Introduction

White adipose tissue is no longer considered an inert energy storage site, but a complex, metabolically active tissue [1]. Excess adipose tissue can be detrimental to human health with regard to common medical disorders associated with obesity. In addition, excess adipose tissue accumulation in livestock animals is costly to producers as wasted feed resources and discounts or loss of premiums for carcass cutability. In ruminant animals, the functional unit of white adipose tissue, the adipocyte, synthesizes and stores fatty acids and secretes hormones. Hormones produced by adipocytes include leptin [2], adipokines [3], and a newly, proposed class of hormone, “lipokine”, in which fatty acids can act as signals to other body tissues [4].

As components of both human and livestock diets, fatty acids have become a recent focus in energy metabolism research. In the public eye, interest in fatty acid composition of meat and milk products has increased in recent years with rising concerns of SFA in the human diet. Generally perceived, SFA are bad for human health; whereas, MUFA and PUFA are beneficial. A lipidomic study by Cao et al. [4] identified palmitoleic acid (16:1 cis-9), a MUFA, as a fatty acid that may act a hormone. They reported palmitoleic acid functioned as a lipokine which increased insulin sensitivity in skeletal muscle and decreased lipogenesis in liver of mice. However, palmitoleic acid is positively correlated with obesity in adults and children [5, 6]. Whether a component of human or livestock diets, palmitoleic acid may act to regulate lipid metabolism.

Previous results from our laboratory showed palmitoleic acid functions to reduce de novo fatty acid synthesis and reduce lipogenic gene expression in cultured bovine adipocytes [7]. Concurrent with 16:1 increasing in palmitoleic acid-treated cells, cis-vaccenic (18:1 cis-11) and eicosenoic (20:1 cis-13) acids also increase. The physiologic consequence, however, of these long-chain MUFA are largely unknown. We hypothesize that 18:1 cis-11 and 20:1 cis-13 are products of palmitoleic acid elongation that are jointly responsible for the lipogenic effects seen with palmitoleic acid supplementation in bovine adipocytes. Therefore, the objectives of this study were to: (1) confirm elongation products of palmitoleic acid (16:1 cis-9) elongation in vitro using stable isotopes and (2) evaluate if exogenous supplementation of palmitoleic acid, elongation products, or both are responsible for decreased desaturation and lipogenesis rates observed with palmitoleic acid supplementation in bovine adipocytes.

Materials and Methods

Cell Culture

Primary bovine stromal vascular cultures were harvested from intermuscular adipose tissue obtained at the lateral edge of longissimus muscle between the longissimus costarum and posterior serratus dorsalis at the 12th rib of two beef carcasses as described by Hirai et al. [8] using slight modifications as described by Pratt et al. [9]. Cells were plated at 1 × 104 cells/cm2 and passaged every 2–4 days when 60 % confluent. Cells were incubated at 37 °C under 5 % CO2 humidified atmosphere with media [Dulbecco’s modified Eagles medium (DMEM) containing 10 % fetal calf serum (FCS), and 2× antibiotic/antimycotic (AB/AM; containing 10,000 U/mL penicillin G, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B)] replacement every 2 days. After four passages, cell lines were stored in liquid nitrogen at 1 × 106 cells/mL in freezing media (DMEM, 20 % FCS, and 10 % dimethyl sulfoxide) for later use.

Treatments

Individual cultures from two beef carcasses were used in duplicate for each of these two experiments (n = 4). Cells were thawed, passaged three times, and seeded in plates at 1 × 105 cells/cm2. Cells were allowed to reach confluence, held for 2 days, and differentiated on day 0 with DMEM containing 5 % FCS, 2× AB/AM, 2.5 μg/mL insulin, 0.5 mM 2-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone (DEX), 5 μM troglitazone (TRO), and 10 mM acetate [8, 9]. Secondary differentiation media (DMEM, 5 % FCS, 2× AB/AM, 2.5 μg/mL insulin, 5 μM TRO, and 10 mM acetate) was applied for 4 (day 6) along with 0 μM additional fatty acids (control) or 150 μM fatty acids. Fatty acids were bound to fatty acid-free bovine serum albumin (2:1, w/w) as described by Van Harken et al. [10] prior to media addition. Control media contained an equal amount of fatty acid-free bovine serum albumin as used in fatty acid treatment media. According to the objectives of the experiment, cells were either harvested on day 6 for fatty acid composition and lipogenic gene expression or treated with stable isotopes on day 6 for enzymatic activity assays. Previously, our stromal vascular cultures displayed characteristic changes in morphology, lipid-filling, and adipogenic gene expression upon hormonal treatment, consistent with adipocyte differentiation by day 6 [7, 9].

Experiment 1

Previously, our palmitoleic acid-treated cultures showed a dramatic increase in 18:1 cis-11 and 20:1 cis-13 [7]. It has been proposed that palmitoleic acid can be elongated into 18:1 cis-11 and potentially elongated further into a 20C fatty acid [11]. To confirm 18:1 cis-11 and 20:1 cis-13 as elongation products and to measure elongation in vitro, we added stable isotope labeled palmitoleic acid [150 μM (U–13C)16:1] on day 6 for 0, 6, 12, 24, and 36 h to adipocytes cultures. At harvest, cells were washed three times with PBS, removed with trypsin, and placed directly into 2:1 chloroform/methanol (vol/vol) to terminate all enzymatic activity. Cellular fatty acids were extracted using Folch et al. [12] and transmethylated according to Park and Goins [13]. Fatty acid methyl esters (FAME) were analyzed using an Agilent 6890N gas chromatograph (GC; Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an Agilent 5973 mass spectrometer (MS) using a 100m Varian CP7489 (Varian Instruments Inc., Walnut Creek, CA, USA) capillary column (0.25 mm i.d. and 0.20 μm film thickness). Samples were run in the chemical ionization mode with He as the carrier gas and CH4 as the reagent gas.

Ions of mass-to-charge ratio (m/z) 268 (m) and 284 (m + 16) were selectively measured to calculate the isotopic enrichments of 16:1. Similarly, ion abundance of 298 (m) and 312 (m + 16) were measured for 18:1 cis-11. We calculated the relative abundance (RA) of labeled (m + 16) to unlabeled (m) isotopomers of 16:1 and 18:1 cis-11. Fatty acid enrichment was calculated as tracer-to-tracee ratio (TTR), according to Wolfe and Chinkes [14].

$$ {\text{TTR}}_{\text{sample}} = \left( {{\text{RA}}_{\text{sample}} -{\text{RA}}_{\text{blank}} } \right). $$

Samples that were not exposed to the labeled isotope served as ‘blanks’. Molar percent excess (MPE) of 16:1 was calculated by TTR/(TTR + 1) and fractional synthetic rate (FSR) of elongation was calculated from TTR18:1 cis-11 and MPE16:1.

$$ {\text{FSR}}_{\text{elongation}} = \frac{{{\text{TTR}}_{{18:1cis {\text{-}} 11,{\text{ time}}1}} -{\text{ TTR}}_{{18:1cis - 11,{\text{ time}}0}} }}{{{\text{MPE}}_{16:1} \times \, \left( {{\text{time}}1 \, -{\text{ time}}0} \right)}}. $$

Experiment 2

The objective of this experiment was to determine if palmitoleic acid or an elongation product was responsible for changes in lipogenesis. Therefore, we treated adipocytes cultures with 0 μM fatty acids (control) or 150 μM palmitic (16:0), 150 μM palmitoleic, or 150 μM cis-vaccenic acids on day 2–6. FAME were prepared from cultures on day 6 as stated above for analysis with an Agilent 6850 GC equipped with an Agilent 7673A automatic sampler. Separations were accomplished using a 100m Supelco SP-2560 (Supelco, Inc., Bellefonte, PA, USA) capillary column (0.25 mm i.d. and 0.20 μm film thickness) according to Duckett et al. [15]. Individual fatty acids were identified by comparison of retention times with standards (Sigma, St. Louis, MO, USA; Matreya, Pleasant Gap, PA, USA). Fatty acids were quantified by incorporating an internal standard, methyl tricosanoic (23:0) acid, into each sample during methylation and expressed as a weight percentage of total fatty acids per well.

Lipogenesis

To measure lipogenesis in vitro, we substituted unlabeled acetate in the culture media on day 6 with stable isotope labeled acetate [10 mM (1-13C)2] to cultures treated with 0 μM fatty acids (control), 150 μM palmitoleic, or 150 μM cis-vaccenic acid for 0, 12, 24, and 36 h. Cells were harvested and FAME prepared as described above. Ions of mass-to-charge ratio (m/z) 270 (m), 271 (m + 1), and 272 (m + 2) were selectively measured to calculate the isotopic enrichments of 16:0, the primary product of de novo lipogenesis.

The formation of a fatty acid polymer, 16:0, from repeating units of acetate is an ideal application of mass isotopomer distribution analysis (MIDA) [14]. Therefore, we used this approach to estimate enrichment in our precursor pool and calculate FSR of lipogenesis. First, we calculated RA of singly (m + 1) and doubly (m + 2) labeled 16:0 to unlabeled (m) 16:0 isotopomers. To account for background noise and natural abundance of 13C isotope when using a singly-labeled tracer, the enrichment of 16:0 was calculated as \( {\text{TTR}}_{ 1 6 : 0} {\text{ = (RA}}_{\text{sample}} - {\text{RA}}_{\text{blank}} )\times ( 1-A)^{N} , \) where A was the natural abundance of 13C, A = 0.011, and N was the number of C in the fatty acid molecule, N = 16 [14]. Samples that were not exposed to labeled isotope served as ‘blanks’. Precursor enrichment in MPE was estimated by:

$$ {\text{MPE}}_{\text{precursor}} = \frac{{ 2 { } \times \, \left( {{\text{TTR}}_{{(m + { 2})}} /{\text{ TTR}}_{{(m + { 1})}} } \right)}}{{\left( {{\text{p }}-{ 1}} \right) \, + \, ( 2 { } \times \, \left( {{\text{TTR}}_{{(m + { 2})}} /{\text{ TTR}}_{{(m + { 1})}} } \right)}} $$

where, p is the number of precursor monomers present in the polymer, p = 8. Finally, our estimate of lipogenesis could be derived by calculating FSR [14].

$$ {\text{FSR}}_{\text{lipogenesis}} = \frac{{{\text{TTR}}_{{16:0,{\text{ time}}1}} - {\text{TTR}}_{{16:0,{\text{ time}}0}} }}{{\left( {p \, \times {\text{ MPE}}_{\text{precursor}} } \right) \, \left( {1 - {\text{ MPE}}_{\text{precursor}} } \right)^{p - 1} \left( {{\text{time}}1 - {\text{time}}0} \right)}}. $$

Desaturation

Stearoyl-CoA desaturase 1 (SCD1) is the enzyme responsible for creating a double bond at the ∆9 position of several fatty-acyl CoA and its preferred substrates are 16:0 and 18:0 [16]. Its transcription and activity can be regulated by certain fatty acids [17], including palmitoleic acid [7]. In order to measure desaturation rates in vitro, we added 100 μM [U–13C]18:0 to cultures treated with 0 μM fatty acid (control) or 150 μM palmitic, palmitoleic, or cis-vaccenic acid on day 6 for 0, 12, and 24 h. Samples were prepared and analyzed as described above for analysis with GC–MS. Ions of mass-to-charge ratio (m/z) 298 (m) and 316 (m + 18) were selectively measured to calculate the isotopic enrichments of 18:0. Similarly, isotope abundance of 296 (m) and 314 (m + 18) was measured for 18:1 cis-9. Since tracer enrichment can be measured in this case and the product of the SCD reaction is not a polymer of the tracer, it was not necessary to use MIDA. The FSR was calculated for rate of desaturation using the precursor enrichment, MPE18:0.

$$ {\text{FSR}}_{\text{desaturation}} = \frac{{{\text{TTR}}_{{18:1cis {\text{-}} 9,{\text{ time}}1}} - {\text{TTR}}_{{18:1cis {\text{-}} 9,{\text{ time}}0}} }}{{{\text{MPE}}_{18:0} \times \left( {{\text{time}}1 - {\text{time}}0} \right)}}. $$

Gene Expression

Total cellular RNA was isolated from cells using the mirVana microRNA Isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions and RNA quality as described by Duckett et al. [18]. Quality was assessed using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Agilent Bioanalyzer 2100. All tcRNA samples used in real-time PCR had a 260:280 absorbance ratio >1.9 on the Nanodrop and RNA integrity number >9.0 (1.0–10.0 scale) using Agilent RNA 6000 Nano kit. Superscript III reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA) was used to synthesize first strand cDNA. Real-time PCR was conducted using an Eppendorf MasterCycler ep realplex (Westbury, NY, USA) with the QuantiTect SYBR Green RT-PCR Two Step Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s directions. Two genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin, were evaluated as housekeeping genes for data normalization [18]. To determine the appropriate housekeeping gene to be used to normalize the data, the cycle threshold values (C T) for GAPDH, β-actin, and all target genes per sample were entered into the BESTKEEPER program (http://www.gene-quantification.info). The program determines the most stable housekeeping gene to be used for normalization by repeated pair-wise correlation and regression analysis [19]. Both GAPDH and β-actin exhibited a correlation coefficient of 0.99 (P < 0.001) in the analysis and were suitable for data normalization. Primers for bovine mRNA were designed using Primer 3 software (http://www.frodo.wi.mit.edu/primer3/). Genes of interest for this study were those involved in fatty acid biosynthesis including acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), fatty acid elongase (ELOVL) -5 and -6, SCD1, sterol regulatory element binding protein-1c (SREBP), and carnitine palmitoyl-transferase 1A (CPT1A). Primer sets were first evaluated according to Duckett et al. [18], with the exceptions of ELOVL5 (forward: 5′-gtcatctggccgtgtacctt-3′; reverse: 5′-gggaagaaaagctgctgatg-3′), ELOVL6 (forward: 5′-ggaagcaacgaaagctgac-3′; reverse: 5′-tgggttgtgtgtttgctcat-3′), and CPT1A (forward: 5′-gtgatgtcgaccatgtacgc-3′; reverse: 5′-agcagcaccttcagggagta-3′).

Statistical Analysis

The data were analyzed using Proc GLM procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA) for treatment comparisons over time. For Experiment 1, an analysis of variance (ANOVA) was performed as a completely randomized design with factor time (0, 6, 12, 24, and 36 h). For Experiment 2, a two-way ANOVA was performed as a completely randomized design with factors fatty acid (control, palmitic, palmitoleic, and cis-vaccenic), time (0, 12, 24, and 36 h), and two-way interaction. Least squares means were computed and separated statistically using Fisher’s protected LSD test. For the analysis of relative gene expression, all C T values for each sample/primer pair combination and the respective primer efficiency were analyzed using the REST-2009 program (http://www.gene-quantification.de/rest-2009.html; [19]) and the data were normalized using GAPDH. This software calculates relative gene expression using Pair-wise Fixed Reallocation Randomization Test and relative expression determined at the 95 % confidence interval.

Results

Experiment 1

Incubating bovine adipocytes with [U–13C] 16:1 resulted in enrichment (P < 0.001) of 16:1 and 18:1 cis-11 fatty acids over time. TTR of 16:1 was greater than 0 (P < 0.01) 6 h after [U–13C] 16:1 addition to the media and remained elevated (P < 0.01) to 36 h (Fig. 1). Enrichment of 18:1 cis-11 as TTR was also greater than 0 (P < 0.05) by 6 h and continued to increase (P < 0.01) above 6 h levels at 24 and 36 h. The FSRelongation increased (P < 0.05) over the 36 h period (Fig. 1).

Fig. 1
figure 1

Elongation of 16:1 to 18:1 cis-11 in bovine adipocyte cultures. a Main effect of time on tracer-to-tracee ratio (TTR) of 16:1 following 150 μM [U–13C]16:1 addition to the media at 0 h. b Enrichment of 18:1 cis-11 displayed as TTR over time in cells. c Fractional synthetic rate of [13C]18:1 cis-11 from [13C]16:1 displayed as molar percent excess (MPE) per h and was calculated over a 36 h period following stable isotope administration. d Enrichment of [13C] 18:2 cis-9, cis-11 displayed as TTR over time in cells. e Enrichment of [13C] 20:1 cis-13 displayed as TTR over time in cells. (n = 4, mean ± SEM). a−cTime points without a common letter differed (P < 0.01)

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In addition to label appearing in 16:1 and 18:1 cis-11 fatty acids following incubation with [U–13C] 16:1, enrichment was also seen in 20:1 cis-13 and cis-9, cis-11 CLA after concentrating the samples. These 20:1 cis-13 and cis-9, cis-11 CLA were present at very low levels in our samples (<1.0/100 g total fatty acids). Fatty acid peaks contained masses consistent with m + 16 enrichment from [U–13C] 16:1. Enrichment of 20:1 cis-13 was present at 24 and 36 h with TTR of 0.15 and 0.20, respectively. Enrichment of cis-9, cis-11 CLA was present at 24 and 36 h with TTR of 0.42 and 0.55 respectively.

Experiment 2

Comparing fatty acid data between control, palmitic, palmitoleic, and cis-vaccenic acid-treated cells, there was a main effect of fatty acid treatment (P < 0.01) for total fatty acids (Table 1). Micrograms of total fatty acids were greatest (P < 0.001) in palmitoleic and cis-vaccenic acid-treated cells at about 2× greater than control cells (Table 1). Palmitic acid-treated cells also had higher (P < 0.05) levels of total fatty acids compared with controls, but lower (P < 0.05) than palmitoleic and cis-vaccenic acid-treated cells, despite being supplemented at the same level.

Table 1 Fatty acid composition of bovine adipocyte cultures treated with 0 μM fatty acids (control), 150 μM palmitic, palmitoleic, or cis-vaccenic acid (n = 4)
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Palmitic acid-treated cells had increased (P < 0.05) levels of 16:0, 18:0, 18:1 cis-9, and 18:2n-6 compared with all other treatment groups (Table 1). Palmitoleic acid-treated cells had increased (P < 0.05) levels of 16:1 compared with all other treatment groups. In addition, palmitoleic acid cells had increased (P < 0.05) levels of 18:1 cis-11 and 20:1 cis-13 compared with controls, consistent with results from our previous work [7]. cis-Vaccenic acid-supplemented cells had slightly elevated (P < 0.05) 16:1 compared with controls, but were not different (P > 0.05) than palmitic acid-treated cells. Also, cis-vaccenic acid-supplemented cells had the greatest (P < 0.05) amount of 18:1 cis-11 and 20:1 cis-13 compared with all other treatment groups. Palmitoleic and cis-vaccenic acid-treated cells had the lowest (P < 0.05) levels of 18:1 cis-9 and 18:2n-6 fatty acids. In addition, C20:4n-6 tended (P = 0.07) to be lower in both MUFA-treatment groups compared with controls.

Despite low levels of 18:1 cis-9 in both MUFA-treated groups, the desaturation index of 18:1 cis-9/18:0 was reduced (P < 0.05) in palmitoleic acid treated cells only compared will all other treatment groups (Table 1). The desaturation index of 18:1 cis-9/18:0 indicates desaturase activity was only impacted by palmitoleic acid supplementation, not cis-vaccenic acid.

Lipogenesis: precursor enrichment, MPEacetate, did not change (P > 0.05) over time or due to treatment in our cultures using MIDA, nor was there a time by treatment interaction (Fig. 2), suggesting we had isotopic steady state in our cultures [20]. There was an interaction (P < 0.01) between fatty acid treatment and length of [13C] 2 incubation on 16:0 enrichment. The TTR16:0 did not differ (P > 0.05) between treatment groups at 0 or 12 h and increased (P < 0.05) in all groups over time. At 24 and 36 h time points, TTR16:0 was decreased (P < 0.05) in palmitic, palmitoleic, and cis-vaccenic acid-treated cells compared with controls. The FSRlipogenesis, calculated from MPEprecursor and TTR16:0, also shows a reduction in lipogenesis in palmitic, palmitoleic, and cis-vaccenic acid-treated cells. The FSRlipogenesis was decreased (P < 0.05) in palmitoleic acid-treated cells between 0 and 12 h and decreased (P < 0.01) in all fatty acid-treated cells compared with controls between the 12–24 and 24–36 h time points (Fig. 2).

Fig. 2
figure 2

Lipogenesis in bovine adipocyte cultures treated with 0 μM fatty acid (control), 150 μM palmitoleic, or 150 μM cis-vaccenic acid. a Estimation of molar percent excess (MPE) of acetate after [1-13C] 2 addition to the media. b Enrichment of 16:0 displayed as tracer-to-tracee ratio of 16:0 over time. c Fractional synthetic rate of [13C] 16:0 from [13C] 2 displayed as molar percent excess (MPE) per h calculated over a 36 h period following stable isotope administration. (n = 4, mean ± SEM). Asterisk indicates within a time point, fatty acid-treated cells differed (P < 0.05) from controls

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Desaturation: because the desaturation index of 18:1 cis-9/18:0 is not always representative of SCD1 activity [21], we supplemented a labeled substrate of the SCD1 reaction [U–13C]18:0 to our bovine adipocyte cultures to serve as a measurement of desaturase activity. There was a treatment by length of incubation interaction for precursor enrichment, TTR18:0. Enrichment of 18:0 was not different (P > 0.05) than 0 at 0 h, but TTR18:0 increased (P < 0.05) over time for each sample. However, there was a differential incorporation of labeled [U–13C] 18:0 into the different treatment groups. Cells treated with palmitoleic and cis-vaccenic acids had increased (P < 0.01) TTR18:0 compared with control and palmitic acid-treated cells (Fig. 3). There was also a treatment by incubation time interaction (P < 0.01) for TTR18:1cis-9. At 0 h, there was no enrichment (P > 0.05) of 18:1 cis-9 in any treatment group; by 12 h, TTR18:1cis-9 of palmitoleic acid-treated cells was lower (P < 0.01) than all other treatment groups and was not different (P > 0.05) than 0. Control, palmitic, and cis-vaccenic acid-treated cells did not differ (P > 0.05) from each other and were enriched (P < 0.05) with 18:1 cis-9 at 12 h. By 24 h, all treatment groups had enrichment (P < 0.05) of 18:1 cis-9 and differed (P < 0.05) from each other. Palmitoleic acid-treated cells had reduced (P < 0.01) TTR18:1cis-9 compared to controls, palmitic, and cis-vaccenic acid treated cells at 12 and 24 h. Palmitic and cis-vaccenic acid-treated cells had higher (P < 0.05) TTR18:1cis-9 at 24 h than controls, which were also higher (P < 0.05) than palmitoleic acid-treated cells. Despite increased TTR18:0 in both palmitoleic and cis-vaccenic acid-treated cells, only palmitoleic acid-treated cells showed less (P < 0.05) formation of the desaturase product, as TTR18:1cis-9, than control cells.

Fig. 3
figure 3

Desaturation of 18:0 to 18:1 cis-9 in bovine adipocyte cultures treated with 0 μM fatty acids (control), 150 μM palmitic, 150 μM palmitoleic, or 150 μM cis-vaccenic acid. a Tracer-to-tracee ratio (TTR) of 18:0 and b 18:1 cis-9 displayed over time following 100 μM [U–13C] 18:0 addition to the media. c Fractional synthetic rate of [13C] 18:1 cis-9 from [13C] 18:0 displayed as molar percent excess (MPE) per h calculated over a 24 h period following stable isotope administration. (n = 4, mean ± SEM). Asterisk indicates within a time point, fatty acid-treated cells differed (P < 0.05) from controls

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The FSRdesaturation over 0–12 h was decreased (P < 0.01) in palmitoleic acid-treated cells compared with all other treatment groups, which did not differ (P > 0.05) from each other (Fig. 3). cis-Vaccenic acid-treated cells tended (P = 0.052) to have a reduced FSRdesaturation from 0 to 12 h compared with controls. From 12 to 24 h, however, all treatment groups had a different FSRdesaturation. Palmitic acid-treated cells had the highest (P < 0.01) rate of desaturation and palmitoleic acid-treated cells had the lowest (P < 0.01) FSRdesaturation from 12 to 24 h (Fig. 3).

Gene expression: palmitic acid supplementation down-regulated (P < 0.05) expression of SREBP and up-regulated (P < 0.05) CPT-1a mRNA expression (Fig. 4). Palmitoleic acid supplementation down-regulated (P < 0.05) FASN, SCD-1, and ELOVL6 mRNA expression and up-regulated (P < 0.05) ACC and CPT-1a mRNA expression compared with controls. cis-vaccenic acid supplementation up-regulated (P < 0.05) ACC and CPT-1a mRNA expression and down-regulated (P < 0.05) FASN and ELOVL6 mRNA expression compare to controls.

Fig. 4
figure 4

Relative gene expression of bovine adipocytes supplemented with 0 μM fatty acids (control), 150 μM palmitic, 150 μM palmitoleic or 150 μM cis-vaccenic acid. a Expression of sterol response element binding protein-1c (SREBP), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and b Acetyl-CoA carboxylase (ACC), fatty acid elongase (ELOVL) 5 and 6, and carnitine-palmitoyl transferase 1A (CPT1A), genes associated with fatty acid biosynthesis and metabolism pathways. All C T values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 4). Asterisk indicates gene expression for this treatment differed (P < 0.05) from controls

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Discussion

Our previous work with palmitoleic acid supplementation in bovine adipocyte cultures resulted in a large increase in 18:1 cis-11 concentration within the cells [7]. Enrichment of 16:1 shortly after [U–13C] 16:1 addition to the media shows that there was rapid incorporation of palmitoleic acid into the adipocytes on day 6. In addition, the enrichment of 18:1cis-11, as shown by TTR18:1cis-11, confirmed 18:1 cis-11 as a direct elongation product of 16:1. Presence of [13C] tracer in 18:1cis-11 fatty acids definitively shows that palmitoleic acid is taken in by bovine adipocytes and elongated to 18:1 cis-11 in vitro. Originally isolated from horse brain [22] and later in intestinal bacteria [23] and a host of mammalian tissues [24], cis-vaccenic acid is a MUFA. It can be derived from the diet or biosynthetic pathways, but its role in metabolism is largely unknown. Elongase enzymes generally have chain-length and saturated bond specificity for their fatty acids substrates [25]. The conversion of 16:1 to 18:1 cis-11 is generally attributed to the ELOVL6 isoform of the mammalian elongase enzyme, which adds an acetate molecule to the carboxylic acid end of the fatty acyl chain. Mice deficient in ELOVL6 show increased levels of 16:0 and 16:1 cis-9 and reduced 18:0, 18:1 cis-9, 18:1 cis-11, and other long chain fatty acids [11]. However, previous work in our lab suggests that ELOVL6 mRNA is down-regulated in response to palmitoleic acid supplementation despite an obvious increase in the ELOVL6 reaction product 18:1 cis-11 [7]. This is the first conclusive study in bovine adipocytes that provides proof of 18:1 cis-11 elongation from 16:1 cis-9. In addition, 18:1 cis-11 subsequent elongation to 20:1 cis-13 and desaturation to cis-9, cis-11 CLA have not been reported to date.

We successfully incorporated our fatty acid supplements into bovine adipocyte cultures based on detection of increased levels (P < 0.01) of treatment fatty acids in cells after all media had been removed. Reduced total fatty acids in palmitic acid-treated cells compared with MUFA-treated cells may be due to an apoptotic effect of palmitic acid supplementation. In murine adipocytes, palmitic acid induces endoplasmic reticulum stress and apoptosis at 100, 250, and 500 μM treatment levels [26]. Similarly, we suspected that palmitic acid-supplementation in our bovine adipocyte cultures induced apoptosis, resulting in reduced total lipid accumulation and treatment fatty acid inclusion compared with MUFA-supplemented cells. To test this hypothesis, we treated differentiated adipocytes with palmitic and palmitoleic acid to day 6 and assayed cell viability using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to manufacturer’s instructions. Relative cell viability was greater (P < 0.05) for palmitoleic acid-treated cells and controls compared with palmitic acid-treated cells (data not shown). Despite a reduction in cell viability, palmitic acid-treated cells had the greatest levels of 18:0 and 18:1 cis-9, which may indicate increased conversion of 16:0 into products with less lipotoxic properties.

Reduced lipogenesis in palmitoleic acid-treated cultures is consistent with our previous results when we supplemented 150 μM palmitoleic acid, which resulted in approximately 45 % reduction in lipogenesis between 0 and 24 h [7]. In the current study, both MUFA induced a repressive effect on lipogenesis of the same magnitude from 12 to 24 h at approximately 50 %. Therefore, this effect of palmitoleic acid supplementation on lipogenesis cannot be solely attributed to palmitoleic acid as its elongated form, cis-vaccenic acid, also displayed anti-lipogenic effects. Palmitic acid-treated cultures also showed a reduction in lipogenesis, but this effect is most likely related to cell viability in palmitic acid-treated cultures discussed previously. The genes that primarily involved in de novo lipogenesis are ACC and FASN. The gene expression results from these two genes are seemingly contradictory as MUFA treatments increase ACC mRNA and decrease FASN mRNA. However, ACC activity can also be regulated allosterically and through phosphorylation. Regulated by the transcription factor SREBP, FASN gene expression was reduced by approximately 40 % in MUFA treatments, similar to FSRlipogenesis results.

The isotopic measure of SCD1 activity, FSRdesaturation, showed a more dramatic effect of fatty acid supplementation on desaturase activity than the 18:1 cis-9/18:0 desaturation index results. The reduction in desaturase activity due to palmitoleic acid was approximately 100 % from 0 to 12 h and approximately 60 % from 12 to 24 h. By contrast, palmitoleic acid reduced the desaturation index by less than 25 % compared with controls (Table 1). The FSRdesaturation also showed a reduction in SCD1 activity by approximately 15 % from 12 to 24 h due to cis-vaccenic acid (Fig. 3), whereas the desaturation index for cis-vaccenic acid-treated cells was not different from controls. Therefore, the FSRdesaturation is a more sensitive measurement of SCD1 activity than the 18:1 cis-9/18:0 desaturation index when fatty acids are supplemented to adipocyte cultures.

Transcription of SCD1 mRNA is controlled primarily by SREBP, which binds to the sterol response element in the promoter region of the SCD1 gene [27, 28]. The promoter region of the bovine SCD1 gene contains a fat specific element, PUFA response element, and SREBP-response region [29]. In addition to PUFA, expression of SCD1 is also regulated by its products and not by the availability of its substrates [29]. For instance, the addition of 18:1 cis-9 and cis-9, trans-11 CLA reduced SCD promoter activity, but 18:1 trans-11 did not [29]. In this study, palmitoleic acid impacts SCD1 activity based on both the FSRdesaturase and desaturase index in addition to gene expression; whereas, cis-vaccenic acid induces a minimal response in rate of desaturation with no effect on the desaturation index or gene expression. In addition, palmitic acid increases the rate of desaturation with no effect on the desaturation index.

Several elongase enzymes are present in mammalian cells and are responsible for adding acetyl-CoA molecules to the carboxylic acid end of a fatty acid hydrocarbon skeleton. Seven elongases have been identified and given the designation of ELOVL for elongation of very long fatty acids (ELOVL 1–7; 30). Elongases can be divided into two groups based on substrate preference: (a) elongation of saturated and monounsatured fatty acids (ELOVL1, 3, and 6) and (b) elongation of polyunsaturated fatty acids (ELOVL2, 4, 5, and 7; 30, 31). ELOVL1 appears to play a role in elongation of very long chain saturated fatty acids (up to 26C) for sphingolipid formation and maintenance of membrane integrity [30]. ELOVL1 is expression in lung, brain, kidney and heart but barely detectable in live, brown adipose tissue, and skin [32]. ELOVL2 has been shown to elongate arachidonic acid and long-chain (20C or greater) omega-3 fatty acids in transfected yeast and mammalian cells but no activity was demonstrated for saturated or monounsaturated fatty acids [31]. Expression of ELOVL3 was only detected in skin [32]; however, others have reported strong correlations with ELOVL3 and brown adipose tissues [31]. ELOVL4 is specifically expressed in human retina where it is believed to have a role in elongation of docosahexaenoic (C22:6) acid [31, 32]. ELOVL5 is involved in the elongation of PUFA 18–20C but cannot elongate beyond 22C [31]. ELOVL5 expression is detected all tissues [32]. ELOVL6 is responsible for the elongation of saturated fatty acids 12–16C to 18C and cannot elongate beyond 18C. ELOVL6 is ubiquitously expressed, especially in tissues with high lipid levels like adipose tissue, liver and brain [31, 32]. ELOVL7 exhibits activity for acyl-CoAs of 16–20C with highest activity for 18:3n-3 and 18:3n-6 [33].

In this study, supplementation of palmitoleic acid to adipocyte cultures resulted in increased levels of 18:1 cis-11 and also 20:1 cis-13 to a more limited extent. Likewise, supplementation of C18:1 cis-11 to adipocyte cultures increased levels of C20:1 cis-13. As FASN is not capable of synthesizing fatty acids greater than 16C in length, acetate molecules were most likely added to 16:1 cis-9 by an elongase. Others [11] have proposed that 16:1 cis-9 can be elongated into 18:1 cis-11 and potentially elongated further into a 20C fatty acid, 20:1 cis-13, but this study is the first to confirm this pathway through the use of stable isotope technology. Elongation of MUFA is reportedly carried out by ELOVL6 but ELOVL5 has been reported to also have some activity for this reaction as well, especially in the absence of ELOVL6 [34]. Green et al. [34] proposed that ELOVL6 preferentially elongates C16 to C18, which then can be further desaturated to C18:1 cis-9, and that ELOVL5 preferentially elongates C16:1 cis-9 to C18:1 cis-11 based on knockdown and overexpression studies in rat insulinoma cell lines (INS-1). These authors propose a coordinated regulation of the elongases in order to balance the levels of C16:1 cis-9, C18:1 cis-11 and C18:1 cis-9 in the cell. Due to increased fatty acid elongation products (18:1 cis-11 and 20:1 cis-13) observed in our study, we know elongation occurs in palmitoleic and cis-vaccenic acid-treated cultures. However, mRNA expression of ELOVL6 was reduced in both palmitoleic and cis-11 vaccenic acid supplemented cells compared to controls; whereas ELOVL5 mRNA expression was not altered with MUFA supplementation. Transcription factors that regulate elongases include SREBP-1c for ELOVL6 [35] and peroxisome proliferator-activated receptor-α (PPARα) for both ELOVL5 and 6 [36]. Our results would indicate that transcription levels may not be directly related to enzyme activity and that further research is needed to examine regulation of elongases in adipocytes.

Carnitine palmitoyl-transferase 1A is responsible for transporting long chain fatty acids through the outer mitochondrial membrane for the purpose of β-oxidation in the mitochondrial matrix. In ruminants, CPT1A is expressed in numerous tissues including adipose tissue in contrast to a more restricted tissue abundance in monogastrics [37]. Inhibition of CPT1A is primarily attributed to malonyl-CoA, the product of the ACC reaction, in monogastric species [38]. Price et al. [37] found N-terminal sequence differences in ovine CPT1 that alter enzyme kinetics for certain substrates, which differs from that of the rat. All fatty acid treatments in this study resulted in increased CPT1A mRNA, which may lead to increase β-oxidation and ATP production.

In this study, we confirmed 18:1 cis-11 and 20:1 cis-13 as products of palmitoleic acid elongation in vitro and that cis-9, cis-11 CLA can also be produced through elongation and desaturation of palmitoleic acid. Supplementation of palmitoleic and cis-vaccenic acids reduces lipogenesis and FASN gene expression. However, palmitoleic acid is largely responsible for reducing SCD1 gene expression and desaturation activity with cis-vaccenic acid contributing a slight reduction in SCD1 activity. A schematic representation of fatty acid biosynthesis and regulation is depicted in Fig. 5. Overall, palmitoleic acid supplementation is directly responsible for decreased desaturation and jointly responsible, with its elongation products, for reduced lipogenesis. However, more research is still needed to determine the mode of action and potential application of these fatty acids to modulate lipogenesis in vivo.

Fig. 5
figure 5

Proposed fatty acid metabolism and biosynthesis pathways in bovine adipocytes supplemented with palmitoleic acid (16:1 cis-9). Dotted lines depict inhibitory effects

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