The thyrotropin receptor (TSHR) is the thyroid autoantigen against which stimulating autoantibodies are directed in Graves' hyperthyroidism. Recent evidence suggests that TSHR may also serve as an orbital autoantigen in Graves' ophthalmopathy (GO) and that expression of this protein is increased in the fatty connective tissues of the orbit in this condition. It has been shown that orbital fibroblasts from patients with GO increase thyrotropin (TSH)-dependent cyclic adenosine monophosphate (cAMP) production and TSHR gene expression when cultured under conditions known to stimulate adipocyte differentiation. In the current study, we wanted to determine whether treatment of these cells with particular cytokines (each 1 ng/mL) during differentiation might further augment TSHR expression. We found that exposure to interleukin (IL)-6 increased TSHR expression above control levels in cells from patients with GO. In contrast, this cytokine did not affect TSHR expression in normal orbital cells. Neither IL-4 nor IL-1alpha had a significant stimulatory effect in either normal or Graves' cultures. These findings suggest that IL-6 may play a role in the pathogenesis of GO by increasing expression of the putative autoantigen within the adipose/connective tissues of the orbit.

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α-CD supplementation did not increase loss of dietary lipid in stool or total fecal fat compared with placebo in healthy adults. Greater stool loss and lesser appearance in plasma TGs of tripalmitin-derived [14C] compared with triolein-derived [3H] TGs imply different metabolic handling of these 2 dietary fat tracers. This trial was registered at www.clinicaltrials.gov as NCT03002168.

The symptoms and signs of Graves' ophthalmopathy (GO) result from both an accumulation of hydrated hyaluronan in the orbital muscles and connective tissues and an expansion of the orbital adipose tissues. Recent studies have suggested a link between the stimulation of adipogenesis within the orbit in GO and the expression in these tissues of TSH receptor (TSHR), the putative orbital autoantigen. To further investigate this association, we treated orbital fibroblasts from patients with GO with rosiglitazone, a thiazolidinedione agonist of the PPARgamma receptor that stimulates adipocyte differentiation. We found this compound to be a potent stimulator of functional TSHR expression as well as TSHR and PPARgamma mRNA levels in differentiated cultures. In addition, rosiglitazone treatment stimulated recruitment and differentiation of a subset of cells within these cultures into mature lipid-laden adipocytes. These results suggest that TSHR expression in GO orbital preadipocyte fibroblasts is linked to adipogenesis, and that ligation of the PPARgamma receptor results in differentiation of these cells. It is possible that endogenous PPARgamma ligands play a role in stimulating orbital adipogenesis in GO, and that future treatments may be aimed at antagonism of various components of the PPARgamma signaling system.

Objective The relationship between inflammation, obesity and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). We compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC) and RT-PCR of ATM markers. Methods We evaluated a new software program (AMCounter) to help measure ATM using IHC and compared this to flow cytometry and RT-PCR. Results IHC had good intra-individual reproducibility for total (CD68), pro-inflammatory (CD14) and anti-inflammatory (CD206) ATM. The AMCounter improved inter-reader agreement and was more time efficient. Flow cytometry had acceptable intra-individual reproducibility for the percent of CD68+ cells that were CD14+ or CD206+, but not for ATM/g tissue. ATM/g 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 ATM’s per 100 adipocytes. Of interest, were also statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14 and adipose flow cytometry measures of CD68+, CD68+/CD14+ and CD68+/CD206+ ATM’s per g tissue. Conclusions The AMCounter software helps reproducibly and efficiency measures of IHC ATM’s. Flow cytometry, immunohistochemistry and RT-PCR measures of adipose inflammation provide somewhat different information.

Morgan-Bathke M, Chen L, Oberschneider E, Harteneck D, Jensen MD. Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3-phosphate acyltransferase activity. Am J Physiol Endocrinol Metab 308: E830 -E846, 2015. First published March 3, 2015 doi:10.1152/ajpendo.00424.2014.-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 ϭ Ϫ0.71, P ϭ 0.003) and subcutaneous (r ϭ Ϫ0.58, P ϭ 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. omental fat; subcutaneous fat; fat distribution; glycerol-3-phosphate acyltransferase 1 BODY FAT DISTRIBUTION PLAYS an important role in the development of the comorbidities associated with obesity. In general, adults with greater amounts of visceral fat have more risk for chronic disease than those with a lesser proportion of visceral fat (14). Although regional balances of fatty acids (uptake vs. release) should determine whether one depot expands at the expense of another, interindividual differences in regional lipolysis (8,13,24) and meal fat storage (21, 26, 31) do not appear to explain differences in body fat distribution. We found that direct free fatty acid (FFA) storage rates in subcutaneous fat are greater in women than men and that the sex-specific variations in direct FFA storage into adipocyte triacylglycerol (TAG) are consistent with body fat distribution patterns (16,18,28). On average, the protein content (CD36 and fatty acid transport protein 1) and enzyme activity [acyl-

Graves' ophthalmopathy (GO) is an autoimmune disease characterized by an increase in the volume of the orbital fatty/connective tissues and extraocular muscles. This volume change is due to expansion of the adipose tissues and to accumulation of glycosaminoglycans and edema within the connective tissues of the orbit. We have shown previously that a subpopulation of confluent human orbital preadipocyte fibroblasts can be induced in vitro to differentiate into cells with morphological features of adipocytes and that these cultures express functional thyrotropin receptor (TSHR). In order to identify and study these cells further, we examined the expression of leptin protein and TSHR and leptin mRNA in these cultures. Using immunocytochemistry with objective measurement of immunofluorescent staining intensity on digitized microscopic images, we determined leptin protein expression to be 6 to 37 times greater in differentiated cultures than in control cultures. In addition, we showed that the expression of both genes is enhanced in differentiated cultures. We suggest that an unknown humoral stimulus, present in Graves' disease, might act to induce the differentiation of normal orbital fibroblasts into TSHR-bearing adipocytes. This process would be expected to result in expansion of the orbital adipose tissues and increased TSHR expression within the orbit.

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.Design and MethodsWe measured direct FFA storage in subcutaneous fat in 13 adults 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.ResultsWe were able to isolate populations of adipocytes 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 (2-4 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.