Hypothyroidism profoundly reduces the capacity of brown adipose
tissue (BAT) to generate CAMP in response to adrenergic stimulation. Evidence obtained with isolated brown adipocytes suggests a postre- ceptor defect that offsets the hypothyroidism-induced increase in /.%- adrenergic receptors. The goal of the present studies was to identify the defect in the CAMP generation pathway for which we studied CAMP generation in isolated cells and purified BAT membranes from normal and hypothyroid rats. Studies with adenosine deaminase and the adenosine receptor-l agonist r-phenyl isopropyl adenosine (R- PIA) show that hypothyroid cells are not more sensitive to adenosine (same EC,,,) but more inhibited by high concentrations of R-PIA. Pretreatment with pertussis toxin reduced the gap in CAMP gener- ation between eu- and hypothyroid cells and the inhibition mediated by R-PIA, but did not normalize the CAMP response to forskolin in hypothyroid cells. Although purified euthyroid BAT membranes in- creased CAMP production with GTP concentrations up to submilli- molar range, to plateau or slightly decrease at higher levels, hypo- thyroid membranes were weakly stimulated by low concentrations of GTP and markedly inhibited (>50%) at concentrations ~10 ’ II. When assayed at 0.3 mw ATP and 1 p%fGTP, hypothyroid membranes actually generated more CAMP in response to forskolin, hut this was reversed when GTP concentration was 1mhl. Immunoblotting studies showed no significant effects of hypothyroidism on the abundance of G,,i or Glj subunits, and ADP ribosylation of G,,i was only 45’~ in- creased in hypothyroidism in contrast to a 2.5-fold increase in hypo- thyroid white adipose tissue membranes from the same rats. Hypo- thyroid membranes also exhibited different kinetics regarding ATP, with higher CAMP generation at submillimolar concentrations but less at >,l rnhl ATP. Actually, at ATP concentrations >0.6 rn>f, CAMP generation was markedly inhibited in hypothyroid membranes. Fix- ing the concentration offree Mg- in these experiments indicates that most of the inhibition seen in hypothyroid membranes is caused by ATP, whereas euthyroid membranes are more sensitive to changes in free Mg Ca ’ I calmodulin did not stimulate adenylyl cyclase (AC) activity. On the contrary, AC activity was inhibited by Ca’ ’ in a concentration-dependent manner, by as low as 100 nM free Ca ’ , and to greater extent in hypo- than in euthyroid membranes (maximal inhibition 60 US.25-30%). Our results suggest that, functionally, hypothyroidism causes a change in the AC of BAT membranes con- sistent with a relative or absolute increase in the type VI AC (AC-VI). The effects on this AC of nuclcotides, Ca , and Mg at concentra- tions prevailing in the hypothyroid brown adipocyte are probably the major factor in the reduced capacity of these cells to generate CAMP. These results also open the possibility of a novel, differential effect of thyroid hormone on AC expression, and support the concept that thyroid hormone affects the adrenergic signal transduction pathways in a tissue-selective manner.
Effects of Hypothyroidism on Brown Adipose Tissue Adenylyl Cyclase Activity.
Suzy D. Carvalhof, Antonio C. Bianco, And J. Enrique Silva. Journal Of Bone And Mineral Research. July 21, 1997.