Harteneck, Debra A.

ObjectiveThe relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real‐time polymerase chain reaction (RT‐PCR) of ATM markers.MethodsA new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT‐PCR.ResultsIHC had good intraindividual reproducibility for total (CD68), proinflammatory (CD14), and anti‐inflammatory (CD206) ATM. The AMCounter improved interreader agreement and was more time efficient. Flow cytometry had acceptable intraindividual reproducibility for the percentage of CD68+ cells that were CD14+ or CD206+, but not for ATMs per gram of tissue. ATMs per gram of tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT‐PCR CD68 and IHC CD68, CD14, and CD206 ATMs per 100 adipocytes. Also of interest were statistically significant correlations between RT‐PCR CD68 and IHC CD68, CD14, and adipose flow cytometry measures of CD68+, CD68+/CD14+, and CD68+/CD206+ ATMs per gram of tissue.ConclusionsThe AMCounter software helps provide reproducible and efficient measures of IHC ATMs. Flow cytometry, IHC, and RT‐PCR measures of adipose inflammation provide somewhat different information.

Adipose tissue fatty acid storage varies according to sex, adipose tissue depot, and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We examined whether differences in adipose tissue glycerol-3-phosphate acyltransferase (GPAT) might play a role in these variations. We optimized an enzyme activity assay for total GPAT and GPAT1 activity in human adipose tissue and measured GPAT activity. Omental and subcutaneous adipose tissue was collected from obese and nonobese adults for measures of GPAT and GPAT1 activities, ex vivo palmitate storage, acyl-CoA synthetase (ACS) and diacylglycerol-acyltransferase (DGAT) activities, and CD36 protein. Total GPAT and GPAT1 activities decreased as a function of adipocyte size in both omental ( r = −0.71, P = 0.003) and subcutaneous ( r = −0.58, P = 0.04) fat. The relative contribution of GPAT1 to total GPAT activity increased as a function of adipocyte size, accounting for up to 60% of GPAT activity in those with the largest adipocytes. We found strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots ( r values 0.58–0.91) and between these storage factors and palmitate storage rates into TAG ( r values 0.55–0.90). We conclude that: 1) total GPAT activity decreases as a function of adipocyte size; 2) GPAT1 can account for over half of adipose GPAT activity in hypertrophic obesity; and 3) ACS, GPAT, and DGAT are coordinately regulated.

Although the autoantigen involved in Graves’ hyperthyroidism is known to be the TSH receptor (TSHr), whether this antigen plays a primary role in the pathogenesis of Graves’ ophthalmopathy (GO) is unclear. We sought to determine whether fibroblasts derived from orbital adipose/connective tissue are capable of differentiating into adipocytes that bear immunoreactive and functional TSHr. In addition, we assessed relative levels of TSHr gene expression in normal and GO orbital adipose/connective tissue specimens.GO and normal orbital preadipocyte fibroblasts, cultured under conditions known to stimulate adipocyte differentiation, showed evidence of adipogenesis and positive immunostaining for TSHr protein. In addition, significantly more cAMP was produced in response to TSH stimulation in the differentiated cultures than in undifferentiated cultures derived from the same individuals’ cells. Other studies demonstrated relatively greater TSHr gene expression in GO than in normal orbital tissue specimens.These results indicate that orbital preadipocyte fibroblasts increase their TSHr expression with differentiation and suggest that these cells play an important role in the pathogenesis of GO. Furthermore, our studies support the concept that TSHr may be an important target antigen in this condition. Factors that stimulate adipocyte differentiation and TSHr expression in the orbit in GO have yet to be defined.

ObjectiveHuman adipocytes take up free fatty acids (FFA) directly from the circulation, even at times of high lipolytic activity. Whether these processes occurs simultaneously within the same cells or are partitioned between different cells, for example large and small cells, is unknown.MethodsThe direct FFA storage in subcutaneous fat in 13 adults were measured using a continuous infusion of [U‐13C]palmitate and a bolus of [1‐14C]palmitate followed 30 min later by abdominal and femoral adipose biopsies. The adipocytes were isolated by digestion procedures and separated into small, medium and large populations by differential floatation.ResultsPopulations of adipocytes were isolated that were statistically and clinically (∼3 fold different) in size. Adipocyte lipid SA was not different between small, medium and large cells, therefore, FFA storage per unit lipid was not different. However, FFA storage rates were significantly (two to four times) greater per cell in large than small cells (P < 0.005). In summary, relative to lipid content, FFA storage rates are not different in large and small adipocytes, however, large cells have greater storage rates per cell.ConclusionsThis suggests that the processes of FFA release and storage are taking place simultaneously in adipocytes.