Bovine Adipose Tissue Explants Research Articles

Hot topic: Ceramide inhibits insulin sensitivity in primary bovine adipocytes

In nonruminants, the sphingolipid ceramide inhibits insulin sensitivity by inactivating protein kinase B (AKT) within the insulin-signaling pathway. We have established that ceramide accrual develops with impaired systemic insulin action in ruminants during the transition from gestation to lactation, dietary palmitic acid supplementation, or controlled nutrient restriction. We hypothesized that ceramide promotes AKT inactivation and antagonizes insulin sensitivity in primary bovine adipocytes. Stromal-vascular cells were grown from bovine adipose tissue explants and cultured in differentiation media. To modify ceramide supply, we treated differentiated adipocytes with (1) myriocin, an inhibitor of de novo ceramide synthesis, or (2) cell-permeable C2:0-ceramide. Insulin-stimulated AKT activation (i.e., phosphorylation) and 2-deoxy-D-[3H]-glucose (2DOG) uptake were measured. Treatment of adipocytes with myriocin consistently decreased concentrations of ceramide, monohexosylceramide, and lactosylceramide. The insulin-stimulated ratio of phosphorylated AKT to total AKT was increased with myriocin but decreased with C2:0-ceramide. Moreover, adipocyte insulin-stimulated 2DOG uptake was decreased with C2:0-ceramide and increased with myriocin. We conclude that ceramide inhibits insulin-stimulated glucose uptake by downregulating AKT activation in primary bovine adipocytes.

Agonists of the G protein-coupled receptor 109A-mediated pathway promote antilipolysis by reducing serine residue 563 phosphorylation of hormone-sensitive lipase in bovine adipose tissue explants

A balanced lipolytic regulation in adipose tissues based on fine-tuning of prolipolytic and antilipolytic pathways is of vital importance to maintain the metabolic health in dairy cows. Antilipolytic pathways, such as the G protein-coupled receptor 109A (GPR109A)-mediated pathway and the insulin signaling pathway in bovine adipose tissues may be involved in prohibiting excessive lipomobilization by reducing triglycerol hydrolysis. This study aimed to evaluate the in vitro antilipolytic potential of the mentioned pathways in bovine adipose tissue explants. Therefore, subcutaneous and retroperitoneal adipose tissue samples (approximately 100mg) of German Holstein cows were treated for 90min ex vivo with nicotinic acid (2, 8, or 32μM), nicotinamide (2, 8, or 32μM), β-hydroxybutyrate (0.2, 1, or 5mM), or insulin (12mU/L), with a concurrent lipolytic challenge provoked with 1μM isoproterenol. Lipolytic and antilipolytic responses of the adipose tissues were assessed by measuring free glycerol and nonesterified fatty acid release. To identify molecular components of the investigated antilipolytic pathways, protein abundance of GPR109A and the extent of hormone-sensitive lipase (HSL) phosphorylation at serine residue 563 were detected by Western blotting. Treatment with nicotinic acid or β-hydroxybutyrate decreased the lipolytic response in adipose tissue explants and concurrently reduced the extent of HSL phosphorylation, but treatment with nicotinamide or insulin did not. Subcutaneous adipose tissue constitutively expressed more GPR109A protein, but no other depot-specific differences were observed. This study provides evidence that the GPR109A-mediated pathway is functionally existent in bovine adipose tissues, and confirms that HSL phosphorylation at serine residue 563 is also important in antilipolytic regulation in vitro. This antilipolytic pathway may be involved in a balanced lipid mobilization in the dairy cow.

Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

ORIGINAL ARTICLE: Putative reference genes for gene expression studies in propionate and β-hydroxybutyrate treated bovine adipose tissue explants

Accurate gene expression normalization using a stable reference gene (RG) improves the reliability of quantitative real-time PCR (qPCR) results. Therefore, a validation of RGs should be done before their use. Only few studies on RGs have been done in cattle, and none in bovine adipose tissue (AT) explants, therefore, we characterize the effects of an in vitro treatment with propionate and β-hydroxybutyric acid (BHB) on the mRNA content of these RGs comparing the output data from the geNorm™ and the Normfinder(©) program. geNorm™ and Normfinder(©) estimated the most stable RGs in the following sequence for subcutaneous and for retroperitoneal AT explants treated with propionate: low density lipoprotein receptor-related protein 10 (LRP10) > hippocalcin-like 1 (HPCAL1) > glyceraldehyde-phosphate-dehydrogenase (GAPDH) > ribosomal protein S9 (RPS9) > RNA polymerase II (Pol II) > beta2 actin (ACTB) > 18S ribosomal RNA (18S rRNA). BHB treated AT explants yielded a different stability ranking for RGs using geNorm™: HPCAL1, GAPDH > Pol II > LRP10 > ACTB > RPS9 > 18S rRNA. Normfinder(©) estimated a different stability ranking for the RGs as shown in the following sequence for subcutaneous and retroperitoneal AT explants treated with BHB: HPCAL1 > Pol II > GAPDH > ACTB > LRP10 > RPS9 > 18S rRNA. Subsequent pairwise analysis of variation of RGs using geNorm™ suggested that LRP10, HPCAL1 and GAPDH should be used for accurate normalization of subcutaneous and retroperitoneal AT explants treated with propionate, while HPCAL1, GAPDH and Pol II should be used for BHB treatment.

Human acylation-stimulating protein and lipid biosynthesis in bovine adipose tissue explants

Human acylation-stimulating protein (hASP) up-regulates triacylglycerol synthesis in human adipocytes. The objectives of this research were 1) to determine the effect of hASP on triacylglycerol synthesis in bovine adipose explants and 2) to determine whether nutritional status influences the sensitivity of adipose tissue to hASP. Fresh s.c. adipose tissue was sectioned into 20- to 30-mg explants and incubated for 1 to 6 h in M199 media containing 3% BSA and either 0.75 mM [1-14C]palmitate, 0.75 mM [9, 10-3H]oleate, or 2.5 mM [1-14C] acetate, as well as hASP and(or) insulin. The explants were extracted, and lipid fractions were separated by TLC and quantified by liquid scintillation. Acetate incorporation into lipids increased 15 to 30%, and palmitate or oleate incorporation increased 10 to 25%, when explants were exposed to hASP, although this response was not significant in every experiment. Insulin increased triacylglycerol synthesis in some experiments, but not in others. Our interpretation is that acylation-stimulating protein (ASP) can mildly enhance triacylglycerol synthesis in bovine adipose tissue. To fulfill the second objective, nine 9-mo-old steers were housed individually for two periods of 3 wk each. During the first period, four of the nine steers were fed to 50% of NEm requirement and the other five consumed the same diet ad libitum. After the first period, all steers consumed feed ad libitum for 2 wk and were assigned the opposite ration for the second period. Steers gained 40.5 kg BW when allowed ad libitum access to feed but lost 30.2 kg BW when feed intake was restricted (SE = 7.84; P < 0.01). At the end of each period, s.c. adipose tissue was sectioned into explants and incubated as described above. Four explants per steer per period were used to test effects of insulin (0 and 1 nM) and hASP (0, 0.01, 0.1, and 1 microM). Insulin did not influence incorporation of acetate or oleate. Acetate incorporation (P < 0.32) was 0.99, 1.03, 1.04, and 1.10 nmol x mg(-1) h(-1) (SE = 0.13) and oleate incorporation (P < 0.01) was 0.347, 0.357, 0.353, and 0.420 nmol x mg(-1)h(-1) (SE = 0.022) for 0, 0.01, 0.1, and 1 microM hASP, respectively. Feed restriction reduced (P < 0.01) acetate and oleate incorporation by 95 and 40%, respectively. No interactions among feed intake, insulin, and hASP were detected. In conclusion, the effect of hASP on fatty acid esterification is not influenced by feed restriction.

Insulin and(or) dexamethasone regulation of glucose and acetate utilization in ovine and bovine adipose tissue explants incubated for seven days.

An in vitro system was used to study the regulation of glucose and acetate utilization by bovine and ovine adipose tissue (AT). Adult nonlactating, nonpregnant cows (n = 5) and ewes (n = 5) were given limited amounts of feed for 8 to 10 d, then overfed for 10 (ewes) or 21 (cows) d. Perirenal AT explants were incubated for 7 d. The effect of 2 mU/mL of insulin on glucose utilization was significant in bovine but not in ovine AT during the 1st d in vitro, whereas the relationship was reversed from the 4th d of incubation. The effect of insulin on acetate utilization was significant in bovine but not in ovine AT. The addition of dexamethasone (DEX, 100 nM) to the insulin-supplemented medium increased (P < .05) glucose and acetate utilization by ovine AT. However, DEX did not affect substrate utilization by bovine AT in the presence of insulin. The DEX alone decreased glucose and acetate utilization by bovine and ovine AT during the first 4 d of incubation. So, the effects of insulin and(or) DEX on substrate utilization differed with species and incubation time.

Lipoprotein lipase and metabolic activities in incubated bovine adipose tissue explants: effects of insulin, dexamethasone, and fetal bovine serum

An in vitro system was used to study the regulation of lipoprotein lipase (LPL) activity in bovine adipose tissue. The utilization of two energetic and lipogenic substrates, acetate and glucose, and the activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme involved in de novo lipogenesis, were also studied. Nine nonlactating, nonpregnant Holstein cows were given limited amounts of feed for 10 d, then they were overfed for 3 to 5 wk. Samples of perirenal adipose tissue were incubated for 24 or 48 h. Insulin (2 mU/mL) increased (P < .001) daily glucose and acetate utilization and attenuated (P < .001) the loss of G6PDH activity detected after 1 or 2 d of incubation. Dexamethasone (DEX, 10 nM) added to the insulin-supplemented medium decreased (P < .02) glucose utilization, but it did not change acetate utilization or G6PDH activity. A higher concentration of DEX (100 nM) potentiated (P < .004) the ability of insulin to attenuate the decrease in G6PDH activity without changing substrate utilization. Under basal conditions, LPL activity was decreased by approximately 66% after 2 d of incubation. The decline in LPL activity was attenuated by insulin addition (P < .02) and was further attenuated (P < .004) by 100 nM of DEX. The addition of 10% fetal bovine serum alone to the medium had no effect on LPL activity, and fetal bovine serum decreased this activity when it was added to the insulin-supplemented medium.

Effects of insulin and fetal bovine serum on lipoprotein lipase and glucose and acetate utilizations by cultured bovine adipose tissue explants

Effects of insulin and fetal bovine serum on lipoprotein lipase and glucose and acetate utilizations by cultured bovine adipose tissue explants

Effects of insulin and fetal bovine serum on lipoprotein lipase and glucose and acetate utilizations by cultured bovine adipose tissue explants

Effects of insulin and fetal bovine serum on lipoprotein lipase and glucose and acetate utilizations by cultured bovine adipose tissue explants