Mature adipocytes store fatty acids and are a common component of tissue stroma. Adipocyte function in regulating bone marrow, skin, muscle, and mammary gland biology is emerging, but the role of ...adipocyte-derived lipids in tissue homeostasis and repair is poorly understood. Here, we identify an essential role for adipocyte lipolysis in regulating inflammation and repair after injury in skin. Genetic mouse studies revealed that dermal adipocytes are necessary to initiate inflammation after injury and promote subsequent repair. We find through histological, ultrastructural, lipidomic, and genetic experiments in mice that adipocytes adjacent to skin injury initiate lipid release necessary for macrophage inflammation. Tamoxifen-inducible genetic lineage tracing of mature adipocytes and single-cell RNA sequencing revealed that dermal adipocytes alter their fate and generate ECM-producing myofibroblasts within wounds. Thus, adipocytes regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes.
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•Inhibiting dermal adipocyte lipolysis reduces inflammatory wound bed macrophages•Wound edge adipocytes dedifferentiate within hours after injury•Adipocyte lipolysis is needed for dedifferentiated adipocytes to populate wound beds•Dedifferentiated adipocytes generate wound bed myofibroblasts after injury
Using genetic mouse models and transcriptomic profiling, Shook et al. show that skin resident adipocytes undergo lipolysis to promote efficient macrophage inflammation after injury. Lipolysis also allows adipocyte-derived cells to dedifferentiate and generate diverse extracellular matrix-producing myofibroblasts in the wound bed.
Intermuscular adipose tissue (IMAT) is negatively related to insulin sensitivity, but a causal role of IMAT in the development of insulin resistance is unknown. IMAT was sampled in humans to test for ...the ability to induce insulin resistance in vitro and characterize gene expression to uncover how IMAT may promote skeletal muscle insulin resistance. Human primary muscle cells were incubated with conditioned media from IMAT, visceral (VAT), or subcutaneous adipose tissue (SAT) to evaluate changes in insulin sensitivity. RNAseq analysis was performed on IMAT with gene expression compared with skeletal muscle and SAT, and relationships to insulin sensitivity were determined in men and women spanning a wide range of insulin sensitivity measured by hyperinsulinemic-euglycemic clamp. Conditioned media from IMAT and VAT decreased insulin sensitivity similarly compared with SAT. Multidimensional scaling analysis revealed distinct gene expression patterns in IMAT compared with SAT and muscle. Pathway analysis revealed that IMAT expression of genes in insulin signaling, oxidative phosphorylation, and peroxisomal metabolism related positively to donor insulin sensitivity, whereas expression of macrophage markers, inflammatory cytokines, and secreted extracellular matrix proteins were negatively related to insulin sensitivity. Perilipin 5 gene expression suggested greater IMAT lipolysis in insulin-resistant individuals. Combined, these data show that factors secreted from IMAT modulate muscle insulin sensitivity, possibly via secretion of inflammatory cytokines and extracellular matrix proteins, and by increasing local FFA concentration in humans. These data suggest IMAT may be an important regulator of skeletal muscle insulin sensitivity and could be a novel therapeutic target for skeletal muscle insulin resistance.
Aims/hypothesis
Subcellular localisation is an important factor in the known impact of bioactive lipids, such as diacylglycerol and sphingolipids, on insulin sensitivity in skeletal muscle; yet, the ...role of localised intramuscular triacylglycerol (IMTG) is yet to be described. Excess accumulation of IMTG in skeletal muscle is associated with insulin resistance, and we hypothesised that differences in subcellular localisation and composition of IMTG would relate to metabolic health status in humans.
Methods
We evaluated subcellular localisation of IMTG in lean participants, endurance-trained athletes, individuals with obesity and individuals with type 2 diabetes using LC-MS/MS of fractionated muscle biopsies and insulin clamps.
Results
Insulin sensitivity was significantly different between each group (athletes>lean>obese>type 2 diabetes;
p
< 0.001). Sarcolemmal IMTG was significantly greater in individuals with obesity and type 2 diabetes compared with lean control participants and athletes, but individuals with type 2 diabetes were the only group with significantly increased saturated IMTG. Sarcolemmal IMTG was inversely related to insulin sensitivity. Nuclear IMTG was significantly greater in individuals with type 2 diabetes compared with lean control participants and athletes, and total and saturated IMTG localised in the nucleus had a significant inverse relationship with insulin sensitivity. Total cytosolic IMTG was not different between groups, but saturated cytosolic IMTG species were significantly increased in individuals with type 2 diabetes compared with all other groups. There were no significant differences between groups for IMTG concentration in the mitochondria/endoplasmic reticulum.
Conclusions/interpretation
These data reveal previously unknown differences in subcellular IMTG localisation based on metabolic health status and indicate the influence of sarcolemmal and nuclear IMTG on insulin sensitivity. Additionally, these studies suggest saturated IMTG may be uniquely deleterious for muscle insulin sensitivity.
Graphical abstract
The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid ...molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.
Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms ...underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.
The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by ...eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study's aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo.
Serum ceramides, especially C16:0 and C18:0 species, are linked to CVD risk and insulin resistance, but details of this association are not well understood. We performed this study to quantify a ...broad range of serum sphingolipids in individuals spanning the physiologic range of insulin sensitivity and to determine if dihydroceramides cause insulin resistance in vitro. As expected, we found that serum triglycerides were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals. Serum ceramides were not significantly different within groups but, using all ceramide data relative to insulin sensitivity as a continuous variable, we observed significant inverse relationships between C18:0, C20:0, and C22:0 species and insulin sensitivity. Interestingly, we found that total serum dihydroceramides and individual species were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals, with C18:0 species showing the strongest inverse relationship to insulin sensitivity. Finally, we administered a physiological mix of dihydroceramides to primary myotubes and found decreased insulin sensitivity in vitro without changing the overall intracellular sphingolipid content, suggesting a direct effect on insulin resistance. These data extend what is known regarding serum sphingolipids and insulin resistance and show the importance of serum dihydroceramides to predict and promote insulin resistance in humans.
The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid ...acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.
•Genetic deletion of cPLA2 resulted in more severe acute murine experimental colitis.•cPLA2−/− mice have reduced levels of eicosanoids as compared to wild-type mice.•Loss of mPGES-1 in mice resulted ...in more severe colitis and impaired recovery.•mPGES-1−/− mice have altered eicosanoid levels compared to wild-types.•DSS administration to APCMin/+; mPGES-1−/− mice reduced intestinal tumor burden.
Release of the free fatty acid arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) and its subsequent metabolism by the cyclooxygenase and lipoxygenase enzymes produces a broad panel of eicosanoids including prostaglandins (PGs). This study sought to investigate the roles of these mediators in experimental models of inflammation and inflammation-associated intestinal tumorigenesis. Using the dextran sodium sulfate (DSS) model of experimental colitis, we first investigated how a global reduction in eicosanoid production would impact intestinal injury by utilizing cPLA2 knockout mice. cPLA2 deletion enhanced colonic injury, reflected by increased mucosal ulceration and pro-inflammatory cytokine expression. Increased disease severity was associated with a significant reduction in the levels of several eicosanoid metabolites, including PGE2. We further assessed the precise role of PGE2 synthesis on mucosal injury and repair by utilizing mice with a genetic deletion of microsomal PGE synthase-1 (mPGES-1), the terminal synthase in the formation of inducible PGE2. DSS exposure caused more extensive acute injury as well as impaired recovery in knockout mice compared to wild-type littermates. Increased intestinal damage was associated with both reduced PGE2 levels as well as altered levels of other eicosanoids including PGD2. To determine whether this metabolic redirection impacted inflammation-associated intestinal tumorigenesis, ApcMin/+ and ApcMin/+:mPGES-1−/− mice were exposed to DSS. DSS administration caused a reduction in the number of intestinal polyps only in ApcMin/+:mPGES-1−/− mice. These results demonstrate the importance of the balance of prostaglandins produced in the intestinal tract for maintaining intestinal homeostasis and impacting tumor development.
Adipose tissue secretes an abundance of lipid and protein mediators, and this secretome is depot‐specific, with local and systemic effects on metabolic regulation. Intermuscular adipose tissue (IMAT) ...accumulates within the skeletal muscle compartment in obesity, and is associated with insulin resistance and metabolic disease. While the human IMAT secretome decreases insulin sensitivity in vitro, its composition is entirely unknown. The current study was conducted to investigate the composition of the human IMAT secretome, compared to that of the subcutaneous (SAT) and visceral adipose tissue (VAT) depots. IMAT, SAT, and VAT explants from individuals with obesity were used to generate conditioned media. Proteomics analysis of conditioned media was performed using multiplex proximity extension assays, and eicosanoid analysis using liquid chromatography–tandem mass spectrometry. Compared to SAT and/or VAT, IMAT secreted significantly more cytokines (IL2, IL5, IL10, IL13, IL27, FGF23, IFNγ and CSF1) and chemokines (MCP1, IL8, CCL11, CCL20, CCL25 and CCL27). Adipokines hepatocyte growth factor and resistin were secreted significantly more by IMAT than SAT or VAT. IMAT secreted significantly more eicosanoids (PGE2, TXB2, 5‐HETE, and 12‐HETE) compared to SAT and/or VAT. In the context of obesity, IMAT is a distinct adipose tissue with a highly immunogenic and inflammatory secretome, and given its proximity to skeletal muscle, may be critical to glucose regulation and insulin resistance.
Intermuscular adipose tissue (IMAT) is interlaced within the muscle compartment and is negatively associated with the metabolic syndrome. This is the first report of the composition of the human IMAT secretome. In obesity, the IMAT secretome is highly immunogenic, secreting a distinct combination of cytokines, chemokines, adipokines and eicosanoids that are associated with insulin sensitivity. This study highlights the potential significance of crosstalk between IMAT and skeletal muscle in the development of type 2 diabetes.
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