Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Dominant Negative Effect of Mutated Thyroid Stimulating Hormone Receptor (P556L) Causes Hypothyroidism in C.RF-Tshr(hyt/wild) Mice

C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.     

A lack of thyroid hormones rather than excess thyrotropin causes abnormal skeletal development in hypothyroidism

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Nerve growth factor concentration in a congenitally hypothyroid mouse model (hyt/hyt) and its responsivity to thyroxine treatment

Earlier our laboratory reported the ontogenic profiles of serum thyroxine (T4) and triiodothyronine (T3) concentrations and nerve growth factor levels in the submandibular gland of Swiss-Webster mice. Further, we demonstrated a responsivity of submandibular gland-nerve growth factor concentrations to exogenously administered T4. To further our understanding of the interactions between thyroid hormones and submandibular gland-nerve growth factor we utilized a congenitally hypothyroid mouse model to examine submandibular gland-nerve growth factor in euthyroid, hypothyroid and hypothyroid-T4 replaced mouse pups. Serum T4 values in the congenitally hypothyroid (hyt/hyt) mice were unmeasurable and their growth in body weight, their incisor eruption, and their eyelid opening were significant delayed. Submandibular gland/body weight ratios were significantly reduced relative to control or heterozygous (+/hyt) animals through 40 days. The increase in submandibular gland-nerve growth factor concentrations observed in normal animals before 20 days was delayed to 35 days in the (hyt/hyt) animals. T4 treatment of (hyt/hyt) animals from 11 or 18 days of age significantly increased mean 40 day submandibular gland/body weight ratios and submandibular gland-nerve growth factor concentrations. However, the 40 day submandibular gland-nerve growth factor levels in treated animals remained significantly below the level of control euthyroid mice. Thus, the possibility of critical time of thyroid hormone replacement for normal submandibular gland maturation has not been shown but must be further explored in this model.     

Hypothalamic CRF immunoreactivity in genetically hypothyroid (hyt/hyt) mice

The induction of hypothyroidism in young rats by feeding thiouracil to their mothers during pregnancy has been shown to depress hypothalamic content of bioactive and immunoactive corticotropin-releasing factor (CRF). The present study was done to determine whether genetically hypothyroid young mice (hyt/hyt) born to euthyroid mothers (+/hyt) exhibited a similar depression in hypothalamic CRF immunoreactivity. Young euthyroid and hypothyroid littermate mice were examined by radioimmunoassay for hypothalamic CRF content at 15, 20, 25, or 30 days of age. Mean CRF content was depressed insignificantly (to about 80% of normal) by hypothyroidism, at 15-25 days of age. However, after weaning by the mother, 30-day-old hypothyroid pups demonstrated significantly depressed hypothalamic CRF levels (71%). It is suggested that maternal factors may be assisting in the maintenance of hypothalamic CRF until after weaning. Furthermore, genetic hypothyroidism does not appear to have nearly as marked an influence as thiouracil feeding on hypothalamic CRF levels.     

Hypomyelination in the Cerebrum of the Congenitally Hypothyroid Mouse (hyt)

2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the cerebrum of the inherited primary hypothyroid mouse (hyt/hyt) is reduced in comparison with the normal heterozygate (hyt/+). No differences were observed with regard to DNA and RNA content and the RNA/DNA ratio. The results of this study indicate that hypomyelination in the hypothyroid mouse is restricted to the cerebrum, and is not related to arrested glial proliferation.     

Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway

Thyroid-stimulating hormone (TSH) has been shown to play an important role in the regulation of triglyceride (TG) metabolism in adipose tissue. Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme controlling the hydrolysis of TG. Thus far, it is unclear whether TSH has a direct effect on the expression of ATGL. Because TSH function is mediated through the TSH receptor (TSHR), TSHR knockout mice (Tshr-/- mice) (supplemented with thyroxine) were used in this study to determine the effects of TSHR deletion on ATGL expression. These effects were verified in 3T3-L1 adipocytes and potential underlying mechanisms were explored. In the Tshr-/- mice, ATGL expression in epididymal adipose tissue was significantly increased compared with that in Tshr+/+ mice. ATGL expression was observed to increase with the differentiation process of 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, TSH significantly suppressed ATGL expression at both the protein and mRNA levels in a dose-dependent manner. Forskolin, which is an activator of adenylate cyclase, suppressed the expression of ATGL in 3T3-L1 adipocytes. The inhibitory effects of TSH on ATGL expression were abolished by H89, which is a protein kinase A (PKA) inhibitor. These results indicate that TSH has an inhibitory effect on ATGL expression in mature adipocytes. The associated mechanism is related to PKA activation.     

Development of an animal model of autoimmune thyroid eye disease

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.     

Interleukin-6 and cachexia in ApcMin/+ mice

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.     

The gender- and fat depot-specific regulation of leptin, resistin and adiponectin genes expression by progesterone in rat

Progesterone affects lipid metabolism in adipose tissue and influences fat distribution in human. The aim of the study was to analyze the effect of progesterone on rat body and fat mass and on expression of genes encoding adipokines involved in the regulation of energy homeostasis. The results presented here indicate that progesterone administration to females caused increase in body and inguinal white adipose tissue mass. The increase of inguinal white adipose tissue mass is associated with the hypertrophy of adipocyte. The same dose of progesterone caused increase of its circulating concentration in males, however it barely reached the value observed in non-treated control females and did not have any effect on body and fat mass. The elevated circulating progesterone concentration was associated with an approximately 6- and 2-fold increase of leptin and resistin mRNA level respectively, and 2-fold decrease of adiponectin mRNA level only in inguinal white adipose tissue of females. RU 486, specific antagonist of progesterone receptor, abolished the effect of progesterone on the adipokine mRNA level in inguinal adipose tissue. In males, the elevated circulating progesterone concentration showed no effects on leptin, resistin or adiponectin mRNA level in inguinal, retroperitoneal or epididymal adipose tissue. Moreover, the results presented in this paper demonstrate a relatively high level of progesterone receptor mRNA in inguinal white adipose tissue of females, which was down-regulated in response to progesterone administration. In retroperitoneal adipose tissue of control females progesterone receptor mRNA level was approximately 3-fold lower as compared to inguinal adipose tissue. In inguinal, epididymal and retroperitoneal white adipose tissue of males progesterone receptor mRNA was hardly detected. Our results suggest that depot- and sex-dependent responsiveness of adipose tissue to the pharmacological dose of progesterone is controlled by both circulating concentration of progesterone and the white adipose tissue progesterone receptor level.     

Impaired fat storage capacity in adipocyte precursors of I versus C57BL mice

I mouse strain displays adipocyte hypoplasia responsible for smaller fat pad size compared with C57BL mice. We investigated possible alterations in the proliferation and/or differentiation capacity of preadipocytes from the stroma-vascular fraction of adipose tissue in the I mouse strain. Control C57BL and I mice were studied at 8 weeks of age, and both adipose and stromal cells were isolated from epididymal and inguinal adipose tissue localizations. Results showed that the lower epididymal adipose mass in I mice was accompanied by a decrease in stromal cell number compared with C57BL mice. In inguinal fat pads, total cell number in the stroma-vascular fraction was unmodified; lipoprotein lipase activity significantly increased in stromal cells from I mice compared with control mice. In this depot, further characterization of cells from the stroma-vascular fraction by separation of cells according to density showed an increased number of preadipocytes in the I mouse whole stromal cell population. These preadipocytes seemed unable to undergo terminal maturation, thus leading to a decrease in the number of mature adipocytes. These results indicated that resistance to fat accumulation in I mice is characterized by site-dependent impairment of both the proliferative rate and the differentiation capacity of adipocyte precursors.     

Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet

We investigated the mechanisms that lead to combined hyperlipidemia in transgenic mice that overexpress human apolipoprotein (apo) A-II (line 11.1). The 11.1 transgenic mice develop pronounced hypertriglyceridemia, and a moderate increase in free fatty acid (FFA) and plasma cholesterol, especially when fed a high-fat/high-cholesterol diet. Post-heparin plasma lipoprotein lipase and hepatic lipase activities (using artificial or natural autologous substrates), the decay of plasma triglycerides with fasting, and the fractional catabolic rate of the radiolabeled VLDL-triglyceride (both fasting and postprandial) were similar in 11. 1 transgenic mice and in control mice. In contrast, a 2.5-fold increase in hepatic VLDL-triglyceride production was observed in 11. 1 transgenic mice in a period of 2 h in which blood lipolysis was inhibited. This increased synthesis of hepatic VLDL-triglyceride used preformed FFA rather than FFA of de novo hepatic synthesis. The 11.1 transgenic mice also presented reduced epididymal/parametrial white adipose tissue weight (1.5-fold), increased rate of epididymal/parametrial hormone-sensitive lipase-mediated lipolysis (1.2-fold) and an increase in cholesterol and, especially, in triglyceride liver content, suggesting an enhanced mobilization of fat as the source of preformed FFA reaching the liver. Increased plasma FFA was reverted by insulin, demonstrating that 11.1 transgenic mice are not insulin resistant. We conclude that the overexpression of human apoA-II in transgenic mice induces combined hyperlipidemia through an increase in VLDL production. These mice will be useful in the study of molecular mechanisms that regulate the overproduction of VLDL, a situation of major pathophysiological interest since it is the basic mechanism underlying familial combined hyperlipidemia.     

Chronic Alcohol Exposure Disturbs Lipid Homeostasis at the Adipose Tissue-Liver Axis in Mice: Analysis of Triacylglycerols Using High-Resolution Mass Spectrometry in Combination with In Vivo Metabolite Deuterium Labeling

A method of employing high-resolution mass spectrometry in combination with in vivo metabolite deuterium labeling was developed in this study to investigate the effects of alcohol exposure on lipid homeostasis at the white adipose tissue (WAT)-liver axis in a mouse model of alcoholic fatty liver. In order to differentiate the liver lipids synthesized from the fatty acids that were transported back from adipose tissue and the lipids synthesized from other sources of fatty acids, a two-stage mouse feeding experiment was performed to incorporate deuterium into metabolites. Hepatic lipids extracted from mouse liver, epididymal white adipose tissue (eWAT) and subcutaneous white adipose tissue (sWAT) were analyzed. It was found that 13 and 10 triacylglycerols (TGs) incorporated with a certain number of deuterium were significantly increased in alcohol induced fatty liver at two and four weeks of alcohol feeding periods, respectively. The concentration changes of these TGs ranged from 1.7 to 6.3-fold increase. A total of 14 deuterated TGs were significantly decreased in both eWAT and sWAT at the two and four weeks and the fold-change ranged from 0.19 to 0.77. The increase of deuterium incorporated TGs in alcohol-induced fatty liver and their decrease in both eWAT and sWAT indicate that alcohol exposure induces hepatic influx of fatty acids which are released from WATs. The results of time course analysis further indicate a mechanistic link between adipose fat loss and hepatic fat gain in alcoholic fatty liver.     

Differences in lipogenesis in tissues of control and gold-thioglucose obese mice after an isocaloric meal

Lipogenesis was measured in 2 and 5 week gold-thioglucose (GTG) obese mice after a single meal of 0.5 g of standard chow. Compared to control mice the rate of lipogenesis in GTG obese mice, was 4-fold higher in liver and 10-fold higher in white adipose tissue (WAT). In brown adipose tissue (BAT) of GTG-injected mice the lipogenic rate was only 50% of that of controls. These results indicate that the increased lipid synthesis observed in GTG-injected mice is not due solely to hyperphagia and that some other stimuli, such as increased basal insulin levels and/or decreased thermogenesis and insulin resistance in BAT, contribute to the high rates of fat synthesis in this animal model of obesity.     

Chronic Glucocorticoid Exposure-Induced Epididymal Adiposity Is Associated with Mitochondrial Dysfunction in White Adipose Tissue of Male C57BL/6J Mice

Prolonged and excessive glucocorticoids (GC) exposure resulted from Cushing''s syndrome or GC therapy develops central obesity. Moreover, mitochondria are crucial in adipose energy homeostasis. Thus, we tested the hypothesis that mitochondrial dysfunction may contribute to chronic GC exposure-induced epididymal adiposity in the present study. A total of thirty-six 5-week-old male C57BL/6J mice (∼20 g) were administrated with 100 µg/ml corticosterone (CORT) or vehicle through drinking water for 4 weeks. Chronic CORT exposure mildly decreased body weight without altering food and water intake in mice. The epididymal fat accumulation was increased, but adipocyte size was decreased by CORT. CORT also increased plasma CORT, insulin, leptin, and fibroblast growth factor 21 concentrations as measured by RIA or ELISA. Interestingly, CORT increased plasma levels of triacylglycerols and nonesterified fatty acids, and up-regulated the expression of both lipolytic and lipogenic genes as determined by real-time RT-PCR. Furthermore, CORT impaired mitochondrial biogenesis and oxidative function in epididymal WAT. The reactive oxygen species production was increased and the activities of anti-oxidative enzymes were reduced by CORT treatment as well. Taken together, these findings reveal that chronic CORT administration-induced epididymal adiposity is, at least in part, associated with mitochondrial dysfunction in mouse epididymal white adipose tissue.     

Browning attenuates murine white adipose tissue expansion during postnatal development

During postnatal development of mice distinct white adipose tissue depots display a transient appearance of brown-like adipocytes. These brite (brown in white) adipocytes share characteristics with classical brown adipocytes including a multilocular appearance and the expression of the thermogenic protein uncoupling protein 1. In this study, we compared two inbred mouse strains 129S6sv/ev and C57BL6/N known for their different propensity to diet-induced obesity. We observed transient browning in retroperitoneal and inguinal adipose tissue depots of these two strains. From postnatal day 10 to 20 the increase in the abundance of multilocular adipocytes and uncoupling protein 1 expression was higher in 129S6sv/ev than in C57BL6/N pups. The parallel increase in the mass of the two fat depots was attenuated during this browning period. Conversely, epididymal white and interscapular brown adipose tissue displayed a steady increase in mass during the first 30 days of life. In this period, 129S6sv/ev mice developed a significantly higher total body fat mass than C57BL6/N. Thus, while on a local depot level a high number of brite cells is associated with the attenuation of adipose tissue expansion the strain comparison reveals no support for a systemic impact on energy balance. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Diversity of SREBP-1 gene expression in rat adipose tissue depots in response to refeeding after food restriction

The SREBP-1c mRNA level and precursor (microsomal) form of SREBP-1 abundance were significantly higher in epididymal and perirenal than in subcutaneous white adipose tissue of control rats. Moreover, the SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were significantly higher in the epididymal and perirenal white adipose tissue of rats maintained on restricted diet and refed ad libitum for 48 h as compared to the control animals. No significant effects of food restriction/refeeding on SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were found in subcutaneous white adipose tissue. The mature (nuclear) form of SREBP-1 was significantly increased in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed animals. The activity, protein level and the mRNA abundance of malic enzyme (one of the target genes for SREBP-1) increased significantly in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed rats as compared to the control animals, however the increase in perirenal and epididymal was higher than in the subcutaneous white adipose tissue. The results presented suggest that SREBP-1c is differently expressed in various rat white adipose tissue depots both under basal (control) and dieting conditions.     

Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts

Controversy continues about whether, and to whatlevels of abundance, thyroid-stimulating hormone receptors (TSHR) arefound in human tissues other than the thyroid gland. Restrictedexpression to the thyroid and orbit would suggest that TSHR representsthe target autoantigen in thyroid-associated ophthalmopathy. A more generalized pattern of tissue expression would be inconsistent withTSHR acting as the autoantigen that is solely responsible forselectively targeting the immune system to the orbit. We have detectedTSHR mRNA in human abdominal adipose tissue by Northern blot analysis.TSHR protein was also detected, by immunoblotting with two differentantibodies, in preadipocytes isolated from human abdominal subcutaneousand omental adipose tissue and in derivative adipocytes differentiatedin primary culture. Preadipocytes treated with thyroid-stimulatinghormone (TSH) exhibited a sevenfold increase in the activity of p70 S6kinase, a serine/threonine kinase recently recognized as a downstreamtarget of TSHR in thyroid cells. Activation of p70 S6 kinase by TSH wasalso observed in orbital fibroblasts. Thus TSHR protein expression isfound in fibroblasts from several anatomic locations, suggesting thatfactors other than site-limited TSHR expression must be involved inrestricting the distribution of Graves' disease manifestations.Furthermore, the presence of functional TSHR in preadipocytes raisesthe possibility of a novel role for TSHR signaling in adipose tissue development.

     

Adipose tissues as an ancestral immune organ: site-specific change in obesity

Close relationships have been demonstrated between adipose tissue and the inflammatory/immune system. Furthermore, obesity is increasingly considered as a state of chronic inflammation. Cytofluorometric analysis reveals the presence of significant levels of lymphocytes in the stroma-vascular fraction of white adipose tissues. In epididymal (EPI) fat, lymphocytes display an "ancestral" immune system phenotype (up to 70% of natural killer (NK), gammadelta+ T and NKT cells among all lymphocytes) whereas the inguinal (ING) immune system presents more adaptive characteristics (high levels of alphabeta+ T and B cells). The percentage of NK cells in EPI fat was decreased in obese mice fed with a high-fat diet, whereas gammadelta positive cells were significantly increased in ING fat. These data support the notion that adipose tissue may elaborate immunological mechanisms to regulate its functions which might be altered in obesity.