In subclinical hypothyroidism (SH), high levels of circulating thyroid stimulating hormone (TSH) maintain normal thyroid hormone levels, despite mild thyroid failure. SH is associated with cardiovascular disease and insulin resistance, although the underlying pathophysiology is not fully understood. We hypothesized that TSH may inhibit insulin action in adipocytes. To investigate this relationship, we studied primary human differentiated adipocytes. Abdominal subcutaneous adipose tissue samples were obtained (approved by OHSN-REB) from 16 weight-stable patients undergoing elective abdominal surgery. We stimulated adipocytes differentiated from stromal preadipocytes with 5 mU/ml TSH and/or 100 nM insulin, and assessed acute insulin signaling, lipogenesis and glucose uptake. Immunoblot analysis revealed that TSH suppressed insulin-stimulated Akt phosphorylation by 45% (n=5; p = 0.01). When adipocytes were pre-incubated with conventional protein kinase C (cPKC) inhibitor Gö6976, TSH inhibition was blocked. Our data indicate that TSH inhibits insulin-stimulated lipogenesis (up to 37%), but depends on BMI. Insulin-stimulated glucose uptake was enhanced by 36% and also correlated with BMI. This data suggests that TSH can modulate adipocyte insulin signaling.

All cells and organisms consume energy for survival. A robust system has evolved in vertebrates to serve as an energy reservoir. In particular, specialized cells, adipocytes, are responsible for the dynamic storage of energy by accumulating and releasing fatty acids. Fluctuating energy demands require adipose tissue to adjust in size, however complications can arise in both extremes giving rise to systemic diseases, such as obesity and diabetes mellitus (T2D). In mammals, leptin production in adipocytes is up-regulated by feeding and insulin to provide long-term post-prandial satiety. Although this regulatory connection is central to all physiological effects of leptin, the molecular mechanism remains unknown for leptin production. Here, we show that the transcription factor Egr1 is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo in a mTORC1-dependent fashion. Induction of Egr1 was immediately followed by an increase in leptin transcription. Chromatin immunoprecipitation and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein Fat specific protein 27 (FSP27) may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knock out of Egr1 along with its over-expression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription. Knockout of the mTORC1-regulated translation repressor 4EBP1/2 increases leptin transcription both in vitro and in vivo. Adipose specific doxycycline-inducible constitutively active Rheb transgenic mouse lines contained higher circulating leptin and transcription of leptin following doxycycline treatment and were able to maintain elevated leptin levels following a 16 hour fast. Thus, insulin and nutrients, such as amino acids and glucose, activate leptin expression via the mTORC1-Egr1 regulatory axis.

Cells can release lipid bilayer vesicles of endosomal and plasma membrane origin, which are known as exosomes or extracellular vesicles (EVs). EVs contain diverse shuttling lipids, RNA and transmembrane proteins, and play an important role in communicating between neighboring or distant cells. Breast cancer is the most commonly diagnosed malignancy, with over 2 million new cases in 2018, and is the leading cause of cancer mortality in women all over the world. Some observational studies have suggested that breast cancer is more likely to develop among women who have type 2 diabetes; the association is clear in postmenopausal women. Moreover, women with type 2 diabetes diagnosed before, at the same time, or after breast cancer diagnosis, have decreased overall survival compared to women without diabetes. The most recent medical studies provide more clues as to why breast cancer is more common and has poorer prognosis in type 2 diabetes patients, by pointing out the role of insulin-resistant adipocytes in the etiopathology. Here, we demonstrate how insulin-resistant adipocytes engage crosstalk with breast cancer cells through EVs in the microenvironment and drive the tumor cells to be more metastatic and aggressive. These progression mechanisms and the effects of insulin-resistant adipocytes on breast cancer cells require Bromodomain and ExtraTerminal (BET) proteins – an important epigenetic pathway. Targeting this pathway may help reduce morbidity and mortality of women with breast cancer and type 2 diabetes.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13055号 / 論農博第2839号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5012(農学部図書室) / 33145 / (主査)教授 河田 照雄, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21159号 / 農博第2285号 / 新制||農||1060(附属図書館) / 学位論文||H30||N5133(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 阪井 康能, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21253号 / 医博第4371号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 浅野 雅秀, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Obesity, a disease characterized by excess adipose tissue (AT), is a growing worldwide epidemic. The Centers for Disease Control and Prevention (CDC), in 2017-2018, reported the prevalence of obesity in adults in the United States was 42.4% . Obesity increases the risk for many other serious health conditions such as type 2 diabetes, cardiovascular diseases, stroke, and some cancers. In individuals with obesity, the hypertrophic expansion of adipocytes, the main cell type within AT, is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Adipocytes are mechanoresponsive and affected by different microenvironmental cues, including hypoxia and mechanical (un)loading. Yet, no study has focused on the role of the aforementioned factors on the adipocyte mechanical response, including actin cytoskeletal remodeling. This dissertation aims to develop an in vitro model of healthy/diseased AT to explore the effect of microenvironmental cues on adipocyte function and actin cytoskeletal remodeling. The first aim is to study (1) the crosstalk between fibroblasts and adipocytes in a co-culture model and (2) the effect of hypoxia on the ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) mechanical pathway and actin cytoskeletal remodeling in adipocytes. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. The second aim explores the effects of mechanical unloading (simulated microgravity) on key adipocyte functions and actin cytoskeletal remodeling. This study demonstrated that mechanical unloading enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling, which together further enhanced glucose uptake. However, disrupting cortical actin remodeling by using inhibitors or exposure to a high concentration of free fatty acids (FFAs) diminished enhanced adipocyte functions observed in simulated microgravity. Overall, the results of these studies support the importance of microenvironmental cues on adipocyte actin cytoskeletal remodeling. Therefore, targeting mechanical pathways that regulate actin cytoskeletal remodeling can be used to improve adipocyte function and AT metabolism and possibly treat related diseases such as type 2 diabetes and obesity. / Bioengineering

Obesity is a disease that increases risk of developing metabolic diseases including insulin resistance (IR), metabolic syndrome (MS), and type 2 diabetes (T2D). Adipose tissue expansion during obesity leads to immune cell infiltration, causing local inflammation and disruption of lipid homeostasis. There is an association between exposure to environmental chemicals, like p,p’-DDE, a metabolite of p,p’-DDT, and diagnosis of obesity, dyslipidemia, IR, and prevalence of MS and T2D. DDE accumulates in fatty tissues and has been shown to have immunomodulatory properties, affecting macrophage and T cell populations. Potential mechanisms were studied by which DDE could modulate adipocyte and immune cell function and facilitate an increased risk of obesity and immune dysregulation, potentially through cyclooxygenase-2 (COX-2). 3T3-L1 preadipocytes and J774A.1 macrophages were studied for the effects of DDE on adipogenesis and macrophage reactivity, respectively. 3T3-L1 cells were induced to differentiate to adipocytes using a sub-optimal differentiation cocktail with increasing concentrations of DDE (0.5uM-100uM). It was determined that DDE enhanced adipogenesis in a concentration dependent manner and the expression of adipogenic and lipogenic genes, indicating that DDE enhances adipogenesis. In J774A.1 cells, the ability of DDE or 10uM NS-398, a specific COX-2 inhibitor, to inhibit the production of the prostaglandins PGE2, PGD2, PGF2a, was assessed in vitro and in a cellree system. DDE or NS-398 followed by immune challenge reduced cellular PG secretion and reduced PG production in a cell free system, indicating that DDE may interfere with lipid mediator signaling. Additionally, DDE or NS-398 exposure altered gene expression in J774A.1 cells following M1 or M2 polarization stimulus. Lastly, male C57Bl mice were exposed to 2mg/kg DDE for 5 days and the macrophage population of the adipose stromal vascular fraction was analyzed by flow cytometry. Adipose from DDE treated animals contained approximately 40% F4/80+CD11b+ macrophages. These results indicate that DDE may alter the homeostasis of adipose tissue by both enhancing adipogenesis and altering the reactivity of the resident macrophage population in a manner that may contribute to adipose dysfunction. These data suggest a possible mechanism by which DDE exposure may contribute to adiposity and adipose tissue dysfunction commonly seen in metabolic disease.

Subcutaneous (sc) and visceral (vis) adipose tissue (AT) contribute to the variability in pathophysiological consequences of obesity and adverse fat distribution. To gain insights into the molecular mechanisms distinguishing vis and sc fat, we compared the transcriptome during differentiation of immortalized adipocytes from murine epididymal (epi) and inguinal (ing) AT. RNA was extracted on different days of adipogenesis (−2, 0, 2, 4, 6, 8) and analyzed using ClariomTM D mouse assays (Affymetrix) covering >214,900 transcripts in >66,100 genes. Transcript Time Course Analysis revealed 137 differentially expressed genes. The top genes with most divergent expression dynamics included developmental genes like Alx1, Lhx8, Irx1/2, Hoxc10, Hoxa5/10, and Tbx5/15. According to pathway analysis the majority of the genes were enriched in pathways related to AT development. Finally, in paired samples of human vis and sc AT (N = 63), several of these genes exhibited depot-specific variability in expression which correlated closely with body mass index and/or waist-to-hip ratio. In conclusion, intrinsically programmed differences in gene expression patterns during adipogenesis suggest that fat depot specific regulation of adipogenesis contributes to individual risk of obesity.

There is concern over the impact that petroleum related compounds (PRCs) associated with mining activity in the Athabasca Oil Sands Region (AOSR) are having on local wildlife. With the increase in oil sands mining activity in the AOSR there has been a corresponding decline in the fertility of indicator species in the AOSR. One of the primary sources of PRCs in the environment is oil sands process affected water (OSPW), which is stored in tailings ponds. Several PRCs, including naphthenic acid fraction components (NAFC), have endocrine disrupting effects, which may, in part, explain reduced fertility in indicator species. For example, male North American river otters (Lontra canadensis) living in areas impacted by mining activity have lower baculum strength those unaffected by mining activity. Weaker baculums are associated with increases in fracture rates and reduced fertility. Baculum strength is maintained throughout life by bone remodeling, a process that requires the differentiation of osteoblasts. NAFCs can impact several pathways integral to the development and path selection of mesenchymal stem cells into osteoblasts or adipocytes. Therefore, the objective of this thesis was to test the hypothesis that NAFCs inhibit osteoblast differentiation and induce adipocyte differentiation from progenitor cells. We exposed osteoblast progenitor cells and adipocyte progenitor cells to NAFCs. We demonstrated that NAFCs inhibit osteoblast differentiation and activate the glucocorticoid receptor pathway. We also found that NAFCs do not induce adipogenesis in adipocyte precursor cells. Lastly, we showed that NAFCs are PPARγ ligands that inhibit the expression of PPARγ associated genes. These insights into the effects of NAFCs on osteoblast and adipocyte progenitor cells suggest NAFCs may contribute to lower baculum strength and impaired adipose tissue function of animals living in the AOSR. These effects my reduce the fertility and population of wildlife in the AOSR. / Thesis / Master of Science (MSc) / There is concern that chemicals from oil sands mining in the Athabasca oil sands region are hurting the reproductive health of animals in the wild. Some of these animals, including bears, wolves, and river otters, need a bone in their penis called a baculum to reproduce. Studies have shown that some chemicals, including those from mining activity can make the baculum bone weaker. For bone to stay strong, bone cells always need to be developing to fix the bone tissue. The goal of our study was to find how chemicals from mining activity can affect the development of bone cells. We found that a group of chemicals that come from oil sands mining called naphthenic acid fraction components (NAFCs) stop bone cells from developing and making new bone. We also know that having more fat cells in bone is associated with weaker bones. We also looked at whether NAFCs could increase the development of fat cells. However, NAFCs did not increase the development of fat cells. Together, this research shows that NAFCs can make bones like the baculum weaker by slowing the development of new bone, but not by increasing fat cells. Our research suggests that exposure to NAFCs may make baculums weaker which may be bad for the reproductive health of animals living near oil sands mining activity.