Social support has been demonstrated to reduce cardiovascular disease morbidity and mortality; however, the mechanisms by which social support reduces disease progression are still unclear. Oxytocin (OT) is a neuropeptide commonly associated with positive social interactions. This series of studies investigated the ability of oxytocin to attenuate atherosclerosis and its putative mediators, pro-inflammatory cytokines. Oxytocin receptors were identified by Western Blot on rat adipose tissue and rat adipocytes. OT receptor mRNA was identified in human adipocyte cDNA. In primary culture of rat abdominal adipocytes, oxytocin (10 pM) decreased LPS-induced IL-6 release by 24.9% after a six hour incubation. Adipose tissue, surgically dissected from ApoE-/- mice chronically infused with OT, secreted less IL-6 than mice infused with a vehicle control. In sum, the presence of OT receptors was demonstrated on adipocytes, OT was shown to reduce IL-6 release in vitro, and chronic OT infusion decreased IL-6 release in adipose explants immediately after sacrifice. Potential mechanisms by which adipose tissue's role in the sympathetic nervous system response may affect inflammation, metabolism, and disease are discussed.

We hypothesized that carcass and fatty acid composition of Angus and Japanese Black (Wagyu) steers would not differ if the steers were fed to a typical U.S. final weight, but that Wagyu steers fed to a typical Japanese endpoint body weight would have greater quality grades and softer fat than Angus steers. Sixteen Angus and 16 Wagyu 8-month-old, weaned steers were assigned to a corn-based diet for 8 or 16 months (n = 4 per breed type and time) or hay-based diet for 12 or 20 months (n = 4 per breed type and time) in a 2 x 2 x 2 factorial arrangement. USDA yield grade was greater at the Japanese endpoint than at the U.S. endpoint in Angus steers (breed x endpoint, P = 0.03). Intramuscular (i.m.) lipid continued to increase to over 20% in the Wagyu steers (P = 0.05), but attained a plateau (14.7%) by 16 months on feed in the Angus steers. These results confirm that Wagyu cattle must be raised to greater physiological maturity before they differ from Angus cattle in M. longissimus thoracis i.m. lipid concentration. Subcutaneous adipose tissue concentrations of oleic (18:1n-9) was greater in Wagyu steers than in Angus steers (P = 0.05). All monounsaturated fatty acids (MUFA) increased between the U.S. and Japanese endpoint, whereas slip points of lipids in s.c. adipose tissue were 10°C lower in Japanese endpoint steers than in U.S. endpoint steers (P = 0.01). Angus adipose tissue exhibited peak SCD enzyme activity at 16 months (corn-based diet) but activity in Wagyu adipose tissue was greatest at 20 months (hay-based diet) (breed x diet x endpoint, P = 0.08). However, SCD gene expression in Angus adipose tissue was maximal at 12 months (hay diet), whereas Wagyu adipose tissue had peak expression at 16 months (corn diet) (P < 0.03). Trans-10, cis-12 CLA has been reported as a potent inhibitor of adipocyte differentiation. CLA (40 µM) strongly decreased SCD and PPARγ expression in bovine adipocytes, even in the presence of 5 mM arginine. It can be concluded that arginine up-regulates bovine preadipocyte differentiation, and CLA antagonizes this effect.

We hypothesized that carcass and fatty acid composition of Angus and Japanese Black (Wagyu) steers would not differ if the steers were fed to a typical U.S. final weight, but that Wagyu steers fed to a typical Japanese endpoint body weight would have greater quality grades and softer fat than Angus steers. Sixteen Angus and 16 Wagyu 8-month-old, weaned steers were assigned to a corn-based diet for 8 or 16 months (n = 4 per breed type and time) or hay-based diet for 12 or 20 months (n = 4 per breed type and time) in a 2 x 2 x 2 factorial arrangement. USDA yield grade was greater at the Japanese endpoint than at the U.S. endpoint in Angus steers (breed x endpoint, P = 0.03). Intramuscular (i.m.) lipid continued to increase to over 20% in the Wagyu steers (P = 0.05), but attained a plateau (14.7%) by 16 months on feed in the Angus steers. These results confirm that Wagyu cattle must be raised to greater physiological maturity before they differ from Angus cattle in M. longissimus thoracis i.m. lipid concentration. Subcutaneous adipose tissue concentrations of oleic (18:1n-9) was greater in Wagyu steers than in Angus steers (P = 0.05). All monounsaturated fatty acids (MUFA) increased between the U.S. and Japanese endpoint, whereas slip points of lipids in s.c. adipose tissue were 10°C lower in Japanese endpoint steers than in U.S. endpoint steers (P = 0.01). Angus adipose tissue exhibited peak SCD enzyme activity at 16 months (corn-based diet) but activity in Wagyu adipose tissue was greatest at 20 months (hay-based diet) (breed x diet x endpoint, P = 0.08). However, SCD gene expression in Angus adipose tissue was maximal at 12 months (hay diet), whereas Wagyu adipose tissue had peak expression at 16 months (corn diet) (P < 0.03). Trans-10, cis-12 CLA has been reported as a potent inhibitor of adipocyte differentiation. CLA (40 µM) strongly decreased SCD and PPAR&#947; expression in bovine adipocytes, even in the presence of 5 mM arginine. It can be concluded that arginine up-regulates bovine preadipocyte differentiation, and CLA antagonizes this effect.

Obesity is associated with numerous co-morbidities, such as chronic low-grade inflammation, oxidative stress, insulin resistance, cardiovascular disease, and some cancers. However, the role of estrogen in the susceptibility to obesity and its co-morbidities is not clear. To determine the role of estrogen in the above morbidities, we used C57BL/6J mice (15/group): 1)males 2)nonovariectomized females (novx) 3)ovariectomized females (ovx) and 4) ovariectomized females supplemented with estrogen (ovx-E), which were randomized to receive a 30% calorie-restricted, low-fat or high-fat diet. Our results showed that male and ovx-female mice were more susceptible to obesity compared to novx-female and ovx-female+E mice. Specifically, we observed that estrogen protected novx-female and ovx-female+E mice from adiposity and glucose intolerance by decreasing adipocyte size and key adipogenic and lipogenic mRNA expression levels. Further experimentation established that estrogen decreased abdominal adiposity by decreasing the number of large adipocytes. Our findings implied that estrogen stimulated lipolysis in novx-female and ovx-female+E mice. Additionally, the enlarged adipocytes observed in the male and ovx-female mice were accompanied with crown-like structures surrounding necrotic adipocytes and F480+ macrophages and elevated mRNA expression levels of CD68, IL6, and TNF[alpha]. Lastly, male and ovx-female mice exhibited liver steatosis, elevated serum ALT levels, and increased insulin resistance. To determine if there were sex differences in oxidative stress, we showed that estrogen protected the novx-female and ovx-female+E mice from adipose tissue oxidative stress as evidenced by fewer γH2AX stained nuclei and lower iNOS, P47x, GP90x, but higher catalase mRNA levels. In order to further understand the role of estrogen in adipocyte inflammation, we differentiated 3t3L1 pre-adipocytes in charcoal-stripped FBS +/- 1nM estrogen. Our findings mimicked our in vivo results; the presence of estrogen significantly decreased adipogenesis and down regulated IL6, TNF[alpha], and GP90x in 3t3L1 adipocytes. Additionally, using 4-hydroxytamoxifen, we demonstrated that the protective effects of estrogen on IL6 and TNF[alpha] mRNA expression were blocked; suggesting that estrogen could mediate its anti-inflammatory effects through ERα. In conclusion, this dissertation demonstrates that estrogen protects female mice from obesity-associated inflammation, oxidative stress, and insulin resistance by altering adipocyte morphology possibly through ER[alpha]. / text

L’os est un tissu dynamique, en remodelage constant grâce à un processus finement régulé impliquant des facteurs locaux et systémiques. Un déséquilibre entre la formation et la résorption osseuses, assurées respectivement par les ostéoblastes et les ostéoclastes, conduit à une altération de la microarchitecture de l’os, une augmentation du risque de fracture, et à l’apparition de pathologies telles que l’ostéonécrose et l’ostéoporose. Ces deux maladies ont pour caractéristiques communes une diminution du nombre de cellules stromales mésenchymateuses (MSC), les progéniteurs des ostéoblastes, et des anomalies fonctionnelles des MSC et des cellules ostéoblastiques (Ob). De surcroit, elles présentent toutes deux une accumulation d’adipocytes au sein de la moelle osseuse. De récentes publications suggèrent que cette accumulation d’adipocytes médullaires pourrait avoir des conséquences délétères sur la physiologie des cellules ostéoformatrices et de leurs progéniteurs, partageant ce même microenvironnement osseux. Une relation inverse entre l’excès d’adiposité médullaire et la masse osseuse est en effet aujourd’hui clairement établie. Par son importante activité sécrétrice de cytokines et d’adipokines ainsi que sa capacité à stocker et libérer des acides gras libres (AGL), l’adipocyte médullaire est capable de modifier la composition du microenvironnement osseux, et ainsi d’influencer le métabolisme et la fonction des cellules avoisinantes, et notamment des cellules osseuses. Afin d’étudier l’influence des AGL sur la survie et la fonction des cellules ostéogéniques, nous avons utilisé un AGL saturé, le palmitate (Palm ;C16 :0) et un AGL monoinsaturé, l’oléate (Ole ;C18 :1), tous deux étant particulièrement abondants dans l’organisme et l’alimentation de l’homme et couramment utilisés dans les études de lipotoxicité. Dans un premier temps, nous avons travaillé sur des MSC isolées de la moelle osseuse de sujets sains (HV-MSC), qui ont éventuellement été différenciées en Ob. Nous avons démontré que l’exposition de ces cellules à des concentrations physiologiques de Palm entraîne une cytotoxicité dose-et temps-dépendante, via l’initiation d’un stress du réticulum endoplasmique (RE) et l’activation des voies ERK et NFκB. En outre, l’AGL saturé induit un état pro-inflammatoire en augmentant l’expression du toll-like receptor 4 et la production des interleukines (IL) 6 et 8. Nous avons montré que les Ob présentent une sensibilité accrue au Palm, associée à une exacerbation du stress du RE et de la réponse pro-inflammatoire. L’Ole n’a pas d’effet délétère sur les MSC et les Ob, et de surcroit, il bloque les différentes voies activées par le Palm, neutralisant ainsi totalement la lipotoxicité induite par l’AGL saturé. Nous avons démontré par ailleurs que l’AGL monoinsaturé favorise l’estérification et le stockage du Palm dans des gouttelettes lipidiques, contrant ainsi ses effets délétères. Nous avons ensuite étudié les effets de ces deux AGL, sur des MSC isolées de patients atteints d’ostéonécrose de la tête fémorale (ON-MSC), comparativement aux HV-MSC. Lors de la procédure d’isolation des MSC, nous avons conservé le surnageant obtenu après la première centrifugation (bone marrow supernatant fluid, BMSF) pour une analyse de sa composition lipidique, celle du sérum étant réalisée en parallèle. Nous avons démontré que l’exposition au Palm favorise la différenciation des MSC vers la lignée adipogénique, au détriment du phénotype ostéoblastique. De plus, nous avons observé que les ON-MSC possèdent une capacité de différenciation adipogénique supérieure à celles des HV-MSC.D’autre part, nos résultats ont montré que les ON-MSC sont plus sensibles à la lipotoxicité que les HV-MSC, cette hypersensibilité étant associée à un dérèglement de plusieurs mécanismes cellulaires impliqués dans la survie ou les processus de protection cellulaire :le niveau d’activation basal de la voie ERK est supérieur à celui des HV-MSC et la régulation de l’expression génique des enzymes stearoyl-CoA desaturase 1 (SCD1) et carnitine palmitoyl transferase 1 (CPT1), favorisant respectivement la désaturation des AGL saturés et leur β-oxydation mitochondriale, est altérée dans les ON-MSC. Par ailleurs, dans ces cellules, la production des cytokines IL-6 et IL-8 est triplée par rapport à celle des HV-MSC.La caractérisation du profil lipidique du sérum n’a pas mis en évidence de différence significative entre les sujets sains et ostéonécrotiques. Cependant, de profondes modifications du contenu en AGL du BMSF ont été observées, montrant un enrichissement important en acide palmitique, palmitoléique, oléique, vaccénique et linoléique chez les sujets ostéonécrotiques. Ces modifications reflètent un changement du microenvironnement osseux chez ces patients qui pourrait être lié à l’activité sécrétrice des adipocytes médullaires et altérer le fonctionnement des cellules voisines.L’ensemble de nos travaux suggère qu’au sein du microenvironnement osseux, l’accumulation d’adipocytes médullaires pourrait avoir un effet délétère sur les cellules responsables de la formation osseuse, notamment via la libération d’AGL. Ces AGL étant cytotoxiques pour les cellules ostéoformatrices mais bénéfiques pour les cellules responsables de la résorption osseuse, ils pourraient favoriser un déséquilibre du remodelage osseux. En lien avec ces observations, nos résultats obtenus avec les ON-MSC suggèrent également que la lipotoxicité pourrait participer aux mécanismes pathogéniques qui initient et/ou entretiennent l’ostéonécrose. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown if these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. The effect of HG on adipogenesis was assessed. Human subcutaneous abdominal preadipocytes were induced to differentiate in HG or NG conditions. HG did not affect adipogenesis. HG increased ChREBP-β mRNA and protein levels, and increased GLUT4 mRNA, in differentiated adipocytes. It did not change mRNA levels of ACC, SCD, and FAS. The increase in ChREBP-β mRNA was positively correlated with HG-induced increase in GLUT4 mRNA. The effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation was compared. Human monocyte-derived macrophages (MDM) were placed in NG or HG glucose for 24 hours to generate MacCM. HG-MacCM, but not NG-MacCM inhibited triacylglycerol accumulation and protein expression of PPARγ during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased MCP-1 and IL-6 and reduced adiponectin mRNA expression. HG increased phosphorylation of IKK-β and decreased protein expression of IκBα in MDMs. In addition, HG reduced protein expression of PPARγ in MDMs. The pro-inflammatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKβ inhibitor). My data demonstrate that HG-induced expression of ChREBP-β in adipocytes may be associated with increased GLUT4 mRNA. The anti-adipogenic and pro-inflammatory effects of HG-MacCM are more potent than NG-MacCM. This suggests the possibility that adipose tissue cellular remodeling in vivo may be altered with hyperglycemia.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18212号 / 薬博第802号 / 新制||薬||237(附属図書館) / 31070 / 京都大学大学院薬学研究科生命薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Insulin resistance, the cause of type 2 diabetes mellitus, is intimately linked to the dysregulation of adipose tissue. Recent decades have witnessed the discovery and characterization of numerous hormones produced by adipocytes, including leptin, adiponectin and resistin, underscoring the endocrine functions of adipose tissue. To better understand the role of the adipocyte in the mediation of obesity-related insulin resistance and inflammation, this study has optimized the primary adipocyte isolation technique to minimize inflammation inherent to the isolation procedure and has analyzed adiponectin levels and insulin sensitivities of various adipocyte size populations both in vitro and ex vivo.The data described herein suggest that cell size plays an important, but not solitary, role in the regulation of insulin action and adiponectin production. It is possible that obesity-related insulin resistance is associated with the failure of a population of small adipocytes to expand and produce the insulin sensitizing protein hormone, adiponectin.

The current epidemic of obesity poses a substantial threat to public health worldwide. Obesity is associated with many deleterious health conditions, including type 2 diabetes, hypertension, dyslipidaemia, respiratory conditions, arthritis, and some forms of cancer. Moreover, the rising prevalence of obesity has been accompanied by a substantial increase in the cost of treating these conditions. Obesity results from a complex interaction between behavioural, environmental, and genetic factors. While the recent increase in the prevalence of obesity is largely due to behavioural factors (for example, physical inactivity); it has also been observed that genetic factors make a large contribution to individual susceptibility. In fact, studies indicate that as much as 50 - 80% of the variation in measures of obesity can be attributed to the effects of genes. Furthermore, closer examination of this genetic component using segregation analysis has indicated the presence of common genes for obesity, with large effects on the phenotype. However, these putative major genes for obesity have not yet been identified. The aim of this thesis was to investigate the role of three distinct genetic loci in obesity and related cardiovascular factors, including type 2 diabetes and dyslipidaemia. The aim of the first investigation was to test whether a common polymorphism (Pro12Ala) in the gene encoding peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) was associated with obesity and other cardiovascular risk factors in a large group of Caucasian subjects. PPAR-γ2 is an adipogenic transcription factor, which also regulates insulin sensitivity in adipose tissue. No association was observed between the Pro12Ala polymorphism and obesity in Caucasians, but obese subjects carrying the Ala allele displayed an altered blood lipid profile compared with obese Pro/Pro subjects. As the Pro12Ala polymorphism may exacerbate the risk of cardiovascular disease by modifying blood lipid profile in obesity, this relationship was examined further in a separate population. The aim of the second investigation was to determine whether the Pro12Ala polymorphism was associated with obesity, dyslipidaemia, diabetes and carotid intima-medial wall thickening in a population at high risk of developing cardiovascular disease. Australian Aboriginal people display high rates of mortality from cardiovascular disease, and it is possible that their increased susceptibility is due to genetic factors. However, the results from the Aboriginal population confirmed the results of the first study: there was no intrinsic association between the Pro12Ala variant and obesity. In addition, the Ala allele was not associated with deleterious changes in blood lipid profile, as it was in Caucasians. The aim of the third investigation was to confirm the presence of a quantitative trait locus (QTL) for obesity on chromosome 20q13. Highly polymorphic genetic markers in this region were tested for linkage and association with several measures of obesity in a Caucasian population. None of the measures of obesity were linked to or associated with markers spanning 20q13, suggesting that this chromosomal region does not contain a major locus for obesity in this Caucasian population. In the fourth investigation, the 5' sequence of Agouti Signalling Protein (ASIP) was identified. ASIP is a candidate gene for obesity, as it is expressed at high levels in adipocytes, and may participate in several obesity-related processes. Three new exons and two alternative promoters were identified for the ASIP gene. These results may lead to greater understanding of the role of ASIP in obesity and adipocyte metabolism; and may also be used to direct further research into genetic variation within this candidate gene. In conclusion, extensive study of two established candidate genetic loci revealed no association with measures of obesity. Therefore, it is likely that loci other than these make significant contributions to obesity in humans. Further investigation of novel candidate genes, such as ASIP, may allow the identification of novel genetic polymorphisms and new pathways important for the genetic basis of obesity.

BACKGROUND: Fat is stored in adipose tissue. In obesity, differentiation of preadipocytes to new adipocytes (fat cells) is required for energy storage. Otherwise fat accumulation in non-adipocytes contributes to fatty liver and diabetes. Our goal was to assess subject characteristics associated with poor in-vitro differentiation capacity of preadipocytes from omental (OM) and abdominal subcutaneous (SC) fat. APPROACH: A convenience sample of, 4 males and 20 females, age 39±2 (range 20-56) years, BMI 42 ± 2 (23-63) kg/m2 (i.e. from lean to obese), 7 Caucasian, 8 Hispanic, 1 other and 8 African Americans) undergoing elective surgery was studied. Fat samples collected during surgery were used for histology and preadipocyte isolation. Fat cell diameters and their distribution (normal or bimodal) were analyzed from histology. Preadipocyte differentiation capacity was measured in vitro. RESULTS: In the OM depot, no effect of ethnicity, sex or HbA1c was found. Unexpectedly, subjects with preadipocytes with poor differentiation capacity tended to be younger (poor differentiation group 36 ± 2 years versus high 43 ± 3 years, p=0.09) and to have lower fasting glucose (poor 97 ± 3.65 mg/dl versus high 111 ± 7.08 mg/dl, p=0.06). In SC, no differences were noted. Fat cell size was not associated with differentiation capacity in either depot. Bimodal distribution, which may show formation of new adipocytes, was seen mostly in Caucasian subjects (5 out of 7) compared to Hispanic (3 out of 8) and African Americans (2 out of 8). CONCLUSION: It is important to investigate the associations between age/ethnicity and OM preadipocyte differentiation/cell distribution in adequately powered cross-sectional studies.