Fifteen steers were used to evaluate the difference of diets (corn-based for 8 mo or hay-based for 12 mo) and breeds (Angus; n = 7 or Wagyu; n = 8) in a completely randomized design with 2 x 2 factorial arrangement of treatments to test the hypothesis that there are differences in fatty acid metabolism and cellularity in subcutaneous (s.c.) and intramuscular (i.m.) adipose tissue between these breeds types. Fat thickness, carcass weight, overall maturity, and yield grade of Angus steers were higher than those of Wagyu steers fed either corn (34%, 22%, 3%, and 8% higher, respectively) or hay diets (20%, 8%, 10%, and 8% higher, respectively) (P < 0.03). Moreover, marbling scores tended (P = 0.70) to be greater in Angus steers than in Wagyu steers fed either diet. Lipogenesis from acetate in both s.c and i.m. adipose tissue was higher in Wagyu steers (212.82 and 86.23 nmol/(105 cells per 2 h)) than in the Angus steers (86.23 and 29.66 nmol/(105 cells per 2 h)). Also, acetate incorporation into fatty acids was greater in s.c. adipose tissue than in i.m. adipose tissue (P < 0.05). Subcutaneous adipose tissue stearoyl-CoA desaturase (SCD) activity was significantly greater in corn-fed steers and than in hay-fed steers (P < 0.05), but there was no difference in SCD activity between Angus and Wagyu steers (P > 0.05). Adipocyte cellularity data demonstrated that both breeds have more cells per gram adipose tissue and smaller cell volumes in i.m. adipose tissue than in s.c. adipose tissue. In s.c. adipose tissue, saturated fatty acids tended to be lower in corn-fed Angus and Wagyu steers than in hay-fed steers (P < 0.06). Similarly, monounsaturated fatty acids were higher in corn-fed Wagyu and Angus steers than in hay-fed Wagyu and Angus steers (P < 0.01). Slip point was positively correlated with percentage stearic acid in corn-fed and hay-fed steers, and there was a negative correlation between slip point and the SCD index. These data demonstrated that corn-based diets provide not only increased contents of monounsaturated fatty acid in Angus and Wagyu adipose tissue but also increased lipogenic activity.

Fifteen steers were used to evaluate the difference of diets (corn-based for 8 mo or hay-based for 12 mo) and breeds (Angus; n = 7 or Wagyu; n = 8) in a completely randomized design with 2 x 2 factorial arrangement of treatments to test the hypothesis that there are differences in fatty acid metabolism and cellularity in subcutaneous (s.c.) and intramuscular (i.m.) adipose tissue between these breeds types. Fat thickness, carcass weight, overall maturity, and yield grade of Angus steers were higher than those of Wagyu steers fed either corn (34%, 22%, 3%, and 8% higher, respectively) or hay diets (20%, 8%, 10%, and 8% higher, respectively) (P < 0.03). Moreover, marbling scores tended (P = 0.70) to be greater in Angus steers than in Wagyu steers fed either diet. Lipogenesis from acetate in both s.c and i.m. adipose tissue was higher in Wagyu steers (212.82 and 86.23 nmol/(105 cells per 2 h)) than in the Angus steers (86.23 and 29.66 nmol/(105 cells per 2 h)). Also, acetate incorporation into fatty acids was greater in s.c. adipose tissue than in i.m. adipose tissue (P < 0.05). Subcutaneous adipose tissue stearoyl-CoA desaturase (SCD) activity was significantly greater in corn-fed steers and than in hay-fed steers (P < 0.05), but there was no difference in SCD activity between Angus and Wagyu steers (P > 0.05). Adipocyte cellularity data demonstrated that both breeds have more cells per gram adipose tissue and smaller cell volumes in i.m. adipose tissue than in s.c. adipose tissue. In s.c. adipose tissue, saturated fatty acids tended to be lower in corn-fed Angus and Wagyu steers than in hay-fed steers (P < 0.06). Similarly, monounsaturated fatty acids were higher in corn-fed Wagyu and Angus steers than in hay-fed Wagyu and Angus steers (P < 0.01). Slip point was positively correlated with percentage stearic acid in corn-fed and hay-fed steers, and there was a negative correlation between slip point and the SCD index. These data demonstrated that corn-based diets provide not only increased contents of monounsaturated fatty acid in Angus and Wagyu adipose tissue but also increased lipogenic activity.

Conjugated linoleic acid (CLA) reduces adipose mass in several species. Studies were conducted to determine: 1) the effect of dietary trans10,cis12-CLA on growth, tissue fatty acid profile, mRNA expression for stearoyl-CoA desaturase (SCD) in adipose and liver, and mRNA expression for fatty acid synthase (FAS) in adipose of mice, 2) the effect of a dietary combination of trans-vaccenic acid (TVA) and trans10,cis12-CLA on delta9- desaturation, and 3) the effect of cis9,trans11-CLA, trans10,cis12-CLA, and carnitine palmitoyltransferase-1 (CPT-1) inhibitors on expression of mRNA for CPT-1 and fatty acid profile in mouse hepatocytes (AML-12) and human hepatoma cells (HepG2). In the first study, male or female mice were fed diets containing 0, 0.15%, or 0.30% trans10,cis12-CLA for 6 wk. Epididymal adipose weights (males) and inguinal adipose weights (females) decreased by 81% and 52%, respectively, in response to 0.30% trans10,cis12-CLA. Dry carcass weights decreased from 4.75 g for the control to 3.62 g for mice fed 0.30% trans10,cis12-CLA and the decrease was due to a reduction in ether extract. Liver weights increased linearly from 0.55 g (control) to 0.65 g (0.30% trans10,cis12-CLA). Dietary trans10,cis12-CLA (0.30%) reduced FAS and SCD mRNA in adipose by 60 and 30 % respectively, compared with the control, suggesting reduced lipogenesis and desaturation might be primary factors responsible for reducing body fat. In the second study, adult male or female mice were fed diets containing 0.40% TVA in combination with 0, 0.15, or 0.30% trans10,cis12-CLA for 10 d. Both TVA and trans10,cis12-CLA were incorporated into plasma, liver, adipose, muscle, and bone lipids proportional to their concentrations in the diets. Desaturation ratios were not affected in adipose, liver, and bone. However, ratios of 16:0 to 16:1 and 18:0 to 18:1 increased from 0.81 to 0.86 and 0.15 to 0.19 respectively, in response to dietary trans10,cis12-CLA (0.30%), suggesting inhibition of delta9 desaturation in muscle. In the third study, AML-12 or HepG2 cells were incubated with control media or media containing 15 uM etomoxir (ETM), 30 uM ETM, 15 uM hemipalmitoylcarnitinium (HPC), 30 uM HPC, 100 uM cis9,trans11-CLA, or 100 uM trans10,cis12-CLA for 24 h. Half the cells were harvested for analysis of fatty acids, mRNA for CPT-1, and cholesterol after 24 h. The remaining cells were incubated for an additional 24 h in control medium. Incorporation (% of total fatty acids) of trans10,cis12-CLA was greater than cis9,trans11-CLA in AML-12 (34 vs 23.6) and HepG2 (28 vs 18) cells. Cells incubated with trans10,cis12-CLA had higher ratios of 16:0 to 16:1, 18:0 to 18:1, and 18:2n6 to 20:4n-6, suggesting inhibition of delta9, delta5 , and delta6 desaturation. Cis9,trans11-CLA also reduced ratio of 18:2n-6 to 20:4n-6 in both cell lines. Trans10,cis12-CLA increased mRNA for CPT-1 in both cell lines compared with the control, suggesting enhanced oxidation of fatty acids. In addition, trans10,cis12-CLA caused a 4-fold and 5-fold increase in free cholesterol content of AML-12 and HepG2 cells, respectively. Overall, results demonstrated that trans10,cis12-CLA modulated lipid metabolism in tissues in vivo and altered fatty acid metabolism, cholesterol synthesis, and CPT-1 mRNA in hepatic cell lines in vitro. / Ph. D.

No description available.

No description available.

CDK4 est une sérine/thréonine kinase qui est largement décrite pour son implication dans le contrôle du cycle cellulaire. Notre laboratoire et d'autres ont montré qu'elle jouait également un rôle majeur dans le contrôle de l'homéostasie du glucose (croissance des cellules β du pancréas et sécrétion d'insuline) et des lipides (adipogenèse). Nous avons montré au cours de cette thèse, par le biais de deux modèles de souris, invalidés pour CDK4 (Cdk4-/- ;cre/cre) ou exprimant un mutant hyperactif de la kinase (Cdk4R24C/R24C), qu'elle est un médiateur important de la voie de l'insuline et régule la lipogenèse et la lipolyse. Les souris Cdk4-/- ;cre/cre ont une diminution significative de la taille des adipocytes et du poids du WAT ou l'inverse est observé sur les souris Cdk4R24C/R24C. CDK4 est activée par l'insuline et va ainsi promouvoir le transport de glucose, la synthèse des lipides de novo et réprimer la lipolyse dans les adipocytes. De plus, nous avons démontré que dans l'adipocyte, cellule non proliférative, CDK4 et son partenaire la cycline D3 sont préférentiellement localisés dans le cytoplasme suggérant un rôle indépendant de leurs fonctions nucléaires. Nous avons identifié deux nouveaux substrats de CDK4 : IRS1 et IRS2. CDK4 phosphoryle IRS1 et IRS2 activant un rétrocontrôle positif permettant le maintien de l'action de l'insuline sur les adipocytes. Nos résultats prouvent un nouveau rôle de CDK4 sur la signalisation de l'insuline et sa fonction dans l'adipocyte. Par conséquent, la modulation de son activité pourrait avoir des conséquences majeures sur le mécanisme de résistance à l'insuline, une complication fréquente dans le développement de pathologies comme le diabète et l'obésité. / CDK4 is a serine/threonine kinase mainly known by its involvement in the control of cell cycle progression. Our laboratory and other laboratories have previously shown a major role for CDK4 in the control of glucose homeostasis (pancreatic β-cell growth) and lipid homeostasis (adipogenesis). In this thesis, we showed that CDK4 is an insulin effector that controls lipogenesis and lipolysis in mature adipocytes. We used Cdk4-/- ;cre/cre mice and Cdk4R24C/R24C mice, carrying a hyperactive mutant Cdk4 allele, for this study. Cdk4-/ - ;cre/cre mice have a manifest adipose tissue phenotype with a significant decrease in body weight and WAT mass. On the other hand, Cdk4R24C/R24C mice show increased body weight and increased adiposity. Furthermore, we demonstrate that CDK4 is activated by insulin to promote glucose transport, lipogenesis and repress lipolysis in adipocytes. Interestingly, we showed that in mature quiescent adipocytes CDK4 and its partner, Cyclin D3, are preferentially localized in the cytoplasm, suggesting a role independent from their nuclear functions. We identified two novel substrates of CDK4: IRS1 and IRS2. CDK4 phosphorylates both IRS1 and IRS2 in order to sustain insulin signaling in adipocytes via a positive feed-back loop. To sum up, our results identify a new function of CDK4 on insulin signaling in adipocyte metabolism. Thus, the modulation of its activity could have consequences on insulin resistance, a common complication of obesity and diabetes.

O uso crônico de altas doses de glicocorticoides (GC) aumenta tecidos adiposos (TA) centrais. Essa pesquisa visou verificar os efeitos do uso contínuo de GC em doses mais baixas sobre diferentes TAs. Ratos Wistar machos com 10 semanas de vida foram divididos em 2 grupos: controle (CON) e cortisol (CORT) e foram implantados com minibomba osmótica, pela qual o grupo CORT recebeu 0,6 mg/kg/d de cortisol e o grupo CON recebeu salina, por 6 semanas. Os resultados mostram um aumento da gordura subcutânea (SC) abdominal dos animais CORT, acompanhada de: aumento da atividade máxima das enzimas ATP-citrato-liase e glicose-6-fosfato desidrogenase; redução da expressão da enzima AMPK; aumento da expressão da enzima 11&#946;HSD1 e das triglicérides circulantes. Esses dados sugerem que a intervenção aumentou a SC abdominal devido maior atividade de enzimas lipogênicas e de um possível aumento da captação de lipídeos séricos por esse tecido, o que pode ter resultado do aumento da concentração local de GC provocado pela expressão aumentada da 11&#946;HSD1. / Chronic use of high doses of glucocorticoids (GC) increases central adipose tissue (AT). This research aimed to verify the effects of continuous use of lower doses of GC on different TAs. Male Wistar rats, 10 weeks old, were divided into 2 groups: control (CON) and Cortisol (CORT), and were implanted with osmotic minipump, whereby CORT group received 0.6 mg/kg/d of cortisol and CON group received saline, for 6 weeks. The results show an increase in abdominal subcutaneous fat (SC) of CORT animals accompanied by: increased maximum activity of ATP citrate lyase and glucose 6-phosphate dehydrogenase enzymes; reduced expression of AMPK enzyme; increased expression of 11&#946;HSD1 enzyme and circulating triglycerides. These data suggest that the intervention increased abdominal SC due to increased activity of lipogenic enzymes and a possible increase in the uptake of serum lipids through this tissue, which may have resulted from increased local GC concentration caused by increased expression of 11&#946;HSD1.

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

The liver maintains energy substrate homeostasis by synchronizing circadian or diurnal expression of metabolic genes with the feeding/fasting state. The activities of hepatic de novo lipogenic gene products peak during feeding, converting carbohydrates into fats that provide vital energy sources for peripheral tissues. Conversely, deregulated hepatic lipid synthesis leads to systemic metabolic dysfunction, establishing the importance of temporal regulation of fat synthesis/usage in metabolic homeostasis. Pharmacological activation of peroxisome proliferator-activated receptor \(\delta / \beta (PPAR \delta / \beta)\)improves glucose handling and systemic insulin sensitivity. However, the mechanisms of hepatic \(PPAR\delta\) actions and the molecular pathways through which it is able to modulate global metabolic homeostasis remain unclear. Here we show that hepatic \(PPAR\delta\) controls the diurnal expression of lipogenic genes in the dark/feeding cycle. Adenovirus mediated liver restricted activation of \(PPAR\delta\) promotes glucose utilization in the liver and fat utilization in the muscle. Liver specific deletion of either \(PPAR\delta\) or the \(PPAR\delta\)-regulated lipogenic gene acetyl-CoA carboxylase 1 (ACC1) reduces muscle fatty acid uptake. Unbiased metabolite profiling identifies 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) as a serum lipid derived from the hepatic \(PPAR\delta\)-ACC1 activity that reduces postprandial lipid levels and increases muscle fatty acid uptake. These findings reveal a regulatory mechanism that coordinates lipid synthesis and utilization in the liver-muscle axis, providing mechanistic insights into the hepatic regulation of systemic energy substrates homeostasis.