorded from birth. Four mice, 15 weeks old, were dissected and their internal organs weighed separately (E). Liver triglycerides (F) and cholesterol (G) were measured in KO and WT mice.

Figure 3

CD24 deficiency and insulin sensitivity. Insulin sensitivity of KO and WT males and females, aged 9–12 weeks, is presented in (A,B), respectively. Following intraperitoneal insulin injection (0.5 unit/kg body weight) of fasted mice, whole-blood glucose was measured at the indicated time points using a glucometer. Values are expressed as a percentage, relative to baseline glucose levels. Asterisks indicate statistically significant differences (p < 0.05) between genotypes at each time point. Each value represents the average of at least three independent measurements.

Figure 4

CD24 deficiency and glucose uptake in 9–12 weeks old mice. After fasting, mice were intraperitoneally injected with glucose (1 g/kg body weight). (A) Baseline insulin levels were measured from mice serum by ELISA assay. (B) Insulin levels were measured 20 min after glucose injection.

Figure 5

PPARγ expression in KO and WT male mice. RNA extraction from kidney adipose tissue from KO and WT mice was prepared and cDNA synthesis was performed. (A). A fragment of the PPARγ gene was amplified by PCR reaction and uniformity of the samples was confirmed by the murine GAPDH housekeeping gene. PPARγ expression levels in samples, shown on the right, were determined by densitometry (TINA 2.0). (B). Quantitative real-time PCR was performed to confirm the results above. (C). Quantitative real-time PCR was performed to determine the levels of perilipin 1, PPARα, and adiponectin in visceral fat.

Figure 6

Adipocytes in KO and WT mice. Adipocytes were significantly larger in KO mice than in WT mice. Brightfield imaging was done with a Nikon microscope at ×10 magnification. * p < 3.51 × 10⁹.

Figure 7

Enteric bacterial populations in young male KO and WT mice. Red circles indicate KO mice, whereas blue squares indicate WT mice. For mice fed a normal diet, n = 8 KO mice, and n = 10 WT mice. For mice fed a high-fat diet, n = 9 KO mice, and n = 11 WT mice. Correlation coefficients and p-values are shown in the figure.