In the past few years, fat grafting has gained immense attention worldwide. Particularly, it is routinely applied in diverse reconstruction surgery, including breast reconstruction after breast cancer mastectomy (Pearl et al., 2012), and contour deformities caused by facial muscle atrophy (Simonacci et al., 2017). The wide applicability of fat grafting is primarily attributed to the accessibility, biocompatibility, and versatility of autologous fat. However, the clinical application of fat grafting is somewhat compromised owing to the unpredictability of volume retention following transplantation. Although an array of surgical techniques, including cell-assisted lipotransfer (CAL) and brava-assisted fat grafting, have been explored to improve the retention of fat grafts (Yoshimura et al., 2008; Khouri et al., 2015), the challenges of long-term volume retention remain unaddressed so far. In addition, unavoidable oil cyst formation and fibrosis hinders the application of fat grafting (Dixit and Wagh, 2013; Abdallah et al., 2020). Therefore, there is an urgent need to devise new strategies for advancing the practical application of fat grafting.

Oil droplets have been previously shown to be closely associated with the occurrence of complications following fat grafting (Chen et al., 2021). Particularly, two main sources of oil droplets have been identified in fat grafts. The mechanical shear force utilized during liposuction induced fragmentation of adipocytes, which inevitably results in redundant oil droplets in the aspirated fat. In addition, the necrotic adipocytes in the hypoxic-ischemic center zone of grafts released oil droplets and initiated lipotoxicity related inflammation (Eto et al., 2012). After fat grafting, bursting of oil droplets in a short time induced the formation of lipid-laden macrophages (Webb and Moore, 2007), and the persistent activation of lipotoxicity associated inflammation, followed by fibrosis and cyst formation (Chen et al., 2021). Therefore, harnessing oil-scavenging capacity is pivotal for the high-quality retention of grafts. Physicians have so far focused on devising different harvesting and processing techniques to remove oil before grafting (Allen et al., 2013). Past studies have primarily focused on adipocyte viability and the number of stem cells (Smith et al., 2006; Kurita et al., 2008; Ferraro et al., 2011). Although the effects of these techniques appeared to be positive, the oil droplets from necrotic adipocytes following fat grafting remained a major challenge, which in turn hindered the clinical application of fat grafting.

Macrophages play an important role in the lipid metabolism (Kim et al., 2018). It has been reported that a HF diet promoted macrophage infiltration in adipose tissue of mice (Tang et al., 2014; Liu et al., 2020). Promoting early macrophage infiltration using used macrophage colony stimulating factor (M-CSF) improved fat graft survival by inducing angiogenesis (Cai et al., 2018). And the administration of HF diet for 8 weeks upregulated the expression of adipose triglyceride lipase (ATGL) and promoted triglyceride hydrolysis in the subcutaneous adipose tissues of mice (Gaidhu et al., 2010). Furthermore, the observations made in clinical practice revealed that the increasing consumption of food leads to the improved volume retention of fat grafts. Hence, it was hypothesized that, in the case of fat grafting, manipulation of macrophage infiltration by a short-term HF diet might reinforce the lipolysis and angiogenesis in grafts, and can assist in achieving fat grafts with decreased fibrosis and oil cyst formation. To prove this hypothesis, the present study established fat grafting models in mice. After fat grafting, the mice were respectively provided a normal diet, a high carbohydrate diet (HC, 80% carbohydrate), or a high-fat diet (HF, 60% fat) for 30 days. The long-term volume retention and quality of the grafts were assessed. In addition, the changes in lipolysis and angiogenesis in fat grafts were evaluated in detail.

The recent decades, accumulated evidence indicated that the adipose tissues act as both an energy reservoir and an endocrine mediation organ (Luo and Liu, 2016). Following sprouting from mesenchymal stem cells, adipocytes deposit lipid droplets under the influence of nutrition and hormones, accompanied by the lipogenic and lipolytic loops. Large lipid droplets formed in mature adipocytes. However, mechanical damage to brittle adipocytes during fat transplantation and the lack of adequate blood and oxygen supply to the grafts during the early stage after fat grafting induced adipocyte necrosis and another wave of oil release (Eto et al., 2012; Sunaga et al., 2013). Next, massive oil droplets get instantaneously accumulated in the fat grafts, causing oil aggregation beyond the scrapable capacity and the formation of oil cysts. The effect incurred by rapidly increasing oil droplets on fat grafts remain unclear. Thus, focus are gained onto lipid metabolism in fat grafting in this study.

The lipolysis procedure involves successive cascades. Particularly, ATGL and HSL act as rate-limiting enzymes for lipolysis and regulate the catabolism of triglycerides in oil droplets (Haemmerle et al., 2006; Haemmerle et al., 2002). These enzymes are responsible for the hydrolysis of about 95% triglycerides (Schweiger et al., 2006). Meanwhile, CGI-58 along with ATGL has been shown to modestly enhance the lipolysis activity (Lass et al., 2006). Each hydrolysis step yielded one fatty acid. Thus, the attenuated expression of these lipolysis genes would induce the accumulation of triglycerides (Radner et al., 2010; Liu et al., 2017). The results for high-throughput sequencing in the present study demonstrated that lipolysis genes, including ATGL, CGI-58, HSL, and MGL, were remarkably downregulated after grafting. Therefore, the formation of oil cysts at a later stage may be directly related to the burst accumulation of oil droplets during the early stage and the attenuated lipolytic capacity. Meanwhile, the oleaginous swamp might evoke enduring inflammation and fibrosis, which drag down the long-term outcomes of fat grafts (Chen et al., 2021). Thus, rejuvenation of the lipolytic activity appears to be a feasible avenue to improve fat grafting, particularly in terms of inhibiting vesicle formation. Interestingly, Gaidhu et al. reported that the expression of ATGL in adipose tissues can be unregulated by HF diet (Gaidhu et al., 2010). In the present study, HF diets could promote the expression of ATGL and HSL in the inguinal fat tissue. This was accompanied by the elevation expression of CD36, FABP4, and CPT1 (Figure 1D), which regulate the uptake of fatty acids (Wolf Greenstein et al., 2017) and the mitochondrial β-oxidation activity (Labrie et al., 2015).

Furthermore, we found that the grafts of the HC and HF groups exhibited lower volume as compared with those of the ND group on day 60. However, the volume of grafts in the HF group on day 90 was larger than that in the ND group. In parallel, HC and HF groups exhibited lesser oil cysts and more smaller preadipocytes on day 30 and 60. On day 90, the previously shrunken adipocytes swelled to a mature size. Weight gain was reported in mice belonging to HC and HF groups by day 30, but after being fed with an ND diet for the next 2 months, no significant differences were recorded in bodyweight among these three groups (Figure 2B). Cumulatively, we presumed that a HF diet increased lipid metabolism, promoted the absorption and scavenging of lipid droplets, and reduced the size of adipocytes. Increased expression of ATGL and HSL was observed in HC and HF grafts on day 30. The HC and HF diets also promoted the expression of CPT1, a key factor that regulats fatty acid metabolism (Chakravarthy et al., 2005). The higher expression of lipolysis genes indicated that the oil droplets in the grafts would be catabolized more quickly. This provided a possible explanation for the existence of less oil cysts and small adipocytes during the early stage of fat grafting in HF grafts.

Macrophages are actively involved in lipid engulfment and degradation (Flaherty et al., 2019). Free fatty acids and glycerol are the hydrolyzed products of triglycerides through lipolysis. In damaged fat tissues, the free fatty acids are shifted to mitochondria, and participate in fatty acid oxidation. Increased angiogenesis and adequate blood supply are critical to the β-oxidation process. With sufficient tissue blood supply and oxygenation, released fatty acids are effectively scavenged (Ahmadian et al., 2007; Onal et al., 2017). However, in fat grafts, early ischemia and hypoxia is inevitable (Kato et al., 2014). The slow vascular regeneration process suppressed the lipolysis and β-oxidation of fatty acids, and resulted in the formation of oil cysts. Conversely, decreased oil cysts were observed in fat grafts assisted by stromal vascular fraction cells or vascular endothelial growth factor (VEGF) (Lu et al., 2009; He et al., 2019). Simultaneously, macrophages act as major immune cells in promoting angiogenesis and vascular regeneration (Godwin et al., 2013; Godwin et al., 2017). As a repetitious propellant for blood supply, vascular regeneration in the early stage seems pivotal for the final volume retention of fat grafts (Mashiko and Yoshimura, 2015). Stimulating macrophage infiltration in fat grafts using macrophage colony stimulating factor was able to improve angiogenesis and fat graft survival (Cai et al., 2018). And increased VEGF expression promoted angiogenesis and decreased hypoxia in adipose tissue. In addition, platelet derived growth factor (PDGF) secreted by macrophages prevented apoptosis of preadipocytes by binding to the PDGF receptor on the surface of preadipocytes (Molgat et al., 2009). Those positive changes can offset the imbalance of lipid metabolism (Sung et al., 2013).

It was previously reported that HF diets promoted macrophage infiltration in adipose tissue (Tang et al., 2014; Liu et al., 2020). In the present study, a short-term HF diet promoted the infiltration of macrophages, and increased the expression of VEGFα and PDGFα in fat grafts. In addition, enhanced ATGL expression was observed around macrophages in Figure 4C. The increased average microvascular density in HF grafts was showed by CD31 staining. Therefore, we were prone to speculate that the macrophages induced by HF diets promoted angiogenesis and vascular regeneration by paracrine excretion of VEGFα and PDGFα. And the increased macrophages activated lipolysis process. The effective vascular regeneration and oxygen supply provided favorable conditions for the enhanced fatty acid metabolism of macrophages. Eventually, the timely clearance of oil droplets mitigated lipotoxicity and reduced fibrosis in grafts. This may provide a possible preliminary explanation for the improvement effects of short-term HF diets on fat grafts. More in-depth studies were needed to explore the specific mechanism between macrophages and lipolysis regulation in fat grafts.

The present results demonstrated that a short-term HF diet stimulated macrophage infiltration, upregulated the lipolytic activity and promoted angiogenesis in the grafts. Moreover, the rapid clearance of oil droplets and timely revascularization further promoted each other, increased the survival rate of transplanted adipocytes, and improved the long-term volume retention of grafts. However, we acknowledge that this study has some limitations as is unavoidable in an animal study, and hence further clinical research is warranted to validate our results. Nonetheless, we demonstrated an intriguing nonsurgical treatment approach through lipolysis regulation that presents a novel direction for improving the outcomes of fat grafting.