There are two distinct types of adipose tissue which have different functions within the body, white (WAT) and brown (BAT). Browning of WAT occurs with increases in the WAT sympathetic nervous system (SNS) drive. In this regard we previously reported that melatonin (MEL) stimulation of MEL receptor 1A (MEL1a) within the SNS outflow to the WAT might be implicated in a naturally-occurring reversal of obesity (by ~30% of total body fat). Therefore, in this study we tested the hypothesis that MEL causes browning of WAT through the stimulation of SNS drive to WAT. This was done by comparing specific browning and lipolytic markers in WAT following 10 weeks of MEL treatment, short day housing (SD), and long day housing with saline injections (LD+VEH). Browning effects of a 5 day treatment of a β3-adrenergeric (β3 AR), CL 316, 243, were also measured. We found that CL 316, 243, MEL treatment, and SD housing had increased expressions of browning markers within WAT and lipolytic activity in MEL treated animals was increased in specific WAT.

Uncoupling protein, UCP, present in the inner mitochondrial membrane of brown adipose tissue (BAT) contributes to adaptive thermogenesis. UCP functions as a proton pore and can dissipate the proton electrochemical gradient established by the respiratory chain during fuel oxidation, and thus generates heat without producing ATP. However, the brown adipose tissue thermogenesis is not likely to be a major mechanism in controlling energy expenditure for humans because adults have only residual amounts of the tissue. Two new members of the UCP family have been identified based on their high sequence homology to UCP in BAT and named UCP2 and UCP3. The original UCP was renamed UCP1. At the amino acid level, human UCP2 and UCP3 are 59% and 57% identical to UCP1, respectively. In contrast to UCP1, UCP2 is expressed in many tissues such as brown adipose tissue, white adipose tissue, muscle, spleen and macrophages. UCP3 is expressed preferentially in skeletal muscle in humans, and brown adipose tissue and skeletal muscle in rodents. Since their identification many functional studies, including transgenic animals and ectopic expression of UCP2 or UCP3 in yeast, showed uncoupling activity of UCP2 and UCP3. A number of studies have been done that show increased expression of UCP2 and UCP3 by fasting, high-fat diets and suckling of newborn mice. A common characteristic of these circumstances is an associated increase in plasma free fatty acid levels. This study aimed to investigate effects of fatty acids, peroxisome proliferator-activated receptors (PPARs) and other transcription factors on UCP2 and UCP3 gene expression and to explore the molecular mechanism of their regulation through analysis of the promoter of the UCP2 and UCP3 genes. The 3.1 kb and 3.2 kb 5�-flanking regions of the mouse UCP2 and UCP3 genes, respectively, were cloned and used to construct promoter reporter gene (firefly luciferase) plasmids. The cloned region of the UCP2 and UCP3 genes contained putative binding motifs for several transcription factors, including PPAR, myogenin, and MyoD. Luciferase assays of both constructs showed basal promoter activity with 20~190-fold induction for the UCP2 promoter and 1.3~23-fold induction for the UCP3 promoter in several transfected cell lines, including 3T3-L1, C2C12, L6, COS7 and HepG2. Oleic acid (0.3 mM) up-regulated endogenous UCP2 mRNA by 2.3-fold in 3T3-L1 preadipocytes but not in C2C12 myotubes, and UCP3 mRNA by 2.5-fold in C2C12 myotubes. Responsiveness of the cloned promoter to oleic acid reflected the tissue-specific responsiveness of their endogenous genes but with less fold induction, 1.4-fold for UCP2 promoter in 3T3-L1 preadipocytes and 1.5-fold for UCP3 promoter in C2C12 myotubes. Forced expression of PPAR isotypes (PPARα, PPAR[delta] and PPARγ) showed tissue and isotype-specific activation of the UCP2 promoter. UCP2 promoter activity was induced by 2-fold by PPARγ in 3T3-L1 and by 2.8-fold by PPAR[delta] in C2C12. Treatment of oleic acid (0.3 mM) brought about further induction of the UCP2 promoter activity only in 3T3-L1. In contrast, all three isotypes induced activation of the UCP3 promoter in 3T3-L1, C2C12 and HepG2 cells. Treatment with oleic acid (0.3 mM) or isotype-specific agonist (10 [mu]M) resulted in further increased activity of the UCP3 promoter in 3T3-L1 and HepG2 cells. In particular, rosiglitazone (10 [mu]M) induced a 41-fold increase in UCP3 promoter activity in PPARγ transfected HepG2 cells, and this induction returned to basal level by treatment with bisphenol A diglycidyl ether (BADGE) (50 [mu]M), an antagonist for PPARγ. In addition, UCP3 promoter activity increased up to 20-fold 4 days after induction of C2C12 myoblasts differentiation, whereas UCP2 promoter activity increased only up to 2-fold. Forced expression of myogenin and MyoD in C2C12 myoblasts to mimic differentiation, induced UCP3 promoter activity in an additive manner, consistent with UCP3 being regulated by muscle differentiation. In the present study, it has been shown that UCP2 and UCP3 genes are regulated differently by fatty acids. The tissue-type dependence in regulation of endogenous UCP2 and UCP3 paralleled the cell type-specific effect of oleic acid on the promoter-reporter constructs, suggesting that fatty acid effects are at the transcriptional level. UCP2 and UCP3 promoters showed differences in their response to PPARs. Mediation of the fatty acid effect through PPARs has been also demonstrated, but direct binding of PPARs and particular regulatory motifs on the cloned promoter region have not yet been investigated.

The obesity epidemic is associated with an increase in the prevalence of a number of chronic diseases including type 2 diabetes, cardiovascular disease, hypertension and some cancers and has been described by the World Health Organisation as one of the greatest public health challenges of the 21st century. Obesity is characterised by excessive expansion of adipose tissue mass underpinned by adipocyte hyperplasia. Central to this is the process of adipogenesis, which encompasses the proliferation and terminal differentiation of fibroblastic preadipocytes, contained within adipose tissue, to mature adipocytes. Despite the pivotal role of this process in obesity our understanding of the regulatory mechanisms governing adipocyte development, either in physiological or pathophysiological settings, is limited. Studies aimed at understanding this complex process are integral to development of more effective strategies for the prevention and/or treatment of obesity and obesity related diseases. Our laboratory recently identified a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in the dynamic regulation of lipid accumulation. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocates to lipid droplets and inhibition of this translocation is correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis, it was hypothesised that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis. Therefore the aim of this project was to extend these investigations to examine the requirement for IMPDH activity during adipogenesis, using the well characterised murine 3T3-L1 cell line and primary human preadipocytes (phPAs). IMPDH expression and activity were transiently increased during differentiation of the 3T3-L1 cells although IMPDH did not associate with lipid droplets under these conditions. Pharmacological inhibition of IMPDH, using mycophenolic acid (MPA; 1 µM), reduced intracellular GTP by 60%, and blocked mitotic clonal expansion (MCE) and adipogenesis. Supplementation with guanosine (60 µM), a substrate in the nucleotide salvage pathway, restored both GTP levels and adipogenesis. These observations indicated that IMPDH activity is required for efficient differentiation of 3T3-L1 preadipocytes. Preliminary studies, involving differentiation of phPAs in standard serum-free medium (SFM) suggested that phPAs were resistant to MPA. To afford better comparison between the phPAs and the 3T3-L1 cells, which are differentiated in serum-containing medium (SCM), a modified 3T3-L1 like protocol facilitating efficient differentiation of the phPAs in SCM was established. Under these conditions phPAs displayed considerable variation in sensitivity to MPA which gave a trend towards decreased differentiation (reduced by 26%; p=0.07). Supplementation with guanosine significantly reduced adipogenesis (by 37%; p<0.05) in the phPAs independent of MPA. Furthermore, cells that were MPA resistant were also refractory to guanosine suggesting greater plasticity of guanine metabolism in phPAs from those subjects. A major difference between the cell types was that phPAs differentiated with high efficiency in the absence of MCE. Collectively, these data indicate that MCE is required for efficient differentiation of 3T3-L1 cells but not phPAs, even when differentiated under similar conditions, and suggest that the involvement of MCE underpins the differences in sensitivity to MPA between cell types. The differential effects of guanosine suggest there are additional differences with respect to the effects of manipulation of guanine nucleotides between cell types. In summary, the work presented in this thesis demonstrated inhibition of IMPDH blocked adipogenesis of murine 3T3-L1 cells and reduced differentiation of phPAs in some subjects. These observations provided novel insights into differences between differentiation of 3T3-L1 cells and phPAs, including their relative sensitivities to alterations in guanine nucleotides, and have implications for adipose tissue biology especially those factors involved in guanine metabolism. Ultimately this knowledge may form the basis for development of novel therapeutic strategies aimed at reduction of obesity and associated complications such as insulin resistance and type 2 diabetes.

Diss. (sammanfattning) Göteborg : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (M.Sc. (Nutrition))--Mahidol University, 1984.