Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis

Biochemical and Biophysical Research Communications 393 (2010) 526–530

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Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis Aziz Elgadi a,*, Helen Zemack a, Claude Marcus a,1, Svante Norgren b,1 a

Department of Clinical Science, Intervention and Technology, Division of Pediatrics, Karolinska Institutet, Endocrine Research Unit B62, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden b Department of Woman and Child Health, Karolinska Institutet, B57, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

a r t i c l e

i n f o

Article history: Received 5 February 2010 Available online 10 February 2010 Keywords: Thyroid stimulating hormone receptor (TSHr) Adipocyte Tissue-specific knockout Cre-lox recombinant

a b s t r a c t Background: The primary function of TSH is to activate TSH receptors (TSHr) in the thyroid gland and thereby stimulate thyroid hormone synthesis and secretion. TSHr are also expressed in other organs, but their physiological importance is still unclear. We have previously shown that TSHr, expressed in adipocytes, are of potential importance for lipolysis and extrauterine adaptation of the neonate. Methodology: To further study the role of TSHr in adipocytes we selectively removed the TSHr gene in mice adipocytes by using the Cre-loxP recombination system (B6.Cg-Tg (Fabp4-Cre) 1Rev/J. TSHr knockout (KO) newborn mice were phenotypically characterized. Isolated adipocytes from 8-week-old male mice were studied in term of adipocyte size and metabolism. Results: Mice lacking TSHr in adipocytes were apparently normal at birth and no differences in thyroid gland function or histology were observed. Sensitivity to TSH-induced lipolysis was ten times lower in adipocytes from targeted animals compared to wild-type. This indicates that adipocytes from targeted animals are refractory to stimulation of physiological concentrations of TSH. Catecholamine-induced lipolysis and insulin-induced inhibition of lipolysis were unaltered. Adipocyte size was increased in the targeted animals. Basal lipolysis was increased as an effect of the increased adipocyte size. Conclusion: Our results indicate that adipocyte TSHr under normal conditions affects adipocyte growth and development. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Adipose tissue is a major metabolic and endocrine organ as well as a target for the action of several hormones, locally secreted cytokines, and paracrine acting substances. Overgrowth of adipose tissue is associated with negative endocrine, metabolic, and cardiovascular effects, and the worldwide obesity epidemic is one of the most important public health care problems due to the associated risks of cardiovascular disease, type 2 diabetes, cancer, arthritis, and a range of psychological problems [1–3]. The key feature of adipocytes is the storage of energy in the form of triglycerides (TGs). As need arises, triglycerides are hydrolyzed by adipocyte lipases leading to the formation of free fatty acids (FFAs) and glycerol for use by other organs as energy substrates. Adipocyte lipolysis is a complex process that is tightly regulated via integration of multiple hormonal and biochemical signals. A large number of substances regulate cAMP levels and thereby lipolysis. In adults, catecholamines are the only hormones * Corresponding author. Fax: +46 8 585 87 370. E-mail address: [email protected] (A. Elgadi). 1 Shared senior authorship. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.042

with a pronounced and immediate lipolytic action [4,5], and the lipolytic action of catecholamine is mediated by three b-adrenergic receptor subtypes: b1, b2, and b3 [6,7]. The lipolytic hormones are balanced by inhibitory factors. Adenosine and nicotinic acid inhibit lipolysis by binding to Gi protein-coupled receptors [8,9]. In man – but not in the mouse – a2 adrenergic receptors modify the lipolytic response of endogenous catecholamine [10–13]. In addition, insulin inhibits lipolysis through multiple pathways distinct from the Gi pathway [4,14]. Thyroid stimulating hormone receptors (TSHr) are expressed in many tissues such as human [15–17] and rat [18,19] adipocytes, heart [17], and bone [20], but the physiological significance of this is still unclear. In human adipocytes, obtained from neonates, TSH is the dominant lipolytic hormone [15,16] since the lipolytic effect of endogenous catecholamines is blocked by an increased alpha-2 adrenoceptor activity [10,15,21,22]. Moreover, recent data suggest a functional role of TSHr in adipocytes. Nannipieri et al. [23] reported less expression of TSHr and higher plasma level of TSH in obese than in lean individuals. These changes reversed after major weight loss. TSH mediates its function by binding to a G-protein-coupled receptor (TSHr), which activates the G-protein which in turn activates adenylyl cyclase and thereby increases the cAMP level [24].

A. Elgadi et al. / Biochemical and Biophysical Research Communications 393 (2010) 526–530

Mutant mouse lines that lack either functional TSH or TSHr display hypothyroidism [25–30]. The phenotypes are complex and reflect the effects of both hypothyroidism and TSHr dysfunction in different tissues. We therefore cannot deduce the specific role of TSHr in adipose tissue in these strains. Thus, in order to study the role of TSHr in regulating adipocyte metabolism, we report the effects of an adipose-tissue-specific TSHr knockout on adipocyte size and metabolism. Materials and methods Study protocol and animal care. The study protocols have been carried out in accordance with Declaration of Helsinki, EC Directive 86/ 609/EEC for animal experiments and approved by the South Stockholm ethics committee. Mice were maintained in standard barrier facilities with a 12-h light/dark cycle and fed ad libitum on a diet of Laboratory Chow (Altromin GmbH, Lage, Germany) according to the regulations of the Federation of European Laboratory Animal Science Association. Mice were weighed at 4, 6, and 8 weeks of age. Means for each group at each time point were calculated. Breeding protocol. Breeding protocols used are summarized as follows. Male chimera mice were first mated with C57BL/6 females (first backcross) and TSHr+/ offspring were mated with a congenic strain C57BL/6 harboring Flip recombinase (second backcross). Sequencing analyses confirmed the removal of neomycin resistance cassette flanked by two FRT sites, with only one FRT site remaining. These mice were then backcrossed to C57BL/6 for five generations. Hybrid offspring were intercrossed to generate TSHr+/+ without FRT. TSHr++ intercrossed to a congenic strain expressing Cre recombinase under the control of the mouse Fabp4 (B6.Cg-Tg (Fabp4-Cre) 1Rev/J Stock Number: 005069 Jax Laboratory). This crossing continues for nine generations to ensure a uniform congenic genetic background. Supplementary data and figures show the methods used for generating adipocyte tissue-specific knockout model. Serum chemical analysis, histology and isolation of white adipose tissue. Eight male homozygous TSHr knockout mice (TSHrloxP/loxP Cre/+) and eight male (TSHrwt/wt) littermates were analyzed. All were weaned at 3 weeks and decapitated at 8 weeks of age. Serum chemical analysis. Blood was collected by cardiac puncture and serum was frozen at 20 °C for later analysis of total T4. Histology. The following tissues were examined: brain, thymus, spleen, pancreas, lymph nodes, liver, kidney, adrenal glands, salivary glands, Harderian gland, trachea, thyroid, esophagus, aorta, lung, testes, epididymis, urinary bladder, ovaries, uterus, oviducts, cervix, urinary bladder, prostate, seminal vesicles, heart, tongue, skeletal muscle, eyes, stomach, small intestine, cecum, colon, rectum, skin, sternum, vertebrae, femur, and spinal cord. Tissues were fixed in buffered aqueous formalin, embedded in paraffin, sectioned at 5 lm, and stained with hematoxylin and eosin (HE). Isolation of adipocytes. Epididymal adipose tissue samples were placed in isotonic saline, and preparation of isolated adipocyte was started within 30 min as described [31]. In brief, adipose tissue was cut into fragments and isolated from stroma by incubation with collagenase (Sigma, St. Louis, MO, USA) for 1 h at 37 °C in Krebs– Ringer phosphate buffer, pH 7.4, containing 40 g/L of bovine albumin. The samples were washed in Krebs–Ringer phosphate buffer, pH 7.4, and aggregated material was removed by filtration through a silk cloth. Determination of adipocyte size. One hundred and fifty microliters aliquots were added to 450 lL of 0.2% methylene blue for nuclei staining and incubated for 15 min at 37 °C in a water bath. About 50–100 lL from the cell suspension was placed on a glass slide, cover-slipped, and measured optically using a Nikon microscope. Next, cells were photographed using a Nikon digital camera attached to the microscope [32]. Cell diameters were measured double blindly by two investigators. Briefly, the recorded images

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were saved in a TIFF format. Using the line tool, the diameters of at least 1000 adipocytes from each animal were manually drawn and individually measured. Southern blot analysis. To estimate the efficiency of tissue specific inactivation of the TSHr gene, Southern blot analysis was performed on 10 lg genomic DNA isolated from adipocytes of TSHr knockout mice (TSHrloxP/loxP Cre/+) and wild-type (TSHrwt/wt) littermates using HindIII and the 30 external probe. Western blot analysis. Proteins isolated from white adipose tissue of TSHr knockout mice (TSHrloxP/loxP Cre/+) and wild-type (TSHrwt/wt) littermates were subjected to electrophoresis in 9% SDSPAGE and transferred to nitrocellulose membranes. Membranes were probed with rabbit polyclonal antibody to the N-terminus of TSHR domain of TSHR (sc-13936 Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1:100. Bound antibodies were detected using Amersham ECL Plus Western blotting detection Reagent according to the manufacturer’s instruction (ECL plus kit; GEhealthcare, UK). Real time quantitative reverse transcription-PCR (real time Q-PCR). A duplicate of total RNA of TSHr knockout mice (TSHrloxP/loxP Cre/+) and wild-type (TSHrwt/wt) littermates were isolated from white and brown adipocytes, heart, liver, muscle, brain, testis and kidney by Aurum total RNA kits according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). A 1 lg aliquot of RNA was used for the reverse transcription reaction with random hexamers, to generate cDNA (promega, Madison, WI, USA). Expression of mRNA was determined by real time RT-PCR using SYBr green fluorescent reaction mixture (Bio-Rad Laboratories) in duplicates. Determination of the lipolysis rate. Adipocyte samples were incubated in duplicates for 2 h at 37 °C in Krebs–Ringer phosphate buffer, pH 7.4, containing bovine albumin (40 g/L), glucose (1 g/L), and ascorbic acid (0.1 g/L). Increasing concentrations of isoprenalin (1013–107 mol/L) or bovine TSH (101–106 mU/L) (Sigma, St. Louis, MO, USA) were added in the absence or presence of 10 mU/L human recombinant insulin (ActrapidÒ Novo Nordisk Scandinavia AB, Malmö, Sweden) at the concentration of isoprenalin (108 mol/L) or bovine TSH (106 mU/L). The final cell concentration was 1% vol/vol, which corresponds to 5000–10,000 cells/mL. At the end of incubation, an aliquot of the medium was removed for the determination of glycerol by a sensitive kinetic bioluminescence method [33].

Results Animal phenotype Congenic heterozygous (TSHrloxP/wtCre+/) and homozygous TSHr knockout mice (TSHrloxP/loxP Cre/+) appeared normal at birth, and analysis of live births showed a genotype distribution that did not differ significantly from Mendelian distribution. Analysis of growth by animal weight showed no differences among the genotypes (data not shown). Weaned at day 21 and maintained on normal diet, TSHr knockout and wild-type mice continued to thrive, and both male and female mice were fertile. Biochemical analysis and histology Serum total T4 did not differ between wild-type (TSHrwt/wt) and knockout (TSHrloxP/loxP Cre/+) mice: 67.55 ± 2.17 and 68.41 ± 2.58 nmol/L, respectively (mean ± SD, P < 0.8). A comprehensive pathologic survey of numerous tissues of TSHr knockout mice (TSHrloxP/loxP Cre/+) and wild-type (TSHrwt/wt) littermates using macroscopic and light microscopic examination of stained sections did not reveal any gross abnormalities, differences in cell type, or morphology.

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A

Somatic TSHr inactivation

Adipocytes size The mean size and size distributions of adipocytes were determined. Cell diameters (n = 1000) were measured in each experiment. A significant difference (P < 0.001) in the mean size of the adipocytes was observed between TSHr knockout (mean ± SEM of eight experiments 53.77 ± 0.18 lm) and wild-type mice adipocytes (49.10 ± 0.227 lm) (Fig. 2A). The distribution of adipocyte sizes was further investigated in both TSHr knockout and wild- type littermates. The distributions showed no significant deviation from the normal distribution in the histograms with a shift in the distribution toward larger cell size in the TSHr knockout mice (Fig. 2B). Lipolysis Basal lipolysis was higher in knockout adipocytes than in wildtype ones when expressed per cell (n = 8, 9.240  109 ± 8.13 

A

B

TSHr knockout

Normalized for expression

C

1.6

TSHr knockout

1.4

Wild-type

1.2 1.0 0.8 0.6 0.4 0.2 0.0

1

2

3

4

5

6

7

8

Fig. 1. Southern, Western blot analysis and QPCR gene expression profiles. (A) Southern blot analysis performed on 10 lg DNA prepared from isolated adipocytes from TSHr knockout (TSHrloxP/loxP Cre/+, lane) and wild (TSHrwt/wt, lane 2) littermates using HindIII restriction enzyme and the 30 external probe. (B) Western blot analysis performed on 40 lg protein prepared from adipose tissue of TSHr knockout (TSHrloxP/loxP Cre/+, lane 1 and 2) and wild (TSHrwt/wt, lane 3 and 4) littermates. (C) Histogram shows eight experiments performed in duplicate. The expression of TSHr mRNA in each knockout and wild-type mouse from different preparations of adipocytes was normalized to G3PDH and B-actin. The data represent the means ± SD. The mean mRNA copy number of TSHr was 75% lower in adipocytes of knockout than that of wild-type, P < 0.0001.

TSHr knockout

B

TSHr Wild-type

50

Wild type

40

% of distribution

Southern blot analysis of DNA from isolated adipocytes demonstrated that the somatic inactivation of the TSHr gene was effective (70–80%), although not complete (Fig. 1A). Western blot analysis of TSHr knockout mice (TSHrloxP/loxP Cre/+) protein did not express detectable amounts of the 115-kDa TSHR, compared to wild-type (Fig. 1B). TSHr transcript was less abundant (reduced by at least 75%) in white and brown adipocytes from TSHr knockout (TSHrloxP/loxP Cre/+) as compared to wild-type (TSHrwt/wt) littermates (Fig. 1C). TSHr expressions in TSHr knockout (TSHrloxP/loxP Cre/+) mice were similar in liver, muscle, heart, kidney, testis and brain compared to wild-type mice. Sequence analysis also confirmed the removal of exon 10.

TSHr knockout

30

20

10

0

0-20

21-40

41-60 61-80 Cell diameter (µm)

81-100

101-120

Fig. 2. Adipocytes sizes and adipocyte diameter distribution. (A) Representative images of isolated adipocytes sizes from TSHr knockout (TSHrloxP/loxP Cre/+) and wild (TSHrwt/wt) littermates. (B) Adipocyte diameter distribution profiles in wildtype and TSHr knockout mice. The size distributions were expressed as percentages of the total number of cells.

1010 vs. n = 8, 6.817  109 ± 6.64  1010, mean ± SEM, P = 0.028, Table 2). However, the basal lipolysis does not differ when expressed per cell surface area (n = 8, 1.0  1012 vs. n = 8, 1.0  1012 lmol/ lm2, mean P = 0.46, Table 1). The maximum TSH-induced lipolysis did not differ between knockout and wild-type adipocytes when expressed per cell or cell surface area (n = 8, 59.4 ± 7.89  109 vs. n = 8, 52.5 ± 5.94  109 lmol/cell, mean ± SEM, P = 0.49) and (n = 8, 5.0 ± 1.0  1012 vs. n = 8, 6.0 ± 1.0  1012 lmol/lm2, mean ± SEM, P = 0.46, Table 1). The TSH dose–response curve was shifted to the right in knockout adipocytes, and the sensitivity to TSH was reduced approximately 10fold (LogEC50 4.57 vs. 3.54, P < 0.0001, Table 2). The lipolytic response to increasing concentrations of isoprenaline in isolated adipocytes from knockout and wild-type littermates displayed a similar dose response pattern. No differences were found in either sensitivity (LogEC50 109 mol/L for both groups) or maximum response (n = 8, 66.6 ± 8.74  109 vs. n = 8, 54.61 ± 6.78  109 lmol/cell, mean ± SEM, P = 0, 29) and (n = 8, 6.0 ± 1.0  1012 vs. n = 8, 6.0 ± 1.0  1012 lmol/lm2, mean ± SEM, P = 0, 46, Tables 1 and 2). Following the addition of a fixed concentration of insulin (10 mU/L) to the incubation buffer containing either isoprenaline (108 mol/L) or TSH (106 mU/L), the mean dose–response of isoprenaline and TSH did not differ significantly between wild-type and knockout adipocytes. In addition, the inhibitory effects of insulin on isoprenaline or TSH-induced lipolysis were similar (Table 1). Discussion In the present study, we demonstrate that adipose-tissue-specific inactivation of TSHr in mice results in a reduced lipolytic effect of TSH and an increased adipocyte size. Our data indicate that the TSHr is of physiological importance for the growth, development,

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Table 1 Adipocytes from 8-week-old males (n = 8) were incubated in a buffer with either increasing concentrations of isoprenalin (mol/L) or TSH (mU/L). A fixed concentration of insulin (10 mU/L) was added to samples containing isoprenalin (108 mol/L) or TSH (106 mU/L). Glycerol release to the medium was determined (lmol/cell or the calculated cell surface area) as the index of lipolysis. The values are presented as the means ± SEM. TSHr knockout

TSHr wild-type

P value

Basal lipolysis Cell (lmol/cell) Cell surface area (lmol/lm2/2 h)

92.40 ± 8.13  1010 1.0  1012

68.17 ± 6.641010 1.0  1012

0.028 0.46

Maximum TSH-induced lipolysis Cell (lmol/cell) Cell surface area (lmol/lm2/2 h) TSH-induced lipolysis + insulin (lmol/cell)

59.4 ± 7.89  109 5.0 ± 1.0  1012 39.8 ± 5.79  109

52.5 ± 5.94  109 6.0 ± 1.0  1012 32.7 ± 4.8  109

0.49 0.50 0.35

Maximum isoprenalin-induced lipolysis Cell (lmol/cell) Cell surface area (lmol/lm2/2 h) Isoprenalin-induced lipolysis + insulin (lmol/cell)

66.68 ± 8.74  109 6.0 ± 1.0  1012 33.9 ± 5.09  109

54.61 ± 6.78  109 6.0 ± 1.0  1012 26.6 ± 3.4  109

0.29 0.84 0.20

Table 2 The mean dose–response with increasing concentrations of TSH (mu/L) and isoprenalin (mol/L)-induced lipolysis in TSHr knockout and wild-type mice adipocytes. Adipocytes were isolated from 8-week-old male TSHr knockout (n = 8) and wild-type mice (n = 8). Glycerol release into the medium was determined (lmol/ml) as the index of lipolysis.

TSH Log EC50 (95% CI) Isoprenalin (95% CI)

TSHr knockout

TSHr wild-type

P value

4.57 ± 0.08 (4.4–4.7) 109.9 (10.5 to 9.4)

3.54 ± 0.2 (3.1–3.9) 1010.0 (10.6 to 9.4)

<0.0001 n.s.

95% CI, confidence interval; n.s., not significant.

and metabolism of adipocytes. Furthermore, the decreased TSH-induced lipolysis in the TSHr knockout mice confirms previous in vitro studies showing that the lipolytic effect of TSH is mediated through activation of the TSHr [16]. Finally, the data indicate that the inactivation of the TSHr gene does not result in any compensatory up regulation of catecholamine-induced lipolysis and that the insulin-induced inhibition of lipolysis is the same in both wildtype and knockout mice. An increased basal lipolysis was observed in the TSHr knockout mice which might be regarded as a compensatory mechanism secondary to the decreased lipolytic effect of TSH. However, it is a well-established fact that larger adipocytes have a higher basal lipolysis when the amount of glycerol release is expressed per cell [10]. When comparing the metabolic events in adipocytes of different sizes it is therefore an advantage to express the results per cell surface area [10]. When the basal lipolysis was expressed in this way, no differences between wild-type and knockout mice were observed, indicating that the increased basal lipolysis is directly related to the increase in adipocyte size. Although we did not explore the mechanism responsible for the increased adipocyte size in the knockout mice, it is likely that removal of the TSHr influences the balance between lipolysis and lipogenesis and thereby enlarges the adipocytes. However, in adenosine A1 knockout mice, no effect on adipose tissue mass was observed despite the fact that adenosine is a very potent inhibitor of lipolysis [34]. Thus, it cannot be ruled out that other effects of TSH are also of importance for the increased adipocyte size. The recent observation that TSHr is important for lipogenesis early on during the differentiation of preadipocytes to adipocytes [35] gives further support for such a possibility. In addition, in a series of studies, Hausman et al. demonstrated increased adipocyte size in hypophysectomized pig fetus. Although the absence of both growth hormone and thyroxin affect adipocyte development [36–38], our present data indicate that the lack of TSH might also contribute. The removal of TSH receptors is only partial which is a common effect when the Cre-lox method is used for the tissue-specific removal of a gene [39,40]. The maximum lipolytic response to

TSH is similar in wild-type and knockout mice, but the sensitivity is ten times lower in the knockout mice. Since the animals are euthyroid with an intact thyroid–pituitary feed-back loop via thyroxin, the reduced sensitivity is sufficient to shift adipocytes outside the physiological range of TSH. There are no indications that mechanisms other than the removal of the TSHr contributed to increased adipocyte size. In addition to our previous reports regarding the importance of TSH in neonate [15], the present findings further support the view that adipocyte TSHr may be of physiological significance for adipocyte growth and development. Since adipocyte size and adipose tissue mass affect the metabolic activity of the adipose tissue [41–43], adipocyte TSHr might be of clinical importance for the development of obesity and obesity related comorbidity. In conclusion, we have demonstrated that the TSHr mediates physiological effects of TSH in adipocytes, but the clinical importance of this remains to be elucidated. Acknowledgments We thank Stephan Teglund at the Karolinska Center for Transgene Technologies (KCTT) for injection of the transgene construct. Rozell Björn, Katarina Carlsson, and Susanne Ohlin for expert assistance. Jan Kowalski for expert advice on statistical analyses. This study was financially supported by the Swedish Research Council (CM), Stockholm County Council, Karolinska institutet Foundations, Frimurare Foundation in Stockholm for Children Welfare, Sällskapet Barnavård, the Samariten Foundation, Wera Ekström, and the Jerring Foundation. Appendix A. Supplementary data Details on the construction of targeting vectors and the generation of adipocyte TSHr-knockout mice are provided in the supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.02.042. References [1] E.E. Kershaw, J.S. Flier, Adipose tissue as an endocrine organ, J. Clin. Endocrinol. Metab. 89 (2004) 2548–2556. [2] M.W. Rajala, P.E. Scherer, Minireview: the adipocyte – at the crossroads of energy homeostasis, inflammation, and atherosclerosis, Endocrinology 144 (2003) 3765–3773. [3] M. Qatanani, M.A. Lazar, Mechanisms of obesity-associated insulin resistance: many choices on the menu, Genes Dev. 21 (2007) 1443–1455. [4] G.Y. Carmen, S.M. Victor, Signalling mechanisms regulating lipolysis, Cell Signal. 18 (2006) 401–408. [5] P. Arner, D. Langin, The role of neutral lipases in human adipose tissue lipolysis, Curr. Opin. Lipidol. 18 (2007) 246–250.

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