The metabolic syndrome (MetS) refers to a cluster of risk factors associated with the onset of two major chronic and lethal diseases, type 2 diabetes mellitus (T2DM) and CVD(Reference Moller and Kaufman1). Although the origins of the MetS remain largely unknown, accumulating evidence indicates that obesity and insulin resistance (IR) are the two critical components of the MetS that are positively associated with the rising prevalence of T2DM and CVD(Reference Moller and Kaufman1–Reference Mottillo, Filion and Genest3). Despite significant advancements in treatment options, lifestyle interventions comprising a combination of diet and physical activity have gained widespread recognition as a means of combating obesity and IR (reviewed in Kimokoti & Brown(Reference Kimokoti and Brown4)). Consequently, these trends necessitate better understanding of the beneficial or adverse effects of diet and its constituents on the MetS. A plethora of both epidemiological and interventional studies have suggested that dietary fats influence metabolic function(Reference Riccardi, Giacco and Rivellese5, Reference Bray, Paeratakul and Popkin6). However, due to the large functional and structural complexity of dietary fats, our current understanding of how and which dietary fat can beneficially modulate metabolic functions remains limited.
Conjugated linoleic acid (CLA; 18 : 2n-7) is one of the dietary fatty acids (FA) present in dairy products and ruminant meats that has been proposed to beneficially influence the characteristics of the MetS(Reference Ryder, Portocarrero and Song7–Reference Declercq, Taylor and Wigle20). However, differences in the metabolic actions of CLA have been linked to the genotype, phenotype and CLA isomer(Reference Tsuboyama-Kasaoka, Takahashi and Tanemura21–Reference de Roos, Rucklidge and Reid24). While these discrepancies require further investigation, research into production practices that enrich the milk content of cis-9, trans-11 (c9, t11)-CLA has resulted in a concurrent elevation of trans-vaccenic acid (VA)(Reference Cruz-Hernandez, Kramer and Kennelly25).
VA (trans-11-18 : 1n-7) is a positional and geometric isomer of oleic acid (18 : 1n-9)(Reference Lock and Bauman26). Biohydrogenation of linoleic acid (18 : 2n-6) and α-linolenic acid (18 : 3n-3) by microbes in the rumen produces VA(Reference Lock and Bauman26). Further metabolism of VA produces either stearic acid (18 : 0) by saturation or c9, t11-CLA by desaturation. The bioconversion of VA to c9, t11-CLA also occurs in mammalian tissues, and is estimated to be 5–12 % in rats(Reference Corl, Barbano and Bauman27) and 19–30 % in humans(Reference Turpeinen, Mutanen and Aro28).
The observational and interventional studies concerning the intake of trans-fats from ruminant sources (TF-R) and the effects on human health are limited. The majority of studies investigating the intake of trans-fats have focused on industrially produced (hydrogenated) trans-fat (IPTF) and have consistently been shown to adversely affect the risk factors of heart disease(Reference Sommerfeld29–Reference Woodside and Kromhout31). Conversely, epidemiological studies have suggested that dietary intake of TF-R has no association with the risk factors of cardiovascular health(Reference Ascherio, Hennekens and Buring32–Reference Sun, Ma and Campos34). Likewise, a recent meta-analysis of cohort studies has indicated that, unlike IPTF, TF-R does not affect the risk factors of CHD(Reference Bendsen, Christensen and Bartels35). However, the number of studies available to reach a firm conclusion on the potential effects of TF-R, including VA, on human health is limited.
On the other hand, studies conducted in experimental animals have demonstrated that dietary supplementation with VA has beneficial effects on lipidaemia(Reference Wang, Jacome-Sosa and Ruth36–Reference Jacome-Sosa, Lu and Wang38) and atherosclerosis(Reference Bassett, Edel and Patenaude39). Both short-term (3-week) and long-term (16-week) dietary supplementation with VA in JCR:LA-cp rats, a model of dyslipidaemia and IR, increased VA in adipose TAG and its bioconversion to c9, t11-CLA, and improved lipidaemia without altering glycaemia(Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37). If the biological effects of VA are due to its conversion to c9, t11-CLA, then the beneficial effects on the components of the MetS would be expected based on the fact that dietary supplementation with c9, t11-CLA attenuates IR and associated metabolic disorders in other rodent models of IR(Reference Ryder, Portocarrero and Song7, Reference Choi, Jung and Park13, Reference Moloney, Toomey and Noone17, Reference Halade, Rahman and Fernandes18, Reference Declercq, Taylor and Wigle20). Hence, it remains unclear whether the effects of VA are direct or indirect, and/or specific to the metabolic characteristics of the experimental model. Given the evidence for bioconversion of VA to c9, t11-CLA(Reference Turpeinen, Mutanen and Aro28, Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37) and our recent finding that dietary c9, t11-CLA (0·4 %, w/w) attenuates blood pressure in fa/fa Zucker rats(Reference Declercq, Taylor and Wigle20), we examined the effects of VA on various MetS characteristics in fa/fa Zucker rats, an animal model that has been well characterised for obesity-mediated IR and metabolic abnormalities(Reference Kurtz, Morris and Pershadsingh40, Reference Kasiske, O'Donnell and Keane41).
Experimental methods
Animals and diet
For the present experiment, 5-week-old fa/fa Zucker male rats (total n 20; Harlan Laboratories) were acclimatised for a week and randomly assigned to the VA group (1·5 % (w/w) trans-11-VA) or the control group (0 % VA). VA (98 % purity as determined by GC) was synthesised by the method of Duffy et al. (Reference Duffy, Quinn and Roche42). The 1·5 % (w/w) dose of VA was based on the study by Wang et al. (Reference Wang, Lu and Ruth37). The diet formulation is shown in Table 1. Both the VA and control diets contained a total of 8·5 % (w/w) fat. Feed intake (corrected for spillage) and weekly body weights (BW) were recorded. The experimental protocol was approved by the University of Manitoba Protocol Management and Review Committee and conducted according to the Canadian Council on Animal Care Guidelines.
Table 1 Diet formulation
VA, trans-11-vaccenic acid; AIN, American Institute of Nutrition.
* Dyets, Inc.
† 200 mg/kg biotin in maize starch as egg white was the protein source.
‡ Sigma-Aldrich.
§ Bunge Canada.
∥ Synthesised by the method of Duffy et al. (Reference Duffy, Quinn and Roche42); 98 % purity by GC.
Blood pressure
At the beginning of week 8 of the dietary intervention, blood pressure was measured in conscious rats by the indirect tail cuff method (IITC Life Sciences blood pressure monitoring system) as described previously(Reference Declercq, Taylor and Zahradka43).
Oral glucose tolerance test
Assessment of glucose responsiveness was determined during the middle of week 8 using a standard method(Reference Noto, Zahradka and Yurkova14). The area under the curve (AUC) for glucose was calculated using the method of Brouns et al. (Reference Brouns, Bjorck and Frayn44) and used as an indicator of glucose tolerance:
$$\begin{eqnarray} Glucose\,AUC = \left (\left (\frac {(G_{15} + G_{0})}{2}\times T_{15}\right ) + \left (\frac {G_{30} + G_{15}}{2}\times T_{15}\right ) + \left (\frac {(G_{60} + G_{30})}{2}\times T_{30}\right ) + \left (\frac {(G_{120} + G_{60})}{2}\times T_{60}\right )\right ), \end{eqnarray}$$
where G0, G15…G120 are glucose concentrations (mmol/l) and T0, T1…T120 indicate time (min).
Blood and tissue collection
After 8 weeks on the diet, the rats were fasted overnight, and asphyxiated with CO2 before collection of blood and tissues. Blood samples were collected and centrifuged at 1500 g for 15 min at 4°C. The serum layer was collected, aliquoted and stored at − 80°C for biochemical analyses. Various organs including adipose tissue (epididymal, perirenal and mesenteric fat pads), liver, heart, kidneys and pancreas were dissected and weighed. A small portion of epididymal adipose tissue was placed in Cryogel embedding media (Instrumedics, Inc.) and frozen immediately in a dry ice–ethanol bath, and then stored at − 80°C. Tissues were flash-frozen in liquid N2 and stored at − 80°C for various analyses.
Serum biochemistry
Enzymatic colorimetric kits were used to quantify glucose, TAG and cholesterol (Genzyme Diagnostics P.E.I. Inc.). Insulin was quantified using an ELISA kit (Alpco Diagnostics). The end points of these assays were detected at the designated wavelength and quantified using a microplate reader (FLUOstar Omega; BMG Labtech). Fasting glucose and insulin values were used to calculate the homeostatic model of assessment of insulin resistance (HOMA-IR), an index of IR:
$$\begin{eqnarray} HOMA\hyphen IR = (insulin\,(\mu U/ml)\times glucose\,(mmol/l))/22\cdot 5. \end{eqnarray}$$
Fatty acid composition of liver and epididymal fat
To verify the presence of VA in the liver and epididymal fat tissue, and to determine whether the bioconversion in the body is contributing to more c9, t11-CLA, the FA composition of liver and epididymal fat was analysed by GC as described previously(Reference Nagao, Inoue and Wang10). Briefly, lipid extracts were separated by TLC to obtain TAG and phospholipid (PL) fractions. Moreover, two methylating agents (sodium methoxide for CLA isomers and methanolic hydrochloric acid for all other FA) were used to prevent isomerisation of double bonds in the FA with conjugated bonds and because no single methylating agent provides adequate methylation of CLA and all other FA(Reference Nagao, Inoue and Wang10). Thus, samples were split for the two methylation procedures and the results combined based on the internal standards (TAG: triheptadecanoin (Nu-Chek Prep, Inc.); PL: 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (Avanti)) added to the samples(Reference Nagao, Inoue and Wang10). The methylated samples were analysed by GC using a Varian WCOT Fused Silica CP-SELECT FAME column (length 100 m, diameter 0·25 mm and film thickness 0·25 μm; Varian Canada, Inc.) and a Varian 450 GC with a flame ionisation detector. The column was operated at 100°C for 2 min and then the temperature was raised to 175°C at 25°C/min, held for 30 min, raised again to 220°C at 15°C/min, held for 10 min, raised again to 240°C at 20°C/min and held for 11 min. The total run time was 60 min and the samples were run with a 10:1 split ratio and a flow rate of 1·8 ml/min. The identity of peaks was verified with standards: Nu-Chek Prep #463 GLC Standard (Nu-Chek Prep, Inc.) and NIST #2377 (National Institute of Standards and Technology) for c9-t11-CLA and t10, c12-CLA isomers.
Adipocyte size
Cryosections (10 μm) of epididymal adipose tissue in Cryogel were fixed with ice-cold acetone for 10 min, rinsed with Tris-buffered saline (50 mm-Tris–HCl, pH 7·4, 150 mm-NaCl) and dehydrated with ethanol and xylene washes. Coverslips were mounted using AquaMount aqueous mounting medium (Lerner Laboratories). Digital images were captured with a light microscope fitted with a camera (Olympus IX81 microscope, Olympus IX2-UCB camera; Olympus America Inc.) at 200 × magnification using Infinity Analyze software (Release 5.0.2; Lumenera Corporation). The cell area (μm2) of approximately 250 adipocytes per rat was measured with ImageJ (National Institutes of Health) as described previously(Reference Noto, Zahradka and Yurkova15).
Western blotting
Proteins were extracted from frozen tissue and quantified as described previously(Reference Noto, Zahradka and Yurkova15). Western blotting was performed by separating proteins (10–20 μg) by SDS–PAGE, transferring to a polyvinylidene fluoride membrane and probing with primary (1:1000 dilution) and horseradish peroxidase-conjugated secondary antibodies (1:10 000 dilution). Quantification of band intensities was carried out using a FluorChem®Q gel scanning system with a charge-coupled device camera (Proteinsimple) and AlphaView®Software (version 1.3.0.6; Alpha Innotech Corporation). Data are expressed as arbitrary units relative to the loading control. Western blot analysis was performed using the following antibodies: Akt, phospho-Akt-Ser473, p42/44 mitogen-activated protein kinase (MAPK), phospho-MAPK-Thr202/Tyr204, β-tubulin, endothelial NO synthase, phospho-endothelial NO synthase-Ser1177, NF-κB p65, phospho-NF-κB p65-Ser536, stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), phospho-SAPK/JNK-Thr183/Tyr185, angiotensin II receptor type (AT) 1, AT2 (Cell Signaling Technologies); angiotensinogen (Fitzgerald); IL-10 (Biosource); adiponectin (Calbiochem); adipophilin (Progen).
Statistical analysis
Experimental data were analysed using SAS statistical software (SAS Institute, Inc.). Student's t test was used to compare the two groups (VA v. control) for end-point data; the χ2 test was used for the adipocyte size distribution. All results are reported as mean values with their standard errors. Differences were considered statistically significant at P< 0·05.
Results
Physical characteristics
Dietary supplementation of VA for 8 weeks did not alter physical measures including final BW, weight gain, total feed intake and body length (Table 2). Likewise, the organ:BW ratios were not different between the VA and control groups for various organs, including liver, visceral fat (sum of epididymal, mesenteric and perirenal fat pads), individual fat pads (mesenteric and perirenal), heart, kidneys and pancreas. However, the VA group had a 14 % lower epididymal:BW ratio than the control group.
Table 2 Physical characteristics of fa/fa Zucker rats fed 1·5 % trans-11-vaccenic acid (VA) for 8 weeks (Mean values with their standard errors; n 10 rats/group)
* Mean value was significantly different from that of the control group (P< 0·05).
† Visceral fat = epididymal fat+mesenteric fat+perirenal fat.
Metabolic parameters
Various indicators of glycaemia/IR (fasting glucose and insulin concentrations, and HOMA-IR) and glucose tolerance (AUC for glucose) at the whole body level were not significantly different from the control group (Table 3). Lipidaemia as assessed by fasting TAG and cholesterol was unchanged. Likewise, there was no difference in total liver lipid concentration, as an indicator of hepatic steatosis. Furthermore, there were no significant differences in both systolic and diastolic blood pressure between the groups.
Table 3 Metabolic parameters of fa/fa Zucker rats fed 1·5 % trans-11-vaccenic acid (VA) for 8 weeks (Mean values with their standard errors; n 6–10 rats/group)
HOMA-IR, homeostatic model assessment of insulin resistance; OGTT, oral glucose tolerance test AUC, area under the curve.
Fatty acid composition of liver
The effect of dietary VA on the liver FA composition of both TAG (intracellular storage) and PL (pool of membrane signalling molecules) in obese IR rats is summarised in Table 4. In both TAG and PL, the total SFA, PUFA, n-3, n-6, n-7 and n-9, and PUFA: SFA (P:S) ratio did not differ between the groups. Notably, PL MUFA was 20 % higher in the VA-fed group compared with the control group. In contrast, the TAG composition of MUFA remained unchanged between the groups. VA in TAG was not different between the dietary groups. VA in PL of the VA-fed group was 50 % less than VA in TAG, while rats fed the control diet had non-detectable levels of VA in PL. In contrast, c9, t11-CLA, a biometabolite of VA, was approximately 2-fold higher in TAG of the VA group compared with the control, whereas there was no difference in PL c9, t11-CLA between the groups.
Table 4 Liver fatty acid composition (g/100 g fatty acids) of fa/fa Zucker rats fed 1·5 % trans-11-vaccenic acid (VA) for 8 weeks (Mean values with their standard errors; n 5 rats/group)
c9, t11-CLA, cis-9, trans-11-conjugated linoleic acid; ND, not detectable.
* Mean value was significantly different from that of the control group (P< 0·05).
Fatty acid composition of epididymal fat
To investigate whether dietary supplementation of VA has any preferential incorporation or bioconversion into other endocrine tissues, we examined the TAG and PL FA composition of epididymal fat (Table 5). The total SFA, MUFA, PUFA, n-3, n-6 and n-9, and P:S ratio in both TAG and PL did not differ between the groups. However, n-7 in TAG was found to be approximately 1·2-fold higher in the VA-fed group than the control group, whereas PL n-7 was not different between the groups. The VA-fed group had higher VA in both TAG (approximately 3-fold) and PL (approximately 17-fold) compared with the control group. The bioconversion of VA to c9, t11-CLA was indicated by the approximately 24-fold elevation of c9, t11-CLA in TAG and the presence of c9, t11-CLA in PL of the VA-fed group compared with the control group. c9, t11-CLA in PL of the VA-fed group was approximately 2-fold higher than c9, t11-CLA in TAG while the control group had non-detectable c9, t11-CLA in PL.
Table 5 Epididymal fat fatty acid composition (g/100 g fatty acids) of fa/fa Zucker rats fed 1·5 % trans-11-vaccenic acid (VA) for 8 weeks (Mean values with their standard errors; n 5 rats/group)
c9, t11-CLA, cis-9, trans-11-conjugated linoleic acid; ND, not detectable.
* Mean value was significantly different from that of the control group (P< 0·05).
Adipocyte size
Adipose tissue function including adipocyte plasticity and expandability has been suggested as one of the firm determinants of obesity-mediated metabolic impairments(Reference Sethi and Vidal-Puig45–Reference Lonn, Mehlig and Bengtsson47). Thus, we examined the effect of VA on adipocyte size. As illustrated in Fig. 1(a), the animals receiving dietary VA had 7 % smaller adipocytes compared with the control group. Based on the size distribution graph (Fig. 1(b)), 66 % of adipocytes in the VA group were < 2500 μm2, whereas only 56 % of cells in the control group were in this range. Conversely, the number of adipocytes of >2500 μm2 was approximately 20 % lower in the VA group compared with the control group (34 v. 43 %, respectively).
Fig. 1 (a) Size and (b) distribution of adipocytes of fa/fa Zucker rats fed the control or trans-11-vaccenic acid (VA) diet for 8 weeks. (a) Representative images with the overall mean cell areas (n 10 rats/group) with their standard errors: control 2449 (se 32) μm2; VA 2284 (se 22) μm2. Mean value of the VA group was significantly different from that of the control group (P< 0·05). (b) Size distribution pattern for six rats per group. 
, Control diet-fed rats; 
, VA diet-fed rats. The distribution of the VA group was significantly different from that of the control group (P< 0·05; χ2 test).
Molecular analyses of adipose
Various molecular effectors in adipose tissue participate in IR, inflammation and hypertension. Specific adipose tissue targets of IR and inflammation that were unaffected by VA supplementation were Akt, p44/42 MAPK, endothelial NO synthase, SAPK/JNK and NF-κB p65 (phosphorylated and total protein for each; data not shown). Conversely, protein levels of IL-10, an anti-inflammatory cytokine, were increased in adipose tissue of the VA group compared with the control group (Fig. 2(a) and (b)) while adiponectin, a cardioprotective and anti-inflammatory adipokine, was unchanged (data not shown). With regard to blood pressure regulation, the VA group had higher levels of the AT2 receptor compared with the control group (Fig. 2(a) and (b)), while the AT1 receptor and angiotensinogen (data not shown) were unchanged. To determine whether the reductions in the epididymal:BW ratio and adipocyte size in response to the VA diet were due do changes in proteins that regulate lipid storage in the adipose, we quantified the abundance of the lipid droplet protein, adipophilin, by Western blotting. Interestingly, the abundance of adipophilin in epididymal adipose tissue was elevated in the VA group (Fig. 2(a) and (b)), indicating a difference in lipid droplet packaging between the two groups.
Fig. 2 (a) Representative Western blots for protein levels of IL-10, angiotensin II receptor type 2 (AT2) receptor, adipophilin and β-tubulin in the epididymal adipose tissue of fa/fa Zucker rats fed the control or trans-11-vaccenic acid (VA) diet for 8 weeks. (b) The relative Western blot band intensities were quantified and normalised to those of β-tubulin. , Control diet-fed rats; , VA diet-fed rats. Quantification of band intensities for adipophilin was based on bands at 48–52 kDa. Values are means (n 9–10 rats/group), with their standard errors represented by vertical bars. * Mean values were significantly different from those of the control group (P< 0·05).
Discussion
The main finding of the present study was that dietary VA supplementation reduced the epididymal fat:BW ratio and adipocyte size and modulated components of the local renin–angiotensin system (AT2 receptor) and inflammation (IL-10); however, these tissue/molecular-level changes were insufficient to improve adipose function or positively alter the whole-body response in terms of IR and hypertension. However, it is also noteworthy that VA, a TF-R present in dairy products, did not exacerbate MetS characteristics including obesity, IR, lipidaemia, hypertension, inflammation and hepatic steatosis.
During the last several years, dietary intake of TF-R has remained fairly constant while there has been a substantial decline in IPTF(Reference Stender, Astrup and Dyerberg48, Reference Mendis, Cruz-Hernandez and Ratnayake49). The amount of VA in ruminant-based foods varies due to factors that have an impact on dairy and beef production, including geographical location, composition of cow feed, season, etc.(Reference Tholstrup, Raff and Basu50). Dietary TF-R is primarily (60–80 %) VA, and TF-R in human diets varies from low (0·8 % of energy; 2·2 g/d) to moderate (1·5 % of energy; 4·2 g/d) to higher (5 % of energy; 11–12 g/d) intakes(Reference Bendsen, Christensen and Bartels35). The amount of VA (1·5 %, w/w) used in the present study is the same as used in the studies with JCR:LA-cp rats(Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37); however, it is unlikely that this level of VA could be attained in the human diet. The present result that VA supplementation has null effects on obesity-mediated metabolic abnormalities parallels an intervention study with TF-R that found no changes in blood lipids and insulin sensitivity in overweight women(Reference Tardy, Lambert-Porcheron and Malpuech-Brugere51), and epidemiological studies have reported neutral health outcomes for VA intake in human subjects(Reference Ascherio, Hennekens and Buring32–Reference Sun, Ma and Campos34). To date, there are no published studies of VA intervention in human subjects with the MetS.
On the other hand, an increasing number of studies have indicated that CLA (a biometabolite of VA) has favourable effects on MetS parameters in human subjects and several rodent models, including genetic and high-fat diet-induced IR(Reference Ryder, Portocarrero and Song7–Reference Declercq, Taylor and Wigle20). Consequently, questions are being raised whether TF-R, including VA, have the same biological effects as CLA or, instead, exert the adverse biological effects of IPTF. In the present study, 8-week dietary VA supplementation elevated c9, t11-CLA in the liver and adipose TAG, thus confirming that VA undergoes endogenous bioconversion as reported previously(Reference Turpeinen, Mutanen and Aro28, Reference Wang, Jacome-Sosa and Ruth36–Reference Jacome-Sosa, Lu and Wang38). These results raise the possibility that both the physiological and molecular effects of VA could be explained by the indirect effects of CLA given that dietary supplementation with c9, t11-CLA attenuates IR, hypertension and other metabolic abnormalities in rodent models(Reference Ryder, Portocarrero and Song7, Reference Choi, Jung and Park13, Reference Moloney, Toomey and Noone17, Reference Halade, Rahman and Fernandes18, Reference Declercq, Taylor and Wigle20). Recently, Wang et al. (Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37) reported in two different studies that dietary supplementation of VA in JCR:LA-cp rats for 3 or 16 weeks increased c9, t11-CLA in the adipose tissue and favourably modified lipidaemia and hepatic steatosis. However, we did not observe any changes in lipidaemia, hepatic steatosis or the liver lipid profile (except MUFA in PL) of fa/fa Zucker rats fed VA for 8 weeks, despite the elevation of c9, t11-CLA in the liver and epididymal fat TAG. Perhaps the JCR:LA-cp model and Western-type background diet (1 % cholesterol and 15 % lipid (w/w); P:S ratio of 0·6) used by Wang et al. (Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37) may be better suited to studying the hypolipidaemic effects of VA compared with the present study with fa/fa Zucker rats and a lower-fat diet (8·5 % lipid, w/w) containing soyabean oil (P:S ratio 4:1). Although the VA-fed group had higher MUFA in hepatic PL, there were no differences in specific MUFA or in the calculated indices for stearoyl-CoA desaturase activity (data not shown) in the present study.
The liver is a major organ for FA metabolism, including elongation and desaturation, and esterification of FA for the synthesis of TAG and their transport in VLDL to peripheral tissues. Perhaps the lack of response on metabolic parameters in the present study is due to insufficient tissue levels of c9, t11-CLA, as in our previous research with dietary supplementation of a CLA mixture, the c9, t11-CLA isomer was 5-fold higher in liver TAG and 2·5-fold higher in adipose TAG compared with the present study(Reference Noto, Zahradka and Yurkova14). Also, we did not observe a consistent elevation of c9, t11-CLA in PL of the liver and adipose tissue of VA-fed fa/fa rats. Perhaps these results reflect the length of the dietary intervention and differences in tissue metabolism and kinetics for the endogenous production of CLA from VA and its partitioning and incorporation into TAG v. PL pools. Furthermore, it is not clear whether CLA in the adipose tissue is from endogenous conversion and/or transport from the liver.
Interestingly, VA in TAG and PL and c9, t11-CLA in TAG were higher in the epididymal fat than in the liver of VA-fed rats, indicating the preferential incorporation of these FA in the adipose tissue of fa/fa rats. This may be linked to the positive effects observed in the adipose tissue. Accumulating evidence indicates that epididymal fat pad mass and adipocyte size are critical components of obesity, T2DM and other metabolic dysfunctions (reviewed in Fruhbeck(Reference Fruhbeck52)). The present results that dietary supplementation of VA reduced the epididymal fat pad mass in fa/fa Zucker rats was not observed in the studies conducted with JCR:LA-cp rats(Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37), although the incorporation of VA and the bioconversion of CLA in the adipose tissue was similar, suggesting model-specific effects.
The architecture of adipose tissue is gaining attention, since adipocyte size is positively correlated with the onset of the MetS(Reference Sethi and Vidal-Puig45–Reference Lonn, Mehlig and Bengtsson47). Azain et al. (Reference Azain, Hausman and Sisk53) reported that dietary supplementation of CLA reduces the fad pad mass by reducing the adipose cell size in female Sprague–Dawley rats. We have previously observed reductions in adipocyte size and positive effects on obesity-associated metabolic abnormalities with CLA supplementation in fa/fa rats despite no changes in BW or adiposity(Reference Noto, Zahradka and Yurkova15, Reference Declercq, Taylor and Wigle20, Reference Declercq, Taylor and Zahradka43, Reference DeClercq, Zahradka and Taylor54). Within this context, the reduction of epididymal fat pad mass and the presence of smaller adipocytes with higher adipophilin protein levels in the adipose tissue of rats fed VA indicate a positive effect. Given that changes in adipocyte size would influence adipokine status(Reference Skurk, Alberti-Huber and Herder55, Reference Kershaw and Flier56), examining the protein biomarkers that are involved in IR and inflammation was relevant to the present study. Although there were no major changes in IR as reflected in the serum glycaemic profile, oral glucose tolerance or critical nodes of insulin signalling (Akt, p42/44 MAPK), IL-10, an anti-inflammatory molecule(Reference Couper, Blount and Riley57), was found to be elevated in the adipose tissue with the VA diet.
It has been suggested that the pathophysiology of obesity and hypertension is highly interconnected and, together, accelerate the progression of T2DM and CVD(Reference Bogaert and Linas58). In this context, we have previously demonstrated that dietary supplementation with c9, t11-CLA (0·4 %, w/w) for 8 weeks attenuates obesity-mediated hypertension in the absence of changes in the renin–angiotensin system in adipose tissue(Reference DeClercq, Zahradka and Taylor54). Thus, given the positive results with c9, t11-CLA, we felt it was reasonable to expect VA would similarly reduce blood pressure. Furthermore, to our knowledge, no studies have investigated the effects of dietary VA on obesity-mediated hypertension. Unlike c9, t11-CLA, dietary supplementation of VA for 8 weeks failed to reduce blood pressure in obese IR rats. These results paralleled the absence of changes in components of the local renin–angiotensin system, except for the elevation in protein levels of the AT2 receptor. The AT2 receptor has been linked to obesity(Reference Yvan-Charvet, Massiera and Lamande59), and it appears to modulate blood pressure via the AT1 receptor(Reference Duke, Evans and Widdop60). Collectively, these molecular changes in adipose tissue indicate that VA positively influences some molecular parameters associated with IR.
The present results that there were no changes in glycaemia and insulinaemia of fa/fa Zucker rats fed VA are in accord with previous studies conducted with JCR:LA-cp rats(Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37). It is noteworthy that fa/fa Zucker rats and JCR:LA-cp rats display an inherent IR and have ‘sturdy’ pancreatic β-cells capable of maintaining insulin-secreting activity and glycaemia throughout life(Reference Srinivasan and Ramarao61). Intriguingly, VA-fed fa/fa Zucker rats had smaller adipocytes and molecular changes in adipose tissue but no reduction in hepatic steatosis, whereas VA-fed JCR:LA-cp rats had beneficial changes in the liver(Reference Wang, Jacome-Sosa and Ruth36, Reference Wang, Lu and Ruth37), suggesting the existence of model-specific effects. Although there were no physiological changes observed in terms of IR, these interesting molecular changes and reduction of adipocyte size may require longer duration (greater than 8 weeks) to produce physiological effects.
In summary, the present study using an obese IR model provides evidence that VA, a dietary trans-FA present in dairy products and ruminant meats, does not exacerbate obesity-mediated IR and metabolic abnormalities. In fact, there were some positive changes in VA-fed rats as reflected by the smaller adipocyte size and higher levels of IL-10, an anti-inflammatory protein. Future studies need to continue to delineate the effects of naturally v. industrially produced trans-FA on MetS parameters and the development of CVD and T2DM.
Acknowledgements
We thank Leslie Rech, Alana Lyons and Jennifer Young for their help with the animal care. We would like to acknowledge Dennis Labossiere for his technical assistance with GC. We also thank St Boniface Hospital Research Foundation for the generous infrastructure support. This study was funded by competitive research grants (to C. G. T. and P. Z.) from the Dairy Farmers of Canada and the Natural Sciences and Engineering Research Council of Canada. The authors' contributions are as follows: S. K. M. participated in the blood pressure data collection, Western blotting, data interpretation and manuscript preparation; D. H. performed the FA composition and serum biochemistry data collection and analysis; L. S. carried out the Western blotting; A. C. and J. E. conducted the adipose tissue morphometry; J. S. and M. R. were involved in the synthesis and analysis of VA for the animal study; P. Z. and C. G. T. contributed to the funding, design, supervision and manuscript editing. The authors have no conflicts of interest to disclose.
