Targeting Inflammation with Dietary ω-3 Polyunsaturated Fatty Acids Improved Lipid Mobilization and Flux in Heat-Stressed Wether Lambs

TL;DR

This study shows that reducing inflammation with dietary ω-3 fatty acids can help heat-stressed lambs better mobilize lipids for energy.

Contribution

The study demonstrates that targeting inflammation with ω-3 fatty acids improves lipid mobilization in heat-stressed lambs.

Findings

Heat stress reduced lipid mobilization from visceral adipose tissue, which was partially or fully resolved by fish oil or ω-3 PUFA.

Fish oil and dexamethasone reduced muscle PPARα, suggesting less lipid utilization.

ω-3 PUFA improved muscle CD36 and PPARγ, indicating better lipid uptake, but did not restore intramuscular lipid content.

Abstract

Background/Objectives: Chronic heat stress impairs lipid mobilization from adipocytes, which reduces substrate availability for muscle metabolism. Systemic inflammation is a key facilitative response to heat stress, and we sought to determine if mitigating inflammation in heat-stressed wether lambs would improve lipid flux. Methods: Two cohorts of commercial feedlot lambs were heat stressed for 30 days. In study 1, heat-stressed lambs received dexamethasone injections every 3 days, fish oil capsules twice daily, or no intervention. In study 2, heat-stressed lambs received daily boluses of ω-3 polyunsaturated fatty acid Ca2+ salts (ω-3 PUFA) or no intervention. Results: In both studies, heat stress reduced ex vivo epinephrine-stimulated free fatty acid and glycerol mobilization from visceral adipose tissue. These deficits were partially resolved by fish oil and fully resolved by ω-3 PUFA. In study 1, fish oil recovered heat stress-induced deficits in circulating triglycerides and HDL-cholesterol but not in circulating free fatty acids. Fish oil and dexamethasone resolved the increase in muscle PPARα, indicating less lipid utilization for metabolism. In study 2, ω-3 PUFA resolved heat stress-induced deficits in muscle CD36 and PPARγ, indicating improved lipid uptake capacity. However, interventions did not resolve reduced intramuscular lipid content in either study. Conclusions: We conclude that inflammation was a primary facilitator of impaired lipid mobilization in heat-stressed lambs but was not the sole driver of lipid dysregulation. Nevertheless, targeting inflammation was a beneficial strategy for improving lipid flux during chronic heat stress.

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1. Introduction

Environmental heat stress is a long-standing barrier to food animal industries, as it limits growth efficiency, carcass merit, and profitability of meat production [1,2]. Biological responses underlying these outcomes include changes in lipid accretion, mobilization, and utilization that compromise metabolic flexibility, particularly when combined with impaired glucose metabolism [3,4]. When sustained, even modest heat stress reduces the availability of fatty acids to be used as energy substrates. Substantial reductions in circulating free fatty acids were observed in pigs after 7 days of heat stress and in sheep after 10 days [5,6,7]. In cows, four weeks of heat stress reduced circulating triglycerides and cholesterol in addition to free fatty acids [8]. Hypolipidemia is associated with an impaired capacity for adipocytes to lipolyze stored triglycerides and mobilize the resulting free (i.e., non-esterified) fatty acids and glycerol into the bloodstream [9]. For cows, the spike in circulating free fatty acids in response to exogenous epinephrine was blunted by almost 40% following 9 days of heat stress [10]. Ex vivo epinephrine-stimulated fatty acid mobilization was likewise reduced in subcutaneous adipose tissue collected from cattle following 30 days of heat stress [11], although the same outcome was not observed following brief periods of heat stress [12,13]. Poor lipid mobilization from subcutaneous adipose coincided with normal or slightly elevated fat deposition [7,14,15], which contributes to the heightened fat-to-lean body composition that is a hallmark of heat-stressed animals [5,6,15,16,17,18]. In skeletal muscle, heat stress diminishes expression of the fatty acid translocase, CD36, and other fatty acid transporters, which limits muscular uptake of lipids [13,19]. This results in less intramuscular fat accretion [14,15,19], despite some studies observing concurrent reductions in muscle fatty acid oxidation [20,21]. A key driver of metabolic dysfunction during chronic heat stress is heightened systemic inflammation [1,22]. Feedlot lambs exposed to several weeks of heat stress exhibited elevated circulating TNFα and IL-6, which coincided with greater circulating monocytes [23,24]. Comparable increases in blood cytokines occurred in cattle transitioning from spring to summer seasons [25,26]. Heat stress also reduced circulating and tissue concentrations of anti-inflammatory ω-3 polyunsaturated fatty acids (ω-3 PUFA) [17,27,28], which may play a role in elevated systemic inflammation and subsequent metabolic dysfunction. Restoring normal inflammatory tone may improve outcomes of heat stress in livestock. Thus, the objective of this study was to determine whether targeting heat stress-induced inflammation could improve lipid homeostasis. Specifically, we hypothesized that ω-3 PUFA nutraceutical interventions would mitigate the adverse effects of chronic heat stress on fatty acid mobilization from adipose tissue and lipid utilization by skeletal muscle.

2. Materials and Methods

2.1. Animals and Experimental Design

This study was approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln, which is accredited by AAALAC International. The samples utilized for this study were derived from two studies of chronic heat stress in feedlot wether lambs at the University of Nebraska-Lincoln Animal Science Complex [23,29,30,31,32]. In brief, animals from study 1 were commercial Polypay lambs (40.8 ± 0.7 kg) that were stratified by bodyweight and randomly assigned to heat stress (40 ± 1 °C, 35 ± 5% relative humidity) for 30 days. These heat-stressed lambs were randomly assigned to receive dexamethasone injections (0.15 mg/kg, IM; MWI Animal Health, Boise, ID, USA) every 72 h (Heat Stress+Dex; n = 11), oral boluses of fish oil capsules (1200 mg; Nature Made Nutritional Products, West Hills, CA, USA) twice daily (Heat Stress+FishOil; n = 11), or placebo injections and capsules (Heat Stress; n = 11). Thermoneutral (19 ± 1 °C, 15 ± 5% relative humidity) lambs (Control; n = 12) were pair-fed to the average daily intake of the heat-stressed lambs. For study 2, commercial Suffolk crossbred wether lambs (40.1 ± 0.5 kg) were stratified by bodyweight and randomly assigned to thermoneutral (n = 12) or heat stress conditions as in study 1. Heat-stressed lambs were supplemented with ω-3 PUFA Ca^2+^ salts (0.21 g/kg, Strata; Virtus Nutrition LLC, Corcoran, CA, USA) twice daily (Heat Stress+ω3; n = 11) or molasses placebo (n = 11). For study 1, blood samples were collected via jugular venipuncture on days −3, 3, 9, 21, and 30. For both studies, lambs were euthanized via barbiturate overdose on the 30th day of heat stress, and visceral adipose tissue and semitendinosus muscles were collected.

2.2. Circulating Lipids

Plasma was isolated from EDTA-treated venous blood via centrifugation (14,000× g, 5 min) and assessed for total triglycerides, cholesterol, and high-density lipoprotein-bound cholesterol (HDL-cholesterol) with a Vitros 250 Analyzer (Ortho Diagnostics, Raritan, NJ, USA) as previously described [33]. Plasma free fatty acids were quantified in duplicate with a commercial ELISA kit (NEFA; Wako Life Sciences, Richmond, VA, USA) as previously described [34], and inter-assay and intra-assay coefficients of variance were less than 15%.

2.3. Ex Vivo Fatty Acid and Glycerol Mobilization

To estimate the capacity for lipid mobilization from fat stores, epinephrine-stimulated release of free fatty acids and glycerol from primary visceral adipose tissue was assessed as previously described [11], with minor modifications. Just after euthanasia, visceral adipose tissue was collected from around the kidney. Small volumes (~5 g) were placed immediately into conical tubes with Modified Krebs Ringer Buffer (i.e., Krebs Ringer Buffer with 15 M NaHCO_3_, 2.5 mM CaCl_2_, 4% fatty acid-free bovine serum albumin; Sigma-Aldrich, St. Louis, MO, USA) that was pre-warmed to 37 °C. The tissue was finely minced at 37 °C and strained through a 200-μm filter. Individual aliquots (400 ± 10 mg) of minced adipose tissue were transferred to pre-warmed 15-mL conical tubes containing 5 mL of Modified Krebs Ringer Buffer. Epinephrine (Sigma-Aldrich) was added to the buffer in final concentrations of 0, 100, or 1000 nM. Tubes were incubated for 2 h at 37 °C in a shaking water bath. After the incubation, tubes were cooled on ice. The media from each tube was filtered through a 2.4-cm glass microfiber filter (Grade 691; VWR, West Chester, PA, USA) and stored at −80 °C. For each lamb, two technical replications per condition were performed and averaged. Free fatty acid and glycerol concentrations in the filtered incubation media were determined using the Free Fatty Acid Quantification Kit (abc65341; Abcam Ltd., Cambridge, UK) and Glycerol Assay Kit (MAK117; Sigma-Aldrich), respectively, according to the manufacturer’s protocols. For both assays, inter-assay and intra-assay coefficients of variance were less than 15%.

2.4. Western Immunoblots

Samples from the mid-portion of the semitendinosus muscle were collected at necropsy and snap-frozen in liquid nitrogen. Protein was extracted as previously described [34,35], with minor modifications. Briefly, 50-mg tissue aliquots were homogenized via sonication in a low salt Tris-HCl buffer with 2.5% protease and 2.5% phosphatase inhibitor for a total of 15 s. Homogenates were then centrifuged (14,000× g, 5 min, 4 °C) to remove debris from the suspension. Total protein concentration within the supernatant of each extracted sample was quantified using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA, USA). Protein immunoblots were performed with antibodies previously validated in sheep [36]. A total of 50 μg of protein for each sample was combined with Bio-Rad 4× Laemmli sample buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA) and heated to 95 °C for 5 min. After the samples were cooled to room temperature, protein was separated by SDS–PAGE and then transferred to Bio-Rad poly-vinylidene fluoride low-fluorescent membranes. Tris-buffered saline containing Tween was used to wash membranes, and Bio-Rad EveryBlot Blocking Buffer was used to minimize non-specific binding. Membranes were incubated with rabbit anti-sera raised against CD36 (1:1000; ab133625, Abcam), PPARα (1:1000; ab126285, Abcam), PPARγ (1:1000; bs-0530R, Bioss Inc., Woburn, MA, USA), or ACSL1 (1:1000; ab177958, Abcam) overnight at 4 °C. After a wash, membranes were incubated with goat anti-rabbit secondary antibodies for 1 h at room temperature in a LI-COR black box (LI-COR Biosciences, Lincoln, NE, USA). Membranes were scanned using a LI-COR Odyssey Infrared System, and the protein bands were analyzed with LI-COR Image StudioLite Software 5.2. Expression of each protein of interest was normalized to the total protein extracted from the sample.

2.5. Lipid Staining

Cross sections of semitendinosus muscles were stained to determine intracellular lipid droplet dynamics as previously described [34]. Mid-portions of the muscle were collected at necropsy, fixed with 4% paraformaldehyde (Sigma-Aldrich), embedded in Tissue-Tek OCT compound (Thermo Fisher), and frozen at −80 °C. Fixed tissues were cryo-sectioned at 10 µm and mounted on charged microscope slides. For staining, slides were rinsed in 60% isopropanol (MilliporeSigma, Burlington, MA, USA) and incubated with Oil Red O (MilliporeSigma) working solution for 15 min. The sections were then washed with 60% isopropanol, rinsed with deionized water, and mounted with National Diagnostics hydromount medium (Thermo Fisher). Images were visualized on an Olympus IX73 and captured by a DP80 microscope camera (Olympus Corp., Center Valley, PA, USA). Mean lipid droplet area, lipid droplet density, and lipid percentage of the total field of view were determined with ImageJ 1.54 Software from 5 non-overlapping fields of view across three cross sections for each muscle. Particle analysis parameters were set to a size range of 1.5–1,000,000 pixels^2^ and circularity of 0.40–1.00. Thresholding was performed in the HSB color space using a red threshold with the following settings: hue (0–255), saturation (125–255), and brightness (0–230).

2.6. Statistical Analysis

All data were analyzed by ANOVA using the mixed procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA) to evaluate the main effect of experimental group. Both studies were performed in multiple blocks, and block was used as a covariate. The individual animal was considered the experimental unit and was treated as a random variable. Ex vivo fatty acid and glycerol mobilization rates were analyzed for the main effects of experimental group, incubation condition, and the interaction using the mixed procedure with repeated measures. For plasma lipids, day was considered a repeated measure. Statistical significance was declared at p ≤ 0.05, with tendencies declared at p ≤ 0.10. Data are presented as means ± standard error.

3. Results

3.1. Inflammatory Mediation

Indicators of heat stress-induced systemic inflammation and inflammatory mediation by the respective interventions measured at the same time points in these same animals were previously reported for both studies [23,30]. Briefly, circulating TNFα concentrations were increased by up to 2.3-fold over the last three weeks of heat stress for lambs produced in the first study. These increases were fully mitigated by fish oil supplementation throughout the study and were mitigated by dexamethasone only after 30 days. In study 2, circulating TNFα was increased by ~70% and circulating IL-6 was increased by ~79% throughout the heat-stress period. The increase in TNFα was partially mitigated, and the increase in IL-6 was fully mitigated by ω-3 PUFA Ca^2+^ salt supplementation.

3.2. Circulating Lipids

For study 1, no experimental group × day interactions were observed for any circulating lipid. Blood plasma triglyceride concentrations were less (p < 0.05) for Heat Stress and Heat Stress+Dex lambs but not Heat Stress+FishOil lambs than for controls (Figure 1A). Free fatty acid concentrations were less (p < 0.05) for Heat Stress, Heat Stress+Dex, and Heat Stress+FishOil lambs than for controls (Figure 1B). Total cholesterol did not differ among groups (Figure 1C). However, HDL-cholesterol was less (p < 0.05) for Heat Stress and Heat Stress+Dex but not Heat Stress+FishOil than for controls (Figure 1D). Circulating lipids were not assessed for lambs in study 2.

3.3. Ex Vivo Fatty Acid Mobilization

3.3.1. Study 1

For the first study, no experimental group × epinephrine concentration interaction was observed for ex vivo free fatty acid mobilization rates or for free fatty acid Δ from basal mobilization (i.e., increase relative to incubations containing no added epinephrine). Free fatty acid mobilization rates tended to be less (p < 0.10) in adipose from Heat Stress and Heat Stress+Dex lambs compared to controls and Heat Stress+FishOil lambs, regardless of media epinephrine concentration (Figure 2A). Additionally, free fatty acid mobilization rates across all groups were greater (p < 0.05) when the media contained 1000 nM epinephrine compared to 0 or 100 nM. Free fatty acid Δ from basal mobilization tended to be less (p < 0.05) in adipose from Heat Stress, Heat Stress+Dex, and Heat Stress+FishOil lambs than from controls, regardless of epinephrine concentration (Figure 2B). Experimental group × epinephrine concentration interactions were observed (p < 0.05) for ex vivo glycerol mobilization rates and for glycerol Δ from basal mobilization. Glycerol mobilization rates did not differ among groups in incubation media containing no epinephrine (Figure 2C). However, rates were less (p < 0.05) for adipose from Heat Stress and Heat Stress+FishOil lambs and were intermediate for adipose from Heat Stress+Dex lambs when incubated in media containing 100 nM epinephrine. Moreover, glycerol mobilization rates were less (p < 0.05) for adipose from Heat Stress, Heat Stress+Dex, and Heat Stress+FishOil lambs than from controls in media containing 1000 nM epinephrine. Glycerol Δ from basal mobilization did not differ among groups in media containing no epinephrine but was less (p < 0.05) for Heat Stress lambs and intermediate for Heat Stress+Dex and Heat Stress+FishOil lambs in media containing 100 or 1000 nM epinephrine (Figure 2D).

3.3.2. Study 2

For the second study, an experimental group × epinephrine concentration interaction was not observed for ex vivo free fatty acid mobilization but was observed (p < 0.05) for free fatty acid Δ from basal mobilization. Free fatty acid mobilization rates tended to be less (p < 0.10) for adipose from Heat Stress lambs but not Heat Stress+ω3 lambs than for adipose from controls, regardless of media epinephrine concentration (Figure 3A). Moreover, free fatty acid mobilization rates across all groups were least (p < 0.05) when media contained no epinephrine, intermediate when media contained 100 nM epinephrine, and greatest (p < 0.05) when media contained 1000 nM epinephrine. Free fatty acid Δ from basal mobilization did not differ among groups in media containing no epinephrine or 100 nM epinephrine (Figure 3B). In media containing 1000 nM epinephrine, however, free fatty acid Δ from basal mobilization was less (p < 0.05) for Heat Stress but not Heat Stress+ω3 lambs than for controls. An experimental group × epinephrine concentration interaction was observed (p < 0.05) for ex vivo glycerol mobilization but not for glycerol Δ from basal mobilization. Glycerol mobilization from adipose did not differ among groups in media containing no epinephrine and was less (p < 0.05) for Heat Stress and Heat Stress+ω3 lambs than for controls in media containing 100 nM epinephrine (Figure 3C). Moreover, glycerol mobilization was less (p < 0.05) for Heat Stress lambs but not Heat Stress+ω3 lambs than for controls in media containing 1000 nM epinephrine. Glycerol Δ from basal mobilization tended to be less (p < 0.10) for Heat Stress lambs but not Heat Stress+ω3 lambs than for controls, regardless of media epinephrine concentration (Figure 3D).

3.4. Muscle Lipid Dynamics

3.4.1. Study 1

For the first study, lipid droplet density within the semitendinosus muscle was less (p < 0.05) for Heat Stress, Heat Stress+Dex, and Heat Stress+FishOil lambs than for controls (Figure 4A). However, average lipid droplet size (i.e., average droplet area) (Figure 4B) and total lipid area (i.e., % of the field of view) (Figure 4C) did not differ among groups.

3.4.2. Study 2

For the second study, lipid droplet density within the semitendinosus muscle (Figure 5A) and average lipid droplet size (Figure 5B) did not differ among experimental groups. However, total lipid area tended to be less (p < 0.10) for Heat Stress and Heat Stress+ω3 lambs than for controls (Figure 5C). Representative images of Oil Red O staining are presented in Supplemental Figure S1.

3.5. Muscle Regulatory Proteins

3.5.1. Study 1

For study 1, muscle CD36 (Figure 6A), ACSL1 (Figure 6B), and PPARγ (Figure 6C) content did not differ among experimental groups. Muscle PPARα content tended to be greater (p < 0.10) for Heat Stress lambs but not Heat Stress+Dex or Heat Stress+FishOil lambs than for controls (Figure 6D). Representative gel images for each protein immunoblot are shown in Supplemental Figure S2.

3.5.2. Study 2

For study 2, muscle CD36 content was less (p < 0.05) for Heat Stress lambs but not Heat Stress+ω3 lambs than for controls (Figure 7A). ACSL1 content did not differ among groups (Figure 7B). PPARγ content tended to be less (p < 0.10) for Heat Stress lambs than for controls and was intermediate for Heat Stress+ω3 lambs (Figure 7C). PPARα content did not differ between Heat Stress lambs and controls but was greater (p < 0.05) for Heat Stress+ω3 lambs than for either other group (Figure 7D).

4. Discussion

In this study, we found that chronic heat stress in wether lambs impaired the capacity for lipids to be mobilized from visceral adipose tissue, particularly in response to adrenergic stimulation. Moreover, systemic inflammation induced by heat stress was the primary facilitator of this disruption, as mitigating it with anti-inflammatory nutrient supplements improved mobilization of both glycerol and free fatty acids. Poor lipid mobilization from visceral fat coincided with diminished circulating triglycerides and free fatty acids and with reduced lipid accrual in skeletal muscle. Reducing inflammation did not resolve the deficiencies in intramuscular lipid content despite rescuing circulating triglyceride concentrations and muscular expression of the fatty acid translocase, CD36, and the transcription factors, PPARγ and PPARα, all of which facilitate lipid uptake and utilization [37,38]. Importantly, all of these changes were independent of any influence from dietary intake, which was controlled by pair feeding in both cohorts of sheep. We previously documented large reductions in muscle glucose metabolism following chronic heat stress [24,39], and the present findings indicate that stored fatty acids are less available to compensate, which helps to explain intake-independent forfeiture of muscle growth [24,32,39]. Moreover, the improvement in lipid mobilization by inflammatory mitigation, which coincided with improved lipidemia, indicates that heightened inflammatory tone is an effective target for improving dysregulation of lipid flux during chronic heat stress.

Reductions in lipid mobilization from visceral fat following chronic heat stress indicated an impaired capacity for adipocytes to lipolyze triglycerides. Fat from heat-stressed animals released only about two-thirds the amount of free fatty acids and glycerol as did fat from unstressed animals, and these deficits were typically more pronounced under epinephrine stimulation. Catecholamines activate triglyceride lipolysis via cyclic AMP/protein kinase A pathways in uncompromised adipocytes [40,41,42]. However, TNFα and other inflammatory cytokines desensitize adrenergic pathways through noncanonical IKKε signaling, which impedes lipolysis substantially [43,44]. Although acute cytokine exposure can actually promote lipolysis [45,46], the indirect inhibitory effects of sustained inflammation during heat stress on epinephrine-stimulated lipolysis appeared to have outweighed any direct stimulation. Suppression of lipolysis and lipid mobilization presumably contributed to corresponding reductions in circulating and intramuscular lipids observed in these heat-stressed lambs. Improved lipid mobilization following ω-3 PUFA intervention highlights the mediating role of inflammation in these deficits. Supplementing fish oil, which delivered about 25 mg of combined EPA and DHA per kg bodyweight daily, recovered more than three-quarters of the heat stress-induced deficit in free fatty acid mobilization. Comparable amounts of ω-3 PUFA Ca^2+^ salts, which are more protected from rumen biohydrogenation [47], fully recovered fatty acid mobilization. This was at least partially due to restoration of adrenergic responsiveness, as improvements were generally most profound in the presence of epinephrine. Similar effects have been observed with other anti-inflammatory agents like IL-33 [48]. Serial treatment with the synthetic corticosteroid dexamethasone failed to improve free fatty acid release and only modestly increased glycerol efflux, which corresponded with less profound reductions in heat stress-induced inflammation [29,30]. Direct effects of corticosteroids on lipolysis in adipocytes also appear to be quite modest. In rats, serial injections that increased circulating corticosterone four-fold above baseline did not induce triglyceride lipolysis in adipose tissue [49]. Moreover, incubation of primary rat adipocytes with dexamethasone increased free fatty acid and glycerol excretion, but only at very high concentrations [50].

Heat stress-induced shortages in intramuscular lipid accretion were not solely attributable to systemic inflammation, as anti-inflammatory interventions failed to curtail losses in lipid droplet density and fractional lipid content within the semitendinosus muscle. The effects of inflammatory cytokines on muscle lipid flux can differ due to the length and magnitude of exposure, as components of the complex signaling pathways are activated at different rates. For example, although chronic infusion of rats with the inflammatory interleukin, IL-25, reduced intramuscular lipid droplet size [51], short infusions of TNFα into adult men had no effect on muscle lipid flux [46]. Likewise, 1-day incubation of myoblasts with inflammatory cytokines did not affect their intracellular lipid content [52]. Conversely, incubation of the isolated soleus muscle with epinephrine for as little as 15 min reduced lipid droplet density and total lipid content [53]. Adrenergic stimulation increases muscular lipolysis by mediating the translocation of hormone-sensitive lipase toward lipid droplets [53,54]. We previously documented elevated circulating epinephrine in heat-stressed animals [24,39], and it is reasonable to speculate that reduced muscle lipid droplet density and lipid content in the present study were at least partially products of heat stress-heightened adrenergic tone.

Diminished intramuscular fat in heat-stressed lambs was associated with indicators of impaired fatty acid uptake and storage but not with indicators of fatty acid esterification. The reduced intramuscular lipid content observed in heat-stressed lambs coincided with a 52% loss in muscular fatty acid translocase, CD36, which facilitates the uptake of long-chain fatty acids for storage or immediate β-oxidation [55,56,57]. The plasticity of CD36 expression is a key part of the balance between muscular glucose and lipid utilization, and cell culture studies show that changes in its expression yield proportional changes in cellular lipid accumulation [37,58,59]. The expression of CD36 typically parallels that of circulating lipids [37,56,57], and thus its reduction in muscle was consistent with the hypolipidemia observed in our heat-stressed lambs. Moreover, inflammatory exposure directly downregulates CD36 content [60,61], which explains its recovery following ω-3 PUFA supplementation. However, the unexpected lack of a corresponding improvement in intramuscular lipid content indicates that its reduction was not due to inflammatory suppression of lipid uptake but rather adrenergic-stimulated utilization of these lipids [24,39]. In fact, studies show that β-adrenergic stimulation increases fatty acid consumption via β-oxidation [53,62,63] despite also reducing CD36 in muscle [64]. Elevation of inflammatory or adrenergic tone can downregulate muscular expression of the transcriptional factor PPARγ [64,65], which was reduced by about 33% in our heat-stressed lambs. PPARγ promotes cellular uptake of fatty acids in part by upregulating CD36 [65]. However, its concurrent upregulation of lipoprotein lipase and other catabolic enzymes promotes large increases in β-oxidation of fatty acids rather than storage [66,67], resulting in a net depletion of intramuscular lipids [68]. Although the deficit in muscle PPARγ content was only modestly improved by ω-3 PUFA supplementation, it is worth noting that both EPA and DHA can activate PPARγ via direct binding [65]. The almost 3-fold increase in muscular PPARα content following heat stress was somewhat unexpected, as previous studies in mice and chickens show that it is often diminished by heat stress [69,70] and by inflammation [61,71,72]. However, it can be upregulated by heightened adrenergic stimulation [73,74,75] or by reduction in circulating free fatty acids [76], and in some cases, chronic heat or cold stress increased PPARα despite concurrently increased inflammation [77,78,79,80]. Regardless, greater muscle PPARα may help explain the loss of intramuscular lipid content in our heat-stress lambs, as it promotes lipid oxidative metabolism as an alternative to glucose [81,82,83,84,85]. Similarly, the reversal of heightened PPARα in response to fish oil presumably coincided with improvements in glucose metabolism [86], which would ease the burden on fatty acid oxidation. Skeletal muscle ACSL1 content did not appear to be affected by heat stress or by any of the anti-inflammatory interventions, despite previous studies documenting its inhibition by inflammatory factors [61,87]. Normal ACSL1 content in skeletal muscle would be consistent with a maintained capacity for fatty acid utilization [88], regardless of exposure to heat stress or anti-inflammatories.

5. Conclusions

We conclude from this study that heat stress-induced systemic inflammation contributes to the impairment of lipid flux between adipose stores and skeletal muscle utilization. Together with diminished glucose metabolism, the lipid dysregulation reported here helps to explain poor growth and body composition in animals experiencing chronic environmental heat stress. Large reductions in glycerol and free fatty acid mobilization from visceral fat, particularly in response to adrenergic stimulation, demonstrate the suppressive effect of heat stress on essential triglyceride lipolysis. However, targeting heat stress-induced inflammation with concurrent supplementation of anti-inflammatory ω-3 PUFA generally restored adipocyte lipolysis, which in turn recovered circulating triglycerides. Reduced CD36 and PPARγ in skeletal muscle from heat-stressed lambs indicate a reduced capacity for fatty acid uptake in concert with the reduced circulating supply. Conversely, the large increase in muscle PPARα content and unaffected ACSL1 content indicates normal or even increased metabolic utilization of intramuscular lipids, which together with less uptake explains the reductions in muscle lipid content. Aberrant muscle CD36, PPARγ, and PPARα and reduced circulating triglycerides were corrected by supplementing ω-3 PUFA, but indicators of diminished intramuscular lipid content were not improved. Thus, although inflammation is a key factor in heat stress-mediated lipid dysfunction, it is not the sole driver. Nevertheless, the observed improvements highlight the potential for anti-inflammatory nutraceutical approaches to mitigate lipid dysregulation and improve metabolic outcomes in heat-stressed livestock. In doing so, it provides insight into practical on-farm strategies to increase the resilience and productivity of livestock in the face of environmental heat events.