Blood albumin levels by Ponceau S staining (loading control). ( A) Serum adiponectin (ACRP30) of Adipoq Cre-/DTA+ control (Con), Adipoq Cre+/DTA+ fat free (FF), and Adipoq knockout (KO) mice by western blot. Adipoq Cre+/mTmG+ mice were housed at 22☌ on a 12 hr/12 hr light/dark cycle. Images representative of the following animals: 4-month-old Adipoq Cre+/mTmG+ male (N = 5) and female (N = 6) 3-week-old Adipoq Cre+/mTmG+ male (N = 5) and female (N = 5). GFP expression is absent in the stroma and on the bone surface. Constitutive BMAT adipocytes (cBMAT) are GFP+ (arrowheads). ( D) Representative tail vertebrae, 3 week old male Adipoq Cre+/mTmG+. Prominent labeling is also observed in the stromal reticular network and on the bone surface.
As in males, regulated bone marrow adipose tissue (rBMAT) adipocytes are GFP+. ( C) Representative proximal tibia, 4 month old, female Adipoq Cre+/mTmG+. All cells, including BMAds, were positive for red fluorescent protein (RFP). ( B) Four month old, male Adipoq Cre-/mTmG+ negative control. GFP expression was also noted in cells lining the bone surface (arrows) and in cells of the stromal reticular network. BMAds were positive for green fluorescent protein (GFP). Bone marrow adipocytes (BMAds) were identified by expression of perilipin (PLIN1, pink, arrowheads). ( A) Representative longitudinal cross-section of the proximal tibial metaphysis and epiphysis at the region of the growth plate (*). ( F) Adipoq Cre-/mTmG+ negative control has RFP+ bone marrow stromal cells and RFP+, PLIN1+ adipocytes. ( E) Day 14 adipogenic colony with uniformly GFP+ stromal cells and PLIN1+ adipocytes. Nearby RFP+ fibroblasts show early signs of conversion (GFP+ perinuclear granules, white arrowheads). ( D) Day 14 adipogenic colony demonstrating PLIN1+ lipid droplets (pink, white arrows) in GFP+ adipocytes. ( C) Representative mixed colony with both GFP+ and RFP+ fibroblasts demonstrating GFP+ perinuclear granules in red cells (white arrowheads), indicating upregulation of adiponectin expression. ( B) Quantification of GFP and RFP expression in fibroblastic stromal cells within each culture dish (n = 3 independent mice, 166 total colonies counted). In addition, spontaneous adipogenesis was assessed based on immunostaining for perilipin 1 (PLIN1, pink). Endogenous fluorescence was then amplified by immunostaining for green fluorescent protein (GFP) and red fluorescent protein (RFP). ( A) Bone marrow stromal cells from 16 week old male Adipoq Cre+/mTmG+ mice were cultured at low density for 14 days to promote formation of colony-forming units (CFUs).
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