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In this study, we generated genetically modified mice and used them to determine the role of TSHr in adipocyte growth, development, and metabolism. We chose to generate tissue-specific knockout mice because such a mouse model would be useful for studying the TSHr cell-specific function since the TSHr is widely expressed in the body. TSHr germline targeting would lead to a complex phenotype reflecting the effects of both hypothyroidism and the TSHr in different tissues, and we therefore could not deduce the specific role of TSHr in adipose tissue in such strains. In addition, for further future uses and studies, our mouse strain can be either converted into a null allele mutant (generalized knockout) by a single cross to a deleter strain expressing Cre recombinase in germ cells, or crossed with transgenes exhibiting tissue-specific promoter activity that can be regulated temporally by adding or removing exogenous agents. This has been demonstrated to be a reliable technique in vivo.

Our results confirmed the deletion of the TSHr gene in an estimated at least 70% of pooled adipocytes from four different knockout mice of the same age (Southern blot, RT-PCR, PCR, and sequencing); these adipocytes were not used for any further experiments. Thereafter, the expression of Cre recombinase and the homozygous floxed exon 10 of TSHr gene were identified by PCR in genomic DNA obtained from

mice tail biopsies. The percentage of TSHr removal was assessed by quantitative TSHr mRNA measurements (RT-PCR). In the eight subsequent lipolysis and adipocytes size measurement experiments, TSHr mRNA was at least 70% less abundant in adipocytes isolated from TSHr knockout strains (RT-PCR).

Mouse strain expressing Cre recombinase only in adipose tissue was used to delete TSHr from adipocytes. The incomplete removal of TSHr from adipocytes and the high expression of Cre recombinase only in adipose tissue indirectly indicate that no major removal of TSHr is possible in tissues other than adipose tissue. Thus, it is unlikely that these mice strains could have suffered from any other endocrine or metabolic disturbances. Moreover, offspring were weaned by day 21 and, on two occasions, at 17 days. No hormone replacement therapy diet or thyroid powder supplements were given. All the animals were viable and fertile, indicating a normal phenotype.

Most of the previous studies use either Cre or the FRT system for the removal of selective markers in ES cells. Cre recombinase is routinely used in ES cells and transgenic mice with high recombination efficiency, whereas the reported use of the FLP/FRT system in ES cells and transgenic mice has not been as efficient [256-258].

We chose to use in vivo manipulation of FRT- flanked selection neomycin gene. The FLP/FRT system was very efficient, and the selective marker neomycin gene was detected neither by Southern blot nor by PCR and sequencing, indicating a complete removal of the neomycin resistance gene. On the other hand, Cre recombinase failed to remove TSHr gene completely. This was not surprising to us; previous animal studies show various efficiencies of in vivo deletion by Cre recombinase [259]. Most likely, several possible explanations contributed to this result.

One explanation is different expression levels of Cre recombinase in individual transgenic lines have been reported as a result of differences in transgene copy number and genomic integration site related to chromatin structure [260-262], transcription rate, DNA methylation [263, 264], or transcript degradation [265].

A second explanation is the effect of the distance between the two loxP sites. Cre recombination efficiency is inversely proportional to the genetic distance between the loxP sites [259, 266, 267]. However, this possibility seems to be unlikely, as the flanked exon 10 of TSHr gene is 1.6 kb, which lies within the range of high Cre recombination efficiency, as previously reported [259, 267].

A third explanation is the intra- and/or extra-uterine environmental factors. Previous studies have demonstrated that Cre recombinase expression operates optimally at 37C [258], and since the Cre strain we used expresses Cre recombinase both in white and brown adipose tissue, removal of TSHr from brown adipose tissue is likely to alter mouse thermogenesis regulation [268] and thereby have an affects on the efficiency of the Cre system. In this respect, additional studies will be required to evaluate whether body temperature has an effect on the efficiency of the Cre system in our model or not.

We demonstrated that adipose-tissue-specific inactivation of the TSHr in mice resulted in a reduced lipolytic effect of TSH and an increased adipocyte size. Our data thus indicate that the TSHr is of physiological importance for the growth and metabolism of adipocytes. Furthermore, the decreased TSH-induced lipolysis in the TSHr knockout mice confirms previous in-vitro studies indicating that the lipolytic effect of TSH is mediated through activation of the TSHr [59].

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 [200]. To compare the metabolic events in adipocytes of different sizes, we used the advantage of expressing the results per cell surface area [200]. 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, but this also further supports our observation of larger adipocytes in TSHr knockout mice.

Although we did not explore the mechanism responsible for the increased adipocyte size in the knockout mice, it is likely that the removal of the TSHr influences the balance between lipolysis and lipogenesis, thereby causing enlargement of 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 [269].

Thus, it cannot be excluded that other effects of TSH also are of importance for the increased adipocytes size. The recent observation that TSHr is of importance for lipogenesis early on in the differentiation of preadipocytes to adipocytes [194] 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 affects adipocyte development [270-272], our present data indicate that the lack of TSH might also contribute.

The sensitivity of adipocytes to TSH is ten times lower in the knockout mice. The reduced sensitivity is sufficient to shift adipocytes from the physiological range of TSH. There are no indications that other mechanisms have caused the reduced TSH sensitivity in the knockout mice. Furthermore, there is no reason to believe that other factors than the removal of the TSHr are causing the increased adipocyte size and increased basal lipolysis in the knockout mice.

It has been shown previously that functional TSHr are present in many tissues including adipocytes in man [55-57]. Therefore, the confirmation that TSHr is active during physiological conditions may be of clinical significance. For example, shortly after birth and before lactation is established, the infant utilizes the stored carbohydrate energy reserves, which develop during gestation, rapidly (12 hr) [273], lipid mobilization via lipolysis in adipocytes become the major energy substrates [274-276].

After delivery newborn serum TSH is clearly elevated for several hours and serum thyroid hormone levels reach a maximum at 24 h [277-281]. Although few data are

available about the regulation of lipolysis during the neonatal period, a few in vitro studies have shown that TSH is the main lipolytic hormone during this critical period of life [282]. Moreover, recent studies suggest an intricate relationship between size, cholesterol concentration, and insulin sensitivity of the adipocyte. Cholesterol accumulates within the adipocyte lipid droplet proportionally to the triglyceride content.

However, hypertrophic adipocytes have reduced membrane cholesterol concentrations compared with smaller adipocytes, and this may explain the insulin resistance in hypertrophic adipocytes such as insulin resistance [283-287].

6 ACKNOWLEDGMENTS

Near the end of this PhD thesis, I suddenly realized that I hade been experiencing a fabulous journey in studying molecular biology and endocrinology. Although the path through the years has been full of tears and struggles, I have gained a lot. I will never regret studying for a PhD, although it was indeed a great challenge to me. I truly believe that such an achievement cannot be brought about by a single person, and everybody mentioned here deserves to share my delight; for this, I have many people to show my deep appreciation.

First, I wish to thank my supervisor, Svante Norgren. Svante has been my main guide starting from the first day to the fight against the clock in finalizing the thesis. Dear Svante, It has been a privilege to work with you; with your sharp mind and clear thoughts, you have provided excellent guidance in molecular genetics. Thank you for being a creative, hardworking scientist, for your detailed analysis of the manuscripts to catch as many mistakes as possible. However, ultimately, the content of each manuscript always improved after your revision. Thanks for your dedicated support both in good and rough times ever since the day I began working on the TSHr. Thank you for your friendship.

Special thanks go to my co-supervisor, Claude Marcus, who taught me how to think in another way, had confidence in me when I doubted myself, and brought out the good ideas in me. Thank you for helping me come up with the thesis topic and guiding me over years of development, and during the most difficult times when writing this thesis, you gave me the moral support and the freedom I needed to move on. Without your encouragement and constant guidance, I could not have finished this dissertation.

Thank you for your friendship.

My special acknowledgment and thanks to our secretary and coordinator Märta Fredriksson, for her hard work and attention to details; her organizational skills are truly some of the best I have ever witnessed. You seemed to never get flustered. Thanks Marta!

Many thanks to Li Tsi, Olle Söder, and Agne Larsson, who provided their supervision, advice, help and expertise without which I would have been lost several times.

Thanks to all my co-authors for sharing the same projects, and for pleasant cooperation:

Dr. Annika Janson, for providing me with all the information about TSHr and for genuine appreciation of different situations; Professor Ulla Berg, for keeping the data bank with all the details and for working on the manuscript until late at night (“It has to be perfect”, is the key for good work); CG Arvidsson; Petra Verbovszki; Jan Zedinius;

Catharina Larsson; Tony Frisk; Anders Höög; and Göran Wallin.

I would like to thank Helen Zemack, Susanne Öhlin and Katarina Carlsson for friendship, encouragement, and helping me at all times and solving any unsolvable problems, and giving me a fine introduction when I joined the lab team. My deepest

knowledge of the technique that I used the most comes from you. Thanks so much Helen for running the RT-PCR, hormonal assay and for always ready to help in difficult time.

Far too many people to mention individually have assisted in so many ways during my work. We’ve all been there for one another and have taught each other and ourselves many tools and issues. I know that I could always ask them for advice and opinions on many issues. They all have my sincere gratitude. In particular, I would like to thank, Barbro Malmgren, Pernilla Danielsson, Jenny Alken, Kerstin Ekbom, Giesla Nyberg, Örjan Ekbolm, Viktoria Svensson, Erika Forsell, Anna Mattsson, Anja Nordenfelt, Ming Chen, Tilda Hultkvist, Micheaela Forssen, Andreas Winkler, and Runa Njalsson.

I thank, Li Villard, Marie Lindefeldt, Gunilla Molinder, Gustav Olsson, Stina Johansson, Mirjam Ekstedt, Maria Westerstahl, Eva Flygare Wallen, Pernilla Hedvall, and Joanna Tingstrom. I haven’t gotten a chance to get to really know you since I’ve been in a hole this past year but you have all been so friendly and personable to me. I also thank, Jan Kowalski for statistical expertise, Eva Sjölin, Kerstin Wåhlén, Stephan Teglund, Björn Rozell, and Agneta Witlock for always being so helpful and friendly.

I thank my family, my parents, my brothers and sisters who always kept me away from family responsibilities and encouraged me to concentrate on my studies, for believing in me, for unconditional support and encouragement to pursue my interests, even when these interests went beyond the boundaries of language, field and geography, for reminding me that my work and research should always be useful and serve good purposes for all humankind.

Last, but not least, to my wife, Rabab, and our children, Ahmed, Zeinab, Ibtihal, and Obada, all I can say is it would take another thesis to express my deep love for you.

Your patience, love and encouragement have upheld me, particularly during those many days on which I spent more time with the computer, articles and books than with you. No more PhD - I promise! now it’s your turn.

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