This study analyzed 70 sphingolipid species, and the concentrations of 46 species were found to be statistically different between RPAT and SCAT (P < 0.1), indicating that the sphingolipid profiles of these two adipose tissue depots were remarkably different. The differential concentration of sphingolipid species can reflect dissimilar sphingolipid pathway activities, affecting downstream signaling function [2]. For instance, ceramide synthesis via the de novo pathway was more related to insulin resistance [21], whereas ceramide formation from the sphingomyelinase pathway was more associated with inflammation, oxidative stress, and apoptosis through the one-step rapid transformation from SM to ceramide [7]. Little was known about the salvage pathway in sphingolipid signaling, but it was reported that ceramides from the salvage pathway could be responsible for the dephosphorylation of the p38 cascade and the PKC signaling pathway [5]. Combining the potential signaling function of various sphingolipids to the sphingolipid profile of RPAT and SCAT, we could better understand the physiologic role of these two adipose tissues. Limitations of this study include the relatively small sampling size, restricting the assessment of any possible dietary influence. Sphingolipid metabolism was shown to be altered by dietary factors in dairy cows [22], however, whether the diet has an effect on adipose dependent distribution of sphingolipids should be tested in further studies. The novelty of this study is confirming that bovine SCAT and RPAT are distinct in their metabolism, by providing the sphingolipid profiles of these two depots in a lipidomics approach.
Backward Accumulation in the De Novo Synthesis Pathway in RPAT
The distribution patterns of the salvage pathway and the sphingomyelinase pathway were more similar than that of the de novo synthesis pathway (Fig. 2). Connecting all sphingolipids with the sphingolipid metabolic map, a unique distribution pattern was shown in Fig. 6. Our results showed that the sphingolipid species that act downstream of 3-ketosphinganine, such as sphinganine, DHCer, and Cer were more concentrated in RPAT (red in Fig. 6), while sphingolipids that act downstream of DHCer and Cer, such as DHSM, Sph, S1P, SM, and glycosphingolipids, were more concentrated in SCAT (blue in Fig. 6). This pattern suggested that there was a ceramide accrual, and even a possible backward accumulation in the de novo synthesis pathway in RPAT either due to a higher influx rate of substrates at the origin of the de novo synthesis, or due to a lower transformation rate of ceramide at the end of the synthesis, or due to a combination of both [2].
The higher substrate influx rate in RPAT could be supported by the greater dynamics of adipose mass, and higher HSL activity in bovine RPAT [23]. It was shown that the adipose mass of RPAT in periparturient German Holstein cows had a greater fluctuation than that of SCAT [13, 24]. Additionally, RPAT had a higher HSL phosphorylation at residues 563 and 660 detected by Western blot analysis, indicating greater enzyme activation [11]. A similar experiment performed in rodents also showed a higher HSL phosphorylation at residues 563 and 660 in the visceral adipose tissue under forskolin stimulation, compared with the subcutaneous adipose tissue [25]. Together with the greater adipose mass in abdominal adipose depot (66.7% of total body fat) than the subcutaneous adipose tissue (17.9% of total body fat) [26], it is suggested that triglycerides stored in RPAT undergo greater facilitated hydrolysis into NEFA than SCAT. Meanwhile, palmitic acid is one of the most prevalent fatty acids among circulating NEFA [27, 28]. The influx of palmitic acid could drain into the de novo synthesis pathway, and result in the accumulation of ceramide [29, 30].
Besides the high influx rate at the origin of the de novo synthesis pathway, the inhibition in the transformation, or a backward synthesis of ceramide should also be considered as a possible explanation for the accrual of ceramides [2]. The balance between ceramides, sphingomyelins, sphingosines, and other sphingolipid metabolites are controlled by the enzymes involved in their biotransformation. For instance, acid sphingomyelinase (ASMase) is the enzyme converting sphingomyelin into ceramide in the sphingomyelinase pathway, under the activation of oxidative stress, pathogens, and the proinflammatory cytokine interleukin-1β (IL-1β) [31, 32]. In dairy cows, Ji et al. demonstrated that the IL-1β mRNA signal was higher in the mesenteric adipose tissue than the subcutaneous depot [10]. Hence, the ceramide accumulation in the RPAT profile could be the consequence of the increased enzyme activity of sphingomyelinase, driven by pro-inflammatory signals. The enzyme that acts in the opposite direction, sphingomyelin synthase (SMS), converts ceramide to sphingolipid in the endoplasmic reticulum [33]. Although the activation mechanism and the physiological role of SMS in adipose tissue in dairy cattle has not yet been identified, it is evident that SMS could downregulate the reactive oxidative species (ROS) level by breaking down sphingomyelin into ceramide, triggering the release of ROS [34, 35], and opposing the action of ASMase. Further mechanistic studies are warranted to elucidate the role of these metabolic pathways in driving adipose depot specific sphingolipid distribution.
Comparing two sources of ceramide accrual, Rico et al. demonstrated that the de novo synthesis pathway might play a more crucial role than the sphingomyelinase pathway in dairy cattle physiology [36]. It was shown that cows with an intravenous triglyceride (TAG) infusion had a higher ceramide synthase 2 (CerS2) mRNA expression, compared with the control. High level of CerS2 indicated an upregulation of the de novo synthesis pathway as CerS2 is the enzyme promoting the synthesis of C22:0- and C24:0 ceramide [37]. Additionally, it was shown that the SM concentration was not altered with the TAG level, indicating that the sphingomyelinase pathway was not involved in the surge of ceramide. Thus, this provided compelling evidence that the de novo synthesis pathway was more important than the sphingomyelinase pathway in contributing to the ceramide accrual [1]. The salvage pathway could be, but to a lesser extent, contributing to the accumulation of ceramide.
Insulin Resistance, Inflammation, and Oxidative Stress in RPAT and SCAT
Our data showed that ceramides were more concentrated in RPAT, and less concentrated in SCAT. As ceramide is the upstream regulator of Akt, confirmed in an ex vivo study in Holstein steers [38], this ceramide distribution suggested that RPAT would be more associated with insulin resistance because of its ceramide profile. Ceramide has been suggested to be a mediator of obesity and insulin resistance. Previously, it was shown that ceramide could bind with SET, releasing its inhibitory function to PP2A for the inactivation of Akt [39]. Recently, it was shown that ceramide could inhibit Akt by dephosphorylating Ser 473 [40], which was in line with bovine adipocytes research using C2:0-ceramide [38]. Besides, it was demonstrated that dairy cows with ceramide accrual in plasma, liver, and skeletal muscle had higher lipolytic activity and lower insulin sensitivity [3]. This provides evidence that adipose tissue with more ceramides would more suppress insulin sensitivity and have an enhanced sphingolipid dynamic. In contrast to SCAT, Kenéz et al. demonstrated that Akt and HSL phosphorylation were greater in the insulin signaling pathway in dairy cow RPAT during the peripartum period [9]. This indicated that RPAT may be more sensitive and responsive in insulin signaling than SCAT. As RPAT had a greater concentration of ceramides, RPAT may likely have a sphingolipid profile more associated with insulin resistance. Hence, RPAT may contribute to the total insulin sensitivity greater in dairy cattle.
Not only insulin sensitivity but also the inflammatory response was found to be different between RPAT and SCAT in cows [10]. Here, we observed that ceramides were more concentrated in RPAT, whereas sphingosines and S1P were more concentrated in SCAT. The distribution of Cer, Sph, and S1P in RPAT and SCAT may provide an explanation in the different expression of pro-inflammatory cytokines in the two adipose depots. Ceramide, derived from the transformation of sphingomyelin, was shown to affect the proinflammatory cytokine IL-1β and tumor necrosis factor (TNF) signaling pathway [41]. Down to the ceramide species level, Brodlie et al. showed that C16:0, C18:0, C20:0-ceramide levels were increased in the lower airway epithelium in human patients with lung inflammation [42]. Furthermore, sphingomyelinase, the enzyme transforming SM to Cer, was shown to be expressed to a greater extent in inflamed adipose tissues than the non-inflamed adipose tissues in humans [6]. Collectively, these studies showed a strong correlation between ceramides and inflammation via the sphingomyelinase pathway. Besides ceramides, Samad et al. demonstrated that Sph and S1P could also increase the mRNA expression of pro-inflammatory proteins (TNF-α, MCP-1, IL-6, and KC) [23]. In particular, Sph induced more TNF-α expression, and S1P induced more IL-6 mRNA expression in 3T3-L1 adipocytes cell culture, compared with C2:0- and C6:0-ceramide. These studies showed that not only ceramide but also its downstream sphingolipid species such as Sph and S1P could be associated with inflammation. In dairy research, Ji et al. demonstrated that the pro-inflammatory cytokines (IL-1β, IL-6R, CCL2, CCL5) mRNA expression of omental and mesenteric adipose tissue was greater than the subcutaneous adipose tissue in overfed dairy cows [10]. As RPAT had a greater pro-inflammatory cytokines signal and a greater concentration of ceramides, it is suggested that RPAT is associated with a stronger inflammatory response, compared with SCAT.
In addition to pro-inflammatory signaling, it was shown that ceramide and sphingosine could also be the downstream mediators of oxidative stress, and regulate the apoptosis signaling pathway in both human and rat cell line [34]. Here, we observed that ceramides were more concentrated in RPAT, whereas sphingosines were more concentrated in SCAT. These are two important mediators in regulating apoptosis under oxidative stress. Goldkorn et al. demonstrated that ROS such as hydrogen peroxide (H2O2) could activate sphingomyelinases and promote the transformation of SM to Cer in the tracheobronchial epithelial cells [43]. The surge of ceramide from the transformation could, therefore, activate cathepsin D and pro-apoptotic protein BID to induce apoptosis [2, 44]. Besides, the elevated ceramide could be converted into sphingosine, and act as a second messenger to induce apoptosis by inhibiting MAP kinase activity [45, 46]. In specific, Osawa et al. demonstrated that C16:0-ceramide induced apoptosis in rat primary hepatocytes [47], and Seumois et al. showed that C16:0 and C24:0-ceramide are pro-apoptotic signals in human blood neutrophil cells [48]. These findings showed that ceramide and sphingosine are important mediators in response to oxidative stress, and therefore induce apoptosis. Although the oxidative stress level of two adipose tissues in dairy cattle was not measured, it is shown that the visceral depots in mice are more sensitive to oxidative stress than the subcutaneous depots by comparing the stress signaling pathway JNK and MAPK [49]. Thus, the visceral adipocytes might be more susceptible to apoptosis than the subcutaneous adipocytes [50]. As ceramide is the precursor of sphingosine, ceramide might take a more important role in apoptosis signaling. Therefore, it is suggested that the sphingolipid profile of RPAT would be more associated with oxidative stress and apoptosis, compared with SCAT.
The Third Ceramide Regulating Pathway: Phosphorylation Pathway
In addition to the ceramide profile, we observed that DHCer1P and C1P were highly concentrated in both adipose tissues, particularly in SCAT. The concentration of DHCer1P was roughly 10 folds higher than that of DHSph, DHCer, and Cer; and the concentration of C1P was roughly 4 folds higher than that of Sph, S1P, and glycosphingolipid. To explain the high concentration of DHCer1P, it seems likely that the DHCer kinase was more active, or the dihydroceramide desaturase (Des1) was less active in the adipocytes [2]. Des1 is the enzyme promoting the transformation from DHCer to Cer [51]. When the kinase activity is higher than Des1, DHCer may shift the synthesis from Cer to DHCer1P. However, the DHCer kinase activity in bovine cells was not reported yet. The physiological role and the downstream signaling pathway of DHCer1P are still elusive. More research has to be done to understand the high concentration of DHCer1P in both RPAT and SCAT. Similarly, the high concentration of C1P might because of a high level of ceramide kinase (CerK), or lower expression of phosphatase [52]. On the whole, as the concentration of DHCer1P, C1P, and SM were similarly concentrated in the sphingolipid profile, the phosphorylated sphingolipids could regulate the synthesis of ceramides through its one-step transformation with phosphatase, like the one-step SM-Cer pathway, to give a rapid response. Thus, besides two major regulating pathways: de novo synthesis pathway and sphingomyelinase pathway, the phosphorylation pathway could be the third pathway in the sphingolipid metabolic network regulating the synthesis of ceramide, in agreement with JW McFadden and JE Rico [1].