We investigated the mechanism of thyroid hormone regulation of osteocalcin (OC) gene expression in osteoblast-like cells (ROS 17/2·8). Treatment with triiodothyronine (T 3 ) (10 8 M) increased OC mRNA levels by 3-fold after 24 h and reached a maximum, 5·4-fold, after 48 h. The mRNA levels of other bonespecific genes, alkaline phosphatase and osteopontin, were not affected by T 3 treatment. Interestingly, T 3 induction of OC mRNA varied according to cell density: 4-fold at 1 10 5 cells/dish and 1·5-fold at 40-60 10 5 cells/ dish. The magnitude of OC mRNA induction by T 3 was 40% lower than induction by 1,25 dihydroxyvitamin D 3 (1,25D 3 ) alone, and the combination of T 3 +1,25D 3 did not further stimulate OC mRNA levels. T 3 induction of OC mRNA was not affected by treatment with cycloheximide (10 µg/ml) for 5 h indicating that new protein synthesis is not required for the response. To study the half-life of OC mRNA, ROS 17/2·8 cells were incubated with actinomycin D. The basal half-life of OC mRNA (means ...) was 6·4 0·2 h which was increased significantly with either T 3 or 1,25D 3 treatment to 10·9 0·6 h and 13·5 0·4 h respectively. T 3 modestly up-regulated the rate of OC gene transcription (1·7 0·2-fold) as determined by run-off assay. T 3 did not induce a reporter construct containing the rat OC gene (rOC) 5 -flanking region (to -1750 bp) or the previously described rOC vitamin D response element, when transfected into ROS 17/2·8 cells. In conclusion, T 3 up-regulates the OC mRNA expression in ROS 17/2·8 cells in a dose-, time-and cell confluence-dependent fashion, and does so by transcriptional and posttranscriptional mechanisms. The greater T 3 induction of OC expression in ROS 17/2·8 cells at low cell density is consistent with findings of thyroid hormone action on bone development.

SummaryVisceral adiposity in elderly is associated with alterations in adipose tissue immune cells leading to inflammation and metabolic dysfunction. The Nlrp3 inflammasome is a critical regulator of macrophage activation, inflammation, and immunometabolism in visceral adipose tissue during aging; however, the potential contribution of adipose tissue B cells is unexplored. Here, we show that aging expands adipose-resident B cells and fat-associated lymphoid clusters (FALCs) in visceral white adipose tissue. Adipose tissue B cells exhibit a memory-like B cell profile similar to the phenotype of aged B cells that are increased in spleen of old mice. Mechanistically, the age-induced FALC formation and adipose B cell expansion, but not B cell transcriptional program, is dependent on the Nlrp3 inflammasome. Furthermore, B cell depletion in aged mice restores lipolysis and defense against loss of core body temperature during cold stress. These data reveal that inhibiting Nlrp3-dependent B cell accumulation can be targeted to reverse metabolic impairment in aging adipose tissue.Highlights- Adipose-resident aged B cells are increased in fat-associated lymphoid clusters (FALC)- FALC formation and adipose-resident B cell expansion during aging are regulated by the Nlrp3 inflammasome- Nlrp3 and B cell depletion in aging restores lipolysis and improves cold tolerancea