Overweight in mice and enhanced adipogenesis in vitro are associated with lack of the hedgehog coreceptor boc

Abstract

Obesity arises from a combination of genetic, environmental, and behavioral factors. However, the processes that regulate white adipose tissue (WAT) expansion at the level of the adipocyte are not well understood. The Hedgehog (HH) pathway plays a conserved role in adipogenesis, inhibiting fat formation in vivo and in vitro, but it has not been shown that mice with reduced HH pathway activity have enhanced adiposity. We report that mice lacking the HH coreceptor BOC displayed age-related overweight and excess WAT. They also displayed alterations in some metabolic parameters but normal food intake. Furthermore, they had an exacerbated response to a high-fat diet, including enhanced weight gain and adipocyte hypertrophy, livers with greater fat accumulation, and elevated expression of genes related to adipogenesis, lipid metabolism, and adipokine production. Cultured Boc(-/-) mouse embryo fibroblasts showed enhanced adipogenesis relative to Boc(+/+) cells, and they expressed reduced levels of HH pathway target genes. Therefore, a loss-of-function mutation in an HH pathway component is associated with WAT accumulation and overweight in mice. Variant alleles of such HH regulators may contribute to WAT accumulation in human individuals with additional genetic or lifestyle-based predisposition to obesity.

Figure 1

Age-related overweight in Boc^−/− mice. A: Time course of body weight in Boc^+/+ (+/+) and Boc^−/− (−/−) mice. Values are means ± SEM; n = 31–42 for Boc^+/+ mice and n = 25–43 for Boc^−/− mice at various time points. *P < 0.01. B and C: Weights of individual organs or tissues dissected from +/+ and −/− mice. Diaph., diaphragm; Ep., epididymal WAT; Kid., kidney; Liv., liver; Sc., subcutaneous WAT; Spl., spleen. Values are means ± SD; n = 12–14 for each tissue. *P < 0.05. D: Dissected BAT from Boc^+/+ and Boc^−/− mice. Note that the Boc^−/− BAT is paler than that of the control and has areas of obvious whitening (arrows). E: H-E–stained sections of BAT from Boc^+/+ and Boc^−/− mice. Bar, 50 μm. F: H-E–stained sections of WAT from Boc^+/+ and Boc^−/− mice. Bar, 50 μm. G: Average area of adipocytes from sections of adult WAT from +/+ and −/− mice. Values are means ± SD; n = 3. H: Distribution of adipocyte areas from sections of adult WAT from +/+ and −/− mice. Values are means ± SD; n = 3. I: Food intake by 24-week-old +/+ and −/− mice. BW, body weight. Values are means ± SD; n = 7.

Figure 2

Alterations in whole-body metabolism of Boc^−/− mice. A: Body weights. B: Body temperature. C: Rearing activity. D: Locomotion. E: O₂ consumption (VO2). F: CO₂ production (VCO2). G: Respiratory quotient (RQ). H: Energy expenditure (EE). Normal chow-fed, 5-month-old Boc^+/+ and Boc^−/− mice were measured with metabolic cages (n = 6). Data in A–H represent means ± SEM. ***P < 0.001; *P < 0.05.

Figure 3

Exacerbated responses to HFD by Boc^−/− mice. Time course of body weight in Boc^+/+ and Boc^−/− mice on normal chow (NC) (A) and HFD (B). Values are means ± SEM; n = 5–7 for each time point. *P < 0.05; **P < 0.005; ***P < 0.001. C: Blood glucose levels in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks and then fasted for 16 h. D: GTT in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks and then fasted for 16 h. Mice were administered glucose intraperitoneally, and blood glucose levels were measured every 15 min for 2 h. Values are means ± SEM; n = 5–7. ***P < 0.01; *P < 0.5. E: ITT in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks and then fasted for 6 h. Mice were administered insulin intraperitoneally, and blood glucose levels were measured every 15 min for 2 h. Values are means ± SEM; n = 5–7. ***P < 0.001; **P < 0.005; *P < 0.05. F: Plasma insulin levels in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks. G: Serum triglyceride (TG) levels in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks. *P < 0.05. H: Blood nonesterified fatty acid (NEFA) levels in Boc^+/+ and Boc^−/− mice on HFD for 8 weeks. **P < 0.005.

Figure 4

Exacerbated liver and WAT phenotypes in Boc^−/− mice on HFD. A: Oil Red O–stained liver sections and H-E–stained WAT sections from Boc^+/+ and Boc^−/− mice on HFD. Bar, 50 μm. B: Average area of adipocytes from sections of adult WAT from Boc^+/+ and Boc^−/− mice. Values are means ± SD; n = 3. *P < 0.05. C: qRT-PCR analysis of expression of lipid metabolism genes in livers of Boc^+/+ and Boc^−/− mice on HFD. Values are means of relative expression levels ± SD; n = 3. ***P < 0.001; **P < 0.005. D: qRT-PCR analysis of expression of adipogenic genes in livers of Boc^+/+ and Boc^−/− mice on HFD. Values are means of relative expression levels ± SD; n = 3. ***P < 0.001; **P < 0.005; *P < 0.05. E: Western blot analysis of expression of lipid metabolism and adipogenic gene in livers of Boc^+/+ and Boc^−/− mice on HFD. F: qRT-PCR analysis of expression of adipogenic, lipid metabolism, and adipokine genes in WAT of Boc^+/+ and Boc^−/− mice on HFD. Values are means of relative expression levels ± SD; n = 3. ***P < 0.001; **P < 0.005; *P < 0.05. G: Western blot analysis of expression of lipid metabolism and adipogenic genes in WAT of Boc^+/+ and Boc^−/− mice on HFD. H: qRT-PCR analysis of expression of uncoupling proteins in BAT of Boc^+/+ and Boc^−/− mice on HFD. Values are means of relative expression levels ± SD; n = 3. *P < 0.05.

Figure 5

Analysis of Boc expression. A: qRT-PCR analysis of Boc expression in various adult tissues. Values are means of relative expression levels ± SD; n = 3. L32 expression is set as 1. B: Alkaline phosphatase staining of WAT from Boc^+/+ and Boc^+/− mice (note the Boc⁻ allele drives expression of a PLAP reporter gene, designated here Boc^PLAP; see text) (a). Alkaline phosphatase staining of isolated WAT adipocytes from Boc^+/PLAP mice (b). Alkaline phosphatase staining of isolated vessels from the SVF from WAT of Boc^+/PLAP mice, and stromal cells are marked by arrows (c). Alkaline phosphatase staining of adherent cells from the SVF from WAT of Boc^+/PLAP mice (d). DAPI staining of the same culture shown in d (e). C: Semiquantitative RT-PCR analysis of Boc and Pparγ expression in WAT, SVF, and adipocytes (Adip.). Gapdh served as a loading control.

Figure 6

Boc^−/− MEFs show enhanced adipogenesis. A: Western blot analysis of various proteins during a time course of adipogenic differentiation of 3T3-L1 cells. Lysates of 3T3-L1 cell cultures in growth medium (G) at 60 and 100% confluence (Cnfl.) or for the indicated number of days in differentiation medium (DM) were blotted with the indicated antibodies. β-Actin served as a loading control. B: Enhanced adipogenesis of Boc^−/− MEFs relative to Boc^+/+ MEFs. Cultures were stained with Oil Red O after 15 days in differentiation medium. C: Quantification of Oil Red O staining by spectrophotometry at a wavelength of 520 nm relative to control sample. Values are means ± SD; n = 3. *P < 0.05. D: Semiquantitative RT-PCR analysis of expression of various genes induced during a time course of adipogenesis by Boc^+/+ and Boc^−/− MEFs. L32 served as a loading control. E: Western blot analysis of various adipogenic proteins in Boc^+/+ and Boc^−/− MEFs at D3 and D6.

Figure 7

HH target gene expression is reduced in Boc^−/− MEFs, and exogenous stimulation of HH pathway activity represses their adipogenic differentiation. A: qRT-PCR analysis of HH pathway component genes in WAT from Boc^+/+ and Boc^−/− mice. Values are means of relative expression levels ± SD; n = 3. **P < 0.005; *P < 0.05. B: Semiquantitative RT-PCR analysis of HH pathway component genes during a time course of adipogenesis by Boc^+/+ (+/+) and Boc^−/− (−/−) MEFs. L32 served as a loading control. C: Western blot analysis of expression of HH pathway components in Boc^+/+ and Boc^−/− MEFs at D3 and D6. D: The SMO agonist purmorphamine (Pur) inhibited adipogenic differentiation of both Boc^+/+ and Boc^−/− MEFs. Cultures were stained with Oil Red O after 15 days in differentiation medium. E: Quantification of Oil Red O staining by spectrophotometry at a wavelength of 520 nm relative to the control sample. Values are means ± SD; n = 3. **P < 0.005; ***P < 0.001. F: qRT-PCR analysis of expression of aP2, Fas, and Scd1 in cultures of +/+ and −/− MEFs cultured in differentiation medium for 6 days plus or minus Pur. Values are means of relative expression levels ± SD; n = 3. ***P < 0.001; **P < 0.005; *P < 0.05. G: Western blot analysis of adipogenic proteins in cultures of +/+ and −/− MEFs cultured in differentiation medium for 3 or 6 days plus or minus Pur. H: SHH inhibited adipogenic differentiation of both Boc^+/+ and Boc^−/− MEFs. Cultures were stained with Oil Red O after 15 days in differentiation medium. I: Quantification of Oil Red O staining by spectrophotometry at a wavelength of 520 nm relative to the control sample. Values are means ± SD; n = 3. **P < 0.005; *P < 0.05. J: qRT-PCR analysis of expression of aP2, Fas, and Scd1 in cultures of +/+ and −/− MEFs cultured in differentiation medium for 6 days plus or minus SHH. Values are means of relative expression levels ± SD; n = 3. ***P < 0.001; **P < 0.005; *P < 0.05.