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Article

Effect of Fatty Acid Mixture on the Hyperplastic and Hypertrophic Growth of Subcutaneous Bovine Stromal Vascular Fraction Cells In Vitro

by
Aliute N. S. Udoka
and
Susan K. Duckett
*
Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634, USA
*
Author to whom correspondence should be addressed.
Lipidology 2025, 2(2), 8; https://doi.org/10.3390/lipidology2020008
Submission received: 13 January 2025 / Revised: 31 March 2025 / Accepted: 1 April 2025 / Published: 7 April 2025
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Abstract

:
Background: Adipose tissue growth follows a biphasic process involving both cellular hyperplasia (an increase in adipocyte number) and hypertrophy (an increase in adipocyte size). Rumen-protected fatty acid supplements have been utilized to alter fat deposition, modify the fatty acid composition of meat, and reduce methane emissions. However, limited research has explored how different fatty acid mixtures influence adipose tissue’s biphasic growth phases. Methods: The objectives of this study are to investigate the effects of fatty acid mixtures (seven different mixtures) on: (1) hyperplasia of undifferentiated stromal vascular fraction (SVF) cells, or (2) hypertrophy of chemically differentiated SVF cells isolated from subcutaneous adipocytes of finished steers. Results: Mixtures containing palmitic and linoleic acids stimulated hyperplasia, enhancing the proliferation of undifferentiated SVF cells, while mixtures with oleic acid (50%) predominantly promoted hypertrophy, driving lipid accumulation and adipocyte maturation. Conversely, mixtures composed solely of saturated fatty acids (50% palmitic and 50% stearic acids) exhibited a profound inhibitory effect on both hyperplasia and hypertrophy, underscoring the importance of fatty acid composition in regulating adipogenesis. Conclusions: These findings demonstrate that the composition of fatty acid mixtures directly influences adipogenesis and lipogenesis in vitro, highlighting their potential role in designing tailored rumen-protected supplements for modifying fat deposition in livestock.

1. Introduction

Adipose tissue is a dynamic and complex tissue that primarily serves as an energy storage reservoir in the body. While energy storage is one of its main functions, in recent years, adipose tissue has been redefined as a tissue that can have a significant influence on overall body metabolism and function [1]. Adipose tissue tends to deposit in specific regions of the body, termed depots, and this deposition is suspected to be a combination of both hypertrophy and hyperplasia of the adipocyte. However, these two processes require additional investigation to uncover how to independently manage these two growth phases that occur under the broad cover of adipogenesis [2]. Overall, efficient fat deposition can directly influence meat palatability, the fatty acid composition of meat, and overall carcass profits [3,4,5,6]. As our consumer behavior begins to transition into wanting a less saturated meat product with the goal of improving human health, new methods for altering ruminant tissue fatty acid composition will be necessary [7,8]. Fatty acids undergo significant biohydrogenation in the rumen, which impacts the overall fatty acid composition of the tissues and places the wants of the consumer on hold [9]. Calcium salts of palm fatty acid distillates are widely utilized in the dairy industry to protect fatty acids from ruminal biohydrogenation and impact overall milk production [10,11,12]. There is limited use of these rumen-protected fat supplements within the beef cattle industry; however, research has shown that these supplements may alter fat composition and deposition [13,14].
In the cattle industry, adipocyte hyperplasia and hypertrophy hold immense significance. They can directly affect meat quality and composition, particularly intramuscular adipocyte hyperplasia, which is positively correlated with marbling, a sought-after trait in beef production [15]. In order to efficiently study the process of hyperplasia, in vitro experiments have been instrumental in understanding the effects of fatty acid supplementation on adipose tissue development. Researchers have explored the impact of various individual fatty acids on intramuscular adipocyte hyperplasia and marbling. In vitro research with individual fatty acids has shown that lipogenesis rates and composition of adipocytes can be altered when individual fatty acids like myristic, palmitic or stearic [16], palmitoleic acid [17], oleic acid [18], and linoleic acid [16] are supplemented, but less is known about how mixtures of these fatty acids at varying concentrations alter adipogenesis and lipogenesis. Rumen-protected fats are available commercially with mixtures of fatty acids depending on the source of oil used for the calcium salt protection process. The objectives of this study are to investigate the effects of fatty acid mixtures on: (1) hyperplasia of undifferentiated SVF cells, or (2) hypertrophy of chemically differentiated SVF cells isolated from subcutaneous adipocytes of finished steers. We hypothesize that certain fatty acid mixtures will enhance SVF hyperplasia and/or hypertrophy to alter adipocyte number and fatty acid content in vitro.

2. Materials and Methods

2.1. Stromal Vascular Fraction Isolation

All animal and experimental protocols were approved by the Clemson University Institutional Animal Care and Use Committee (AUP2021-0045). Subcutaneous adipose tissue was collected from Angus-cross yearling steers (n = 4) that were finished on a high concentrate diet for 124 d (16.6 mo.; BW = 636 kg; avg. Choice) at the Clemson University Piedmont Research and Education Center. Steers were transported to the Clemson University Meat Lab for slaughter, where subcutaneous adipose tissue that covered the longissimus muscle (LM) from 10th to 12th rib was collected and placed into a 5X antibiotic/antimycotic (AB/AM; Gibco ThermoFisher Scientific 15070063, Waltham, MA, USA) and Hanks Balanced Salt Solution (HBSS; Hyclone SH30268.02, Logan, UT, USA) were used for transport to the research laboratory for cell isolation. Subcutaneous adipose tissue was digested, and stromal vascular fraction (SVF) cells were isolated, according to Hirai et al. [19]. SVF cell populations from an individual animal were isolated, and then SVF from two different steers were pooled to provide two combined SVF cell populations for replicated experiments, as described below. The same SVF cell populations were used for experiment 1, hyperplasia, and experiment 2, hypertrophy.

2.2. Experiment 1—Hyperplasia

Replicated SVF cell populations (n = 4) were propagated in basal media (Dulbecco’s Modified Eagle Medium/Nutrient mixture F-12 [DMEM/F12] with 10% fetal bovine serum, 1% AB/AM, 10 mM N-2-hydroxyethylpiperazine-N-2-ethane Sulonic acid [HEPES]). After two passages (F2), SVF cell populations were subsequently seeded into replicated ventilated T25 flasks at a density of 2500 cells per cm2 for each mixture. Once seeded, the cells were allotted 24 h to fully plate down prior to any further manipulation. At the 24 h mark, the basal media was removed, the cells were washed with 1X phosphate-buffered saline (1X PBS) and were treated with one of the eight different fatty acid mixture treatments (200 uM total; see Table 1 for specific mixtures) for a period of four days. Saturated fatty acids were bound to 2% fatty-acid-free BSA to ensure solubility in media [20]. For this study, two technical replicates of each SV population were sampled per treatment. Over the four-day treatment period, the treatment media were changed daily after a wash with 1X PBS. Flasks (T25) were harvested for RNA utilizing 1 mL of Trizol Reagent (ThermoFisher Scientific 15596018, Waltham, MA, USA) or for fatty acid composition utilizing 1 mL trypsin (0.25% Trypsin-2.21 uM EDTA, Corning, 25-053-CI) at the end of the four-day period. Multiple images were taken using the Cytation (BioTek, Agilent, Santa Clara, CA, USA) from several locations in the T25 flask on d 1 and d 4 of the study to determine the cell number (hyperplasia; 10 ug/mL Hoechst dye; ThermoFisher Scientific #62249, Waltham, MA, USA) and accumulation of neutral lipids (hypertrophy; 1:1000 LipidTOX dye; ThermoFisher Scientific H34477, Waltham, MA, USA) over time.

2.3. Experiment 2—Hypertrophy

Replicated SVF cell populations (n = 4) were propagated in basal media (DMEM/F12 with 10% fetal bovine serum, 1% AB/AM, 10mM HEPES) for two passages (F2) and subsequently seeded into ventilated T25 flasks at a density of 2500 per cm2. At 60% confluence, the cell cultures were differentiated for 2 d, according to Liu et al. [21]. Following differentiation, the cell cultures were placed into a post-differentiation media (DMEM/F12, 10% fetal bovine serum, 1 ug/mL insulin, 1% AB/AM) and were treated with one of eight different fatty acid mixtures (200 uM total; see Table 1 for specific mixtures) for a period of four days. The treatment media were changed daily after a wash with 1X phosphate-buffered saline (1X PBS). The fatty acid treatments consisted of varying combinations of palmitic, stearic, oleic, and linoleic acid (Table 1). Saturated fatty acids were bound to 2% fatty-acid-free bovine serum albumin (BSA) to ensure solubility in media [20]. For this particular study, two technical replicates for each SVF cell population were sampled per treatment. T25 flasks were harvested for RNA utilizing 1 mL of Trizol Reagent (ThermoFisher Scientific 15596018, Waltham, MA, USA) or for fatty acid composition utilizing 1 mL trypsin at the end of the four-day period. Multiple images were taken using the Cytation (BioTek, Agilent, Santa Clara, CA, USA) from several locations in the T25 flask on d 1 and d 4 of the study to determine cell number (hyperplasia; 10 ug/mL Hoechst dye; ThermoFisher Scientific #62249, Waltham, MA, USA) and accumulation of neutral lipids (hypertrophy; 1:1000 LipidTOX dye; ThermoFisher Scientific H34477, Waltham, MA, USA) over time.2.4. mRNA Isolation
Total RNA was extracted using the TriZol (ThermoFisher Scientific 15596018, Waltham, MA, USA) isolation method in combination with the Norgen RNA Clean-up and Concentration Kit (Norgen Biotek Corporation #43200, Thorold, ON, USA) according to the manufacturer’s specifications. Isolated RNA was quantified using a Nanodrop 1 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), followed by storage at −80 °C for downstream applications.

2.4. mRNA qPCR

Isolated RNA was converted to cDNA utilizing the qScript cDNA (Quanta 95048, Beverly, MA, USA) supermix according to the manufacturer’s instructions. cDNA was used to analyze fatty acid binding protein-4 (FABP4), stearoyl-coA desaturase-1 (SCD-1), fatty acid synthase (FASN), peroxisome proliferator-activated receptor gamma (PPARy), sterol regulatory element-binding protein (SREBP-1c), SREBP cleavage-activating protein gene (SCAP), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) and platelet-derived factor alpha (PDGFRA) via quantitative real-time RT-PCR (qPCR) using the PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA) and the QuantStudio 3 (Applied Biosystems by Thermo Fisher Scientific). The samples were run under the following protocol: 50 °C for 2 min, 95 °C for 2 min, followed by 95 °C for 15 s and 60 °C for 1 min, repeated for 40 cycles. Bos taurus glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 3 subunit K (EIF3K), and ubiquitously expressed prefoldin-like chaperone (UXT) were evaluated as housekeeping genes. The geometric mean of EIF3K and UXT was selected as the most stable gene via RefFinder. All samples were normalized (ΔCT= Gene CT − Geometric Mean CT). Relative gene expression levels were calculated utilizing the ΔΔCT method and were analyzed in Log2 fold change [22]. Gene expression is presented as a Log2 fold change from the control treatment.

2.5. Fatty Acid Analysis

The samples were extracted with 2:1 chloroform to methanol, according to Folch et al. [23]. The lipid-containing chloroform layer was subsequently dried under nitrogen gas, followed by transmethylation, according to Park and Goins [24]. Fatty acid methyl esters (FAME) were analyzed using Agilent 6850 (Agilent, San Fernando, CA, USA) gas chromatography equipped with an Agilent 7673A (Agilent) automatic sampler. Separations were achieved with a DB-FastFAME (Agilent J&W GC column, Agilent Tech., Santa Clara, CA, USA) capillary column (30 m, 0.25 um i.d., 0.25 μm film thickness). Column oven temperature rose from 150 °C to 225 °C at a rate of 2 °C/min and was held for 5 min. Both the injector and detector were sustained at 250 °C. The samples were injected at a volume of 1 μL. Hydrogen was used as the carrier gas, with a flow rate of 1 mL/min and a split ratio of 10:1. Fatty acids were identified by evaluating the retention times of known standards (Matreya, Pleasant Gap, PA, USA: GLC10, GLC50, GLC70, GLC80, 18912, Sigma, St. Louis, MO, USA; Supelco, Bellefonte, PA, USA: C16:1 cis-9, 18917, PUFA3, C18:1 trans-11). Quantification of fatty acids in each sample was accomplished by adding an internal standard, methyl tricosanoic (C23:0), during methylation and expressed on a gravimetric basis (ug) per flask (T25).

2.6. Statistical Analysis

Data were analyzed using the GLM procedure of SAS (SAS 9.4, SAS Inst. Inc., Cary, NC, USA) with the fatty acid mixture in the model. Statistical significance was set at p ≤ 0.05 and trends at p ≤ 0.10.

3. Results

3.1. Hyperplasia

The cell number differed (p < 0.05) between the different fatty acid treatments at the end of the 4 d treatment period (Figure 1A). The fully saturated treatment (M5: 50% C16:0/50% C18:0) had the lowest (p < 0.05) cell number in comparison to all other treatments, including the CON treatment. M7 (50% C16:0/50% 18:2) generated the highest (p < 0.05) cell number over the four-day period. M6 (75% C16:0/25% C18:2) and M8 (75% C18:1/25% C18:2) had an increased (p < 0.05) cell number compared to the control, but their cell number remained below that of M7. M1 (50% C16:0/50% C18:1) did not alter (p > 0.05) the cell number. M2 (50% C18:0/50% C18:1), M3 (75% C16:0/25% C18:1), and M4 (75% C18:0/25% C18:1) decreased (p < 0.05) the cell number in comparison to the CON.
Although fatty acid supplementation concentration was controlled, the total fatty acid content of SVF differed (p < 0.05) between the treatments (Figure 1B). M5 (50% C 16:0/50% C 18:0) did not differ (p > 0.05) from the control. M8 (75% C18:1/25% C18:2) had the highest total fatty acid content in comparison to all other treatments. M7 (50% C16:0/50% C18:2) with linoleic acid had a greater (p < 0.05) fatty acid content than M1 (50% C16:0/50% C18:1), which contained oleic acid. M1 had a higher (p < 0.05) fatty acid content than M6, which contained higher palmitic acid (75%) and lower linolenic acid (25%) concentrations. M6’s (75% C16:0/25% C18:2) fatty acid content was greater (p < 0.05) than those of M2 (50% C18:0/50% C18:1), M3 (75% C16:0/25% C18:1), and M4 (C75% C18:1/25% C18:1), which were greater (p < 0.05) than CON and M5.
Fatty acid composition also differed (p < 0.05) depending upon treatment and seemed to generally mimic treatment (Table 2). The palmitic acid (C16:0) content of M2 (50% C18:0/50% C18:1), M4 (C75% C18:1/25% C18:1), M5 (50% C 16:0/50% C 18:0), and M8 (75% C18:1/25% C18:2) did not differ (p > 0.05) from that of CON. The C16:0 content did not differ (p > 0.05) between the M1 (50% C16:0/50% C18:1), M3 (75% C16:0/25% C18:1), M6 (75% C16:0/25% C18:2), and M7 (50% C16:0/50% C18:2) treatments. The stearic acid (18:0) content was greater in the M2, M4, and M8 cultures in comparison to CON. The C18:0 content in the CON, M1, M3, M5, M6, and M7 treatments did not differ (p > 0.05). The oleic acid (C18:1cis-9) content varied widely across the treatments. CON had low (p < 0.05) levels of C18:1cis-9, while M8 had the highest (p < 0.05) levels of fatty acid. M5, M6, and M7 had C18:1cis-9 levels comparable (p > 0.05) to those of CON. Treatments M1 and M2 had the next highest oleic contents, followed by M3 and M4. The linoleic acid (C18:2) content was relatively low in most treatments, aside from M6, M7, and M8, in comparison to CON and the other treatments (p < 0.05).
SCAP, FASN, PGC1a and PPARy mRNA expression did not differ (p < 0.05) across treatments. SCD-1’s relative expression in the M1 (50% C16:0/50% C18:1), M6 (75% C16:0/25% C18:2), M7 (50% C16:0/50% C18:2), and M8 (75% C18:1/25% C18:2) treatments did not differ (p > 0.05) from that in the CON treatment (Figure 2A). Treatments M2 (50% C18:0/50% C18:1), M3 (75% C16:0/25% C18:1), and M4 (C75% C18:1/25% C18:1) had an elevated (p < 0.05) SCD-1 expression compared to that of CON, but did not differ (p > 0.05) from that of the M1, M6, M7, and M8 treatments. The treatment M5 (50% C16:0/50% C18:0) had the highest (p < 0.05) SCD-1 relative expression relative to all other treatments. All treatments had an elevated (p < 0.05) FABP4 relative gene expression in comparison to that of CON (Figure 2B). M5 had the next highest FABP4 expression compared to that of CON. The relative expression of the M5 treatment did not differ from that of M2, M4, and M6 treatments. M8 had the highest FABP4 relative expression, although it did not differ from the FABP4 relative expression of M1, M3, and M7. PDGFRa relative expression was down-regulated (p < 0.05) across all treatments in comparison to CON (Figure 2C). Treatment M5 had the lowest PDGFRa relative expression, but its expression did not differ from that of M1. The relative expression of treatment M1 also did not differ (p > 0.05) from that of M2, M3, M4, M6, and M7. The PDGFRa relative expression of M8 did not differ from that of treatments M2, M3, M4, M6, and M7. The SREBP-1c relative expression of treatments M1, M2, M3, M6, M7, and M8 did not differ (p > 0.05) from that of the CON treatment (Figure 2D). M4 had a lower (p < 0.05) SREBP-1c relative expression than M8, but its expression also did not differ (p > 0.05) from that of the CON, M1, M2, M3, M6, and M7 treatments. M5 had the lowest (p < 0.05) SREBP-1c relative expression.

3.2. Hypertrophy

The adipocyte cell number was greater (p < 0.05) for all mixtures containing linoleic acid (M6, M7, and M8) compared to all other treatments (Figure 3A). The adipocyte cell number for CON, M1, M2, and M3 was greater (p < 0.05) than for the M5 fully saturated mixture. The adipocyte cell number for M4 did not differ from that for CON, M1, M2, M3 or M5.
The fatty acid content differed (p < 0.05) depending upon treatment (Figure 3B). The total fatty acid content was highest (p < 0.05) for M1 (50% C16:0/50% C18:1) and lowest (p < 0.05) for M5 (50% C16:0/50% C18:0). The fatty acid content for M2 (50% C18:0/50% C18:1) was greater (p < 0.05) than M7’s (50% C16:0/50% C18:2) and M8 (75% C18:1/25% C18:2), which were greater than M6 (75% C16:0/25% C18:2). M3 (75% C16:0/25% C18:1) and M4 (75% C18:0/25% C18:1) had a greater fatty acid content than CON, which was greater than M5’s (50% C16/50% C18:0).
The fatty acid composition differed depending upon treatment (p < 0.05) across key fatty acids (Table 3). The palmitic acid (C16:0) content varied substantially (p < 0.05) between the treatments. M1 (50% C16:0/50% C18:1) displayed the highest concentration of palmitic acid, followed by M6 (75% C16:0/25% C18:2), M3 (75% C16:0/25% C18:1), and M7 (50% C16:0/50% C18:2; p < 0.05). The palmitic acid content in treatments M2 (50% C18:0/50% C18:1), M4 (75% C18:0/25% C18:1), and M5 (50% C16/50% C18:0) did not differ (p > 0.05) from that of CON. Treatments M2, M4, M5, and M8, along with CON, had much lower levels (p < 0.05) of palmitic acid. The palmitoleic acid (C16:1) content displayed very low levels, and the content amount did not differ (p > 0.05) across the treatments. M5 had no detectable levels of palmitoleic acid. The stearic acid (C18:0) content was notably greater (p < 0.05) in M2, followed by M4, compared to CON and the other treatments. The M3, M5, M6, M7 and M8 treatments had lower levels of stearic acid, while M1 and CON displayed intermediate interactions. The oleic acid (C18:1cis-9) amounts were greater (p < 0.05) in treatments M1, M2, and M8. Treatments M3, M4, and M7 exhibited moderate oleic acid quantities, followed by CON, M5, and M6, with the lowest oleic acid amount. Linoleic acid (C18:2) was significantly elevated (p < 0.05) in the M7, M8 and M6 samples when compared to the CON. CON and treatments M1, M2, M3, M4, and M5 displayed relatively minimal linoleic acid amounts.
The relative SCD-1 expression of treatments M7 (50% C16:0/50% C18:2) and M1 (50% C16:0/50% C18:1) did not differ (p > 0.05) from that of CON. The relative SCD-1 expression of M2 (50% C18:0/50% C18:1), M4 (75% C18:0/25% C18:1), and M6 (75% C16:0/25% C18:2) did not differ (p > 0.05) from that of M7 or M1 (Figure 4A). M5 (50% C16/50% C18:0) had the highest (p < 0.05) relative expression, but it did not differ (p > 0.05) from the M3 treatment’s relative SCD-1 expression. The M3 treatment did not differ (p > 0.05) from tM2, M4, and M8 treatments as regards relative SCD-1 expression. Relative FABP4 expression was elevated across all treatments in comparison to the CON treatment (Figure 4B). M3 had the highest (p < 0.05) relative FABP4 expression, but it did not differ (p > 0.05) from that of the M1 or M7 treatments. The M1 and M7 treatments also did not differ (p > 0.05) from that of the M2, M4, M5, M6, and M8. Relative PPARy expression was lowest (p < 0.05) in M5 in comparison to all other treatments. Relative PPARy expression did not differ (p > 0.05) across all other treatments (Figure 4C). Relative PGC-1a expression of CON did not differ (p > 0.05) from all treatments except for M5. M5 possessed the lowest (p < 0.05) relative PGC-1a expression, although it did not differ (p > 0.05) from M8’s relative expression (Figure 4D). Treatment M8’s relative PGC1a expression also differed (p < 0.05) from that of the M1 and M3 treatments. The relative FASN expression of M1, M4, M6, and M7 did not differ (p > 0.05) from that of the CON treatment (Figure 4E). M3’s relative FASN expression did not differ (p < 0.05) from the numerically higher expressing M5 and M8 or the numerically lower expressing M2, M4, M6, and M7. M3’s relative FASN expression did differ (p < 0.05) from that of the M1 and CON treatments. The relative FASN expression of M5 and M8 did not differ (p > 0.05). The mRNA relative expression of PDGFRa, SCAP or SREBP1c did not differ between the fatty acid mixtures.

4. Discussion

This study examines how different fatty acid mixtures alter two distinct adipocyte growth phases, hyperplasia and hypertrophy. Mixtures containing linoleic acid at 25 to 50% of total supplemental fatty acids (M6, M7, M8) enhanced SVF proliferation in both hyperplasia and hypertrophy experiments. The total number of cells was greatest for the mixtures containing linoleic acid at 50% (M7) for hyperplasia, and at any level (M6, M7, M8) for hypertrophy experiments after 4 d of supplementation. In the hyperplasia experiment, the linoleic acid content of the preadipocytes was greatest for M7 (50% C18:2/50% C16:0); however, the total fatty acid content was greatest for M8 (25% C18:2/75% C18:1), followed by M7. These increases in proliferation with linoleic acid occurred without changes in the expression of known adipogenic (PPARg, PDGFRa) or lipogenic (SCD1, FABP4, SREBP1c) genes compared to other supplemental mixtures. However, despite these transcriptional observations, the presence of lipid accumulation, visualized via lipidTOX staining across both hyperplasia and hypertrophy experiments, provides compelling evidence of adipogenic differentiation. Furthermore, the induction and expression of FABP4, a late-stage adipocyte marker, supports the idea that these cells have progressed beyond proliferation and into functional differentiation, even in the absence of early gene expression changes. It is worth noting that the data may suggest that fatty-acid-induced differentiation may involve non-canonical pathways or that post-transcriptional regulation may not be captured by early adipogenic markers alone. Others [25] have shown that linoleic acid supplementation to 3T3-L1 preadipocyte can prevent differentiation, whereas another study [26] suggest that linoleic acid can stimulate differentiation without changes in known lipogenic genes. This discrepancy across studies may stem from differences in cell models, species-specific responses, or dose-dependent effects of linoleic acid; our findings support a proliferative response. The down-regulation of relative PDGFRA expression for all mixtures in the hyperplasia experiment would support the idea that the SVF cells successfully differentiate into mature adipocytes in order to store the fatty acids being supplied to them. The PDGFRA gene has been related to fibro-adipocyte progenitor maintenance and is known to contribute to white adipose tissue (WAT) formation in humans [27]. Researchers have shown that mature adipocytes can dedifferentiate during in vitro culture and return to a proliferative state [15,28,29]. Wei et al. [29] also suggested that this increase in proliferation may be related to the purity of isolation and SVF cells that contain progenitor cells that remain proliferative and active when stimulated with linoleic acid in this case.
In contrast, mixtures containing 50 to 75% oleic acid stimulated hypertrophy or lipid filling during hyperplasia (M8, 75% C18:1) and hypertrophy (M1 and M2, 50% C18:1) experiments. These changes in total lipid content occurred when the oleic acid content was above 38 ug/flask in culture. Yanting et al. [16] also reported that lipid content of bovine adipocytes in vitro was greatest for oleic and lowest for palmitic acid supplementation. Tian et al. [30] found that oleic acid supplementation at varying concentrations stimulated intramuscular preadipocyte differentiation in goats. Mixture 8 (75% C18:1 and 25% C18:2) up-regulated FABP4 expression compared to other mixtures with a high stearic acid content (M4, M5) during hyperplasia. Mixture 3 (75% C16 and 25% C18:1) up-regulated FABP4 expression compared to all other mixtures during hypertrophy. FABP4, or AP2, is a fat-specific protein that comes from a family of homologous small cytoplasmic proteins that aid in the transport of fatty acids. Specifically, FABP4 is highly expressed in macrophages, as well as adipose tissue [31,32]. Given FABP4’s role in fatty acid transport and metabolism, these findings suggest that oleic acid may enhance the lipid-handling capacity of adipocytes, which is reflected in both gene expression and lipidTOX staining. An elevated FABP4 expression can be indicative of increases in fatty acid uptake, transport, and metabolism [33]. A recent study suggested that FABP4 stores may regulate both adipocyte size as well as adipocyte recruitment [34]. All hyperplastic and hypertrophic treatments displayed a higher expression of FABP4 in comparison to the control, potentially indicating that there was an increase in fatty acid uptake in wells containing fatty acid supplementation treatments. This would align with the current literature, where overexpression of FABP4 in vitro was suggested to be indicative of increased fatty acid transport, uptake, and storage [35,36]. The visual increase in red lipidTOX stain across all treated wells visually could be indicative of increased lipid storage and potential lipogenic activity taking place. In addition, it has been proposed that the type of fatty acid may play a larger role than the total fatty acid content in the expression and activity of fatty acid-binding proteins [37].
Interestingly, mixtures composed exclusively of saturated fatty acids (50% palmitic and 50% stearic acids) showed a profound inhibitory effect on both hyperplasia and hypertrophy. The saturated fatty acid mixture (M5) had the lowest number of cells and total lipid content in both experiments compared to all mixtures, indicating potential apoptosis of the cells. SCD-1 and SREBP-1c mRNA expression was up-regulated and down-regulated, respectively, in the saturated fatty acid mixture (M5) in attempts to desaturate and restore cell viability. Stearoyl-CoA desaturase-1 is a key lipogenic enzyme that catalyzes the conversion of saturated fatty acids—such as palmitic and stearic acids—into their monounsaturated counterparts, including palmitoleic and oleic acid. This desaturation step is critical for facilitating lipid droplet formation and preventing lipotoxicity. The observed up-regulation of SCD-1 may therefore represent the intrinsic defense mechanism against saturated-fat-induced stress. Collins et al. [38] found that exogenous palmitic acid supplementation (0–200 uM) up-regulated de novo lipogenesis and increased desaturation (SCD-1) and elongation in human preadipocytes. Das et al. [39] reported that endoplasmic reticulum stress is induced by palmitic acid (0.25 or 1.0 mM) supplementation but not by oleic acid supplementation in human HepG2 cells. Others [40] have shown that palmitate supplementation to 3T3-L1 cells or rat primary preadipocytes induces apoptosis. The expression of SCD-1 and FASN was up-regulated, and PPARy and PGC-1a were down-regulated in the M5 SVF culture. This could suggest a suppressed metabolic response and potential metabolic dysfunction. Excessive lipid accumulation coupled with impaired oxidative capacity could lead to events such as cell stress and cell death. These findings are particularly noteworthy as they highlight the detrimental impact of certain fatty acid profiles on adipose tissue development. This negative effect could stem from metabolic stress or altered cellular signaling pathways.
Additionally, PPARy and PGC-1a were down-regulated in M5 (50% palmitic and 50% stearic acid) compared to all other mixtures, suggesting a coordinated response to regulate lipid storage and oxidation. As somewhat of a master regulator of adipogenesis and lipid metabolism, PPARy up-regulation could signify lipid uptake promotion and increased triglyceride storage, while PGC-1a is a transcriptional coactivator that interacts with PPARy and regulates the expression of genes involved in energy metabolism. The simultaneous activation of PPARy and PGC-1a could reflect an adaptive mechanism to balance lipid accumulation with oxidative metabolism, preventing lipotoxicity and maintaining energy efficiency. Fatty acid synthase (FASN) expression was up-regulated in M8 and M5 compared to the control during hypertrophy. FASN is a key enzyme in de novo lipogenesis, primarily by functioning as a catalyst in the conversion of malonyl-CoA into palmitate [41]. Additionally, mixtures containing stearic acid (M2, M4, M5) instead of palmitic acid appeared to limit adipocyte proliferation and, in some cases, lipid accumulation. However, the inclusion of an unsaturated fatty acid helped restore balance and prevent potential apoptosis or cell death. Collins et al. [38] found that exogenous palmitic acid supplementation (0–200 uM) up-regulated de novo lipogenesis, and increased desaturation (SCD-1) and elongation in human preadipocytes.

5. Conclusions

The supplementation of fatty acid mixtures significantly altered the biphasic growth phases of stromal vascular fraction in culture, with distinct effects observed between hyperplasia and hypertrophy. Mixtures containing palmitic and linoleic acids stimulated hyperplasia, enhancing the proliferation of undifferentiated SVF cells, while mixtures with oleic acid (50%) predominantly promoted hypertrophy, driving lipid accumulation and adipocyte maturation. Conversely, mixtures composed solely of saturated fatty acids (50% palmitic and 50% stearic acids) exhibited a profound inhibitory effect on both hyperplasia and hypertrophy, underscoring the importance of fatty acid composition in regulating adipogenesis. These findings demonstrate that the composition of fatty acid mixtures directly influences adipogenesis and lipogenesis in vitro, highlighting their potential role in designing tailored rumen-protected supplements for modifying fat deposition in livestock. As consumer demand shifts toward healthier, leaner meat products, understanding the differential effects of fatty acid mixtures on adipose tissue growth provides a foundation for optimizing beef production strategies using nutritional interventions. Future research should explore the in vivo applications of these mixtures to validate their effects on carcass quality, fatty acid composition, and overall production efficiency.

Author Contributions

Conceptualization, A.N.S.U. and S.K.D.; methodology, A.N.S.U. and S.K.D.; formal analysis, A.N.S.U. and S.K.D.; data curation, A.N.S.U.; writing—original draft preparation, A.N.S.U.; writing—review and editing, S.K.D.; funding acquisition, S.K.D. All authors have read and agreed to the published version of the manuscript.

Funding

Technical contribution number 7393 of the Clemson Experiment Station. This material is supported by NIFA/USDA, under project number SC-1700580. This work was supported by Volac Wilmar feed ingredient and Noble Herd.

Institutional Review Board Statement

All animal and experimental protocols were approved by Clemson University Institutional Animal Care and Use Committee (AUP2021-0045).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AB/AMantibiotic and antimycotic
Ctcycle threshold
DMEMDulbecco’s Modified Eagle Medium
EIF3Keukaryotic translation initiation factor 3 subunit K
FABP4fatty acid binding protein 4
FAMEfatty acid methyl esters
FASNfatty acid synthase
GAPDHGlyceraldehyde-3-phosphate dehydrogenase
GLUT4Glucose transporter type 4
HBSSHanks Balanced Salt Solution
HEPES4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid
LMlongissimus muscle
mRNAmessenger RNA
PBSphosphate-buffered saline
PDGFRAplatelet-derived growth factor receptor alpha
PGC-1aperoxisome proliferator-activated receptor gamma coactivator 1-alpha
PPARyperoxisome proliferator-activated receptor gamma
RT-qPCRquantitative reverse transcription polymerase chain reaction
SCAPsterol regulatory element-binding protein cleavage-activating protein
SCD1stearoyl coA desaturase
SREBP-1csterol regulatory element-binding protein 1c
SVstromal vascular
SVFstromal vascular fraction
UXTubiquitously expressed prefoldin-like chaperone
WATwhite adipose tissue

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Lipidology 02 00008 g001aLipidology 02 00008 g001b
Figure 1. Effect of exogenous fatty acid mixture (see Table 1) supplementation on cell number (A), total fatty acid content (B), and stained (C; blue = nuclei, Hoechst; red = neutral lipid, LipidTOX) stromal vascular fraction cells from bovine subcutaneous adipose tissue during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDEFGMeans with uncommon superscripts differ (p < 0.05). Scale bar = 1000 µm.
Figure 1. Effect of exogenous fatty acid mixture (see Table 1) supplementation on cell number (A), total fatty acid content (B), and stained (C; blue = nuclei, Hoechst; red = neutral lipid, LipidTOX) stromal vascular fraction cells from bovine subcutaneous adipose tissue during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDEFGMeans with uncommon superscripts differ (p < 0.05). Scale bar = 1000 µm.
Lipidology 02 00008 g001aLipidology 02 00008 g001b
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Figure 2. Effect of exogenous fatty acid mixture (see Table 1) on SCD-1 (A), FABP4 (B), PDGFRa (C), and SREBP-1c (D) gene expression of stromal vascular fraction from bovine subcutaneous adipose tissue during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCD Means with uncommon superscripts differ (p < 0.05).
Figure 2. Effect of exogenous fatty acid mixture (see Table 1) on SCD-1 (A), FABP4 (B), PDGFRa (C), and SREBP-1c (D) gene expression of stromal vascular fraction from bovine subcutaneous adipose tissue during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCD Means with uncommon superscripts differ (p < 0.05).
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Figure 3. Effect of exogenous fatty acid mixture (see Table 1) supplementation on cell number (A), total fatty acid content (B), and stained (C; blue = nuclei, Hoechst; red = neutral lipid, LipidTOX) bovine subcutaneous stromal vascular fraction cells after differentiation during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDEFGMeans with uncommon superscripts differ (p < 0.05). Scale bar = 1000 µm.
Figure 3. Effect of exogenous fatty acid mixture (see Table 1) supplementation on cell number (A), total fatty acid content (B), and stained (C; blue = nuclei, Hoechst; red = neutral lipid, LipidTOX) bovine subcutaneous stromal vascular fraction cells after differentiation during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDEFGMeans with uncommon superscripts differ (p < 0.05). Scale bar = 1000 µm.
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Figure 4. Effect of exogenous fatty acid mixture (see Table 1) SCD-1 (A), FABP4 (B), PPARy (C), PGC-1a (D), and FASN (E) gene expression composition of bovine subcutaneous stromal vascular fraction after differentiation during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDE Means with uncommon superscripts differ (p < 0.05).
Figure 4. Effect of exogenous fatty acid mixture (see Table 1) SCD-1 (A), FABP4 (B), PPARy (C), PGC-1a (D), and FASN (E) gene expression composition of bovine subcutaneous stromal vascular fraction after differentiation during culture for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12. ABCDE Means with uncommon superscripts differ (p < 0.05).
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Table 1. Fatty acid composition (% of total) of the mixtures supplemented at 200 uM for experiment 1 and experiment 2.
Table 1. Fatty acid composition (% of total) of the mixtures supplemented at 200 uM for experiment 1 and experiment 2.
TreatmentC16:0C18:0C18:1 cis-9C18:2 cis-9,12
CON0%0%0%0%
M150%0%50%0%
M20%50%50%0%
M375%0%25%0%
M40%75%25%0%
M550%50%0%0%
M675%0%0%25%
M750%0%0%50%
M80%0%75%25%
Table 2. Effect of exogenous fatty acid composition mixture (see Table 1) on fatty acid composition (ug/flask [T25]) of stromal vascular fraction cells collected from bovine subcutaneous adipose tissue after 4 d in culture. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12.
Table 2. Effect of exogenous fatty acid composition mixture (see Table 1) on fatty acid composition (ug/flask [T25]) of stromal vascular fraction cells collected from bovine subcutaneous adipose tissue after 4 d in culture. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12.
TreatmentCONM1M2M3M4M5M6M7M8SEp Value
C16:04.95 a27.03 b4.03 a29.17 b5.74 a5.42 a29.87 b32.30 b8.96 a3.790.0093
C18:05.33 a7.68 a19.93 b6.61 a22.32 b7.56 a6.95 a6.97 a22.75 b2.770.0153
C18:1 cis-92.44 a20.93 c22.66 c9.80 b10.44 b0.92 a2.21 a2.55 a46.73 d1.690.0001
C18:20.25 a0.54 a0.54 a0.55 a0.45 a0.12 a13.79 b33.57 d24.19 c0.68<0.0001
abcd Means with uncommon superscripts in the same row differ (p < 0.05).
Table 3. Effect of exogenous fatty acid composition mixture (see Table 1) on fatty acid composition (ug/flask [T25]) of bovine subcutaneous stromal vascular fraction cells differentiated and cultured for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12.
Table 3. Effect of exogenous fatty acid composition mixture (see Table 1) on fatty acid composition (ug/flask [T25]) of bovine subcutaneous stromal vascular fraction cells differentiated and cultured for 4 d. Mixtures: M1 = 50% C16:0/50% C18:1 cis-9; M2 = 50% C18:0/50% C18:1 cis-9; M3 = 75% C16:0/25% C18:1 cis-9; M4 = 75% C18:0/25% C18:1 cis-9; M5 = 50% C16:0/50% C18:0; M6 = 75% C16:0/25% C18:2 cis-9,12; M7 = 50% C16:0/50% C18:2 cis-9,12; M8 = 75% C18:1 cis-9/25% C18:2 cis-9,12.
TreatmentCONM1M2M3M4M5M6M7M8SEp Value
C16:016.67 a57.56 c13.40 a30.98 b9.63 a12.74 a36.82 b31.27 b9.05 a3.36<0.0001
C16:10.42 a0.33 a0.61 a0.19 a0.09 a0 a0.23 a0.33 a0.34 a0.210.6547
C18:011.46 ab15.26 b33.61 d5.49 a23.42 c9.49 ab8.40 ab10.08 ab6.08 a2.280.0002
C18:1 cis-95.82 ab38.09 c45.87 c8.79 ab13.29 b1.67 a2.39 a4.15 ab38.52 c3.22<0.0001
C18:21.18 a0.81 a0.94 a0.47 a0.48 a0.11 a12.62 b25.29 d21.03 c0.28<0.0001
abcd Means with uncommon superscripts in the same row differ (p < 0.05).
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Udoka, A.N.S.; Duckett, S.K. Effect of Fatty Acid Mixture on the Hyperplastic and Hypertrophic Growth of Subcutaneous Bovine Stromal Vascular Fraction Cells In Vitro. Lipidology 2025, 2, 8. https://doi.org/10.3390/lipidology2020008

AMA Style

Udoka ANS, Duckett SK. Effect of Fatty Acid Mixture on the Hyperplastic and Hypertrophic Growth of Subcutaneous Bovine Stromal Vascular Fraction Cells In Vitro. Lipidology. 2025; 2(2):8. https://doi.org/10.3390/lipidology2020008

Chicago/Turabian Style

Udoka, Aliute N. S., and Susan K. Duckett. 2025. "Effect of Fatty Acid Mixture on the Hyperplastic and Hypertrophic Growth of Subcutaneous Bovine Stromal Vascular Fraction Cells In Vitro" Lipidology 2, no. 2: 8. https://doi.org/10.3390/lipidology2020008

APA Style

Udoka, A. N. S., & Duckett, S. K. (2025). Effect of Fatty Acid Mixture on the Hyperplastic and Hypertrophic Growth of Subcutaneous Bovine Stromal Vascular Fraction Cells In Vitro. Lipidology, 2(2), 8. https://doi.org/10.3390/lipidology2020008

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