Dietary lipid is largely deposited in skin and rapidly affects insulating properties

Abstract

Skin has been shown to be a regulatory hub for energy expenditure and metabolism: mutations of skin lipid metabolism enzymes can change the rate of thermogenesis and susceptibility to diet-induced obesity. However, little is known about the physiological basis for this function. Here we show that the thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We found that skin was the largest target in a mouse body for dietary fat delivery, and that dietary triglyceride was assimilated both by epidermis and by dermal white adipose tissue. Skin from mice calorie-restricted for 3 weeks did not take up circulating lipids and showed a highly depleted stratum corneum. Dietary triglyceride acyl groups persist in skin for weeks after feeding. Using multi-modal lipid profiling, we have implicated both keratinocytes and sebocytes in the altered lipids which correlate with thermal function. In response to high fat feeding, wax diesters and ceramides accumulate, and triglycerides become more saturated. In contrast, in response to the dramatic loss of adipose tissue that accompanies restriction of the branched chain amino acid isoleucine, skin becomes more heat-permeable, resisting changes induced by Western diet feeding, with a signature of depleted signaling lipids. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.

Fig. 1.. High fat diet consumption rapidly results in lower heat loss through skins.

Male and female BALBc/J mice (10–15 weeks old) were switched to HFD for 3 days, and daily food consumption assessed together with their body weight (BWt; n=6; A,B). C. Representative H&E-stained skin sections are shown for male and female BALB/cJ mice (see also Fig.S1 for comparison of skins from mice fed HFD for 3 days). * marks sebaceous glands, filled with sebocytes. Distinct tissue layers are marked. Scale bar=100μm. Skin thermal properties were measured by TEWL assay of skin (D) and pelt (E; n=6), and by FLIR assay of relative surface temperature, where °C below adjacent heat block is used to indicate relative insulation (F; n=5). To establish this response for C57BL/6J mice, males and females were fed HFD for 3 days and assayed for their relative food consumption (F; n=3–4), and the properties of their skins (assayed by TEWL of skin and pelt; G,H; n=6). * p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 2.. Skins assimilate more dietary acyl lipid than any other organ.

BALBc/J female mice were administered 3H-triolein by gavage and assessed 24 hours later (24h post-gavage, pg) for incorporation of radiolabel into the tissues indicated (pgWAT, perigonadal (visceral) WAT; iWAT, inguinal (subcutaneous) WAT; BAT, brown adipose tissue). Mice were housed either at thermoneutrality (TMN, 29°C) or cool (10°C); n≥3. A. Radiolabel assimilation was calculated as dpm/mg tissue for the tissues indicated. B. Average size of each organ was measured and expressed as mgs/g BWt. C. Calculated per organ, skins assimilated more label than any other tissue. Assimilation into iWAT was measured for the pair of iWAT depots, noting that there are 10 fat pads altogether in this class. With thermogenic fatty acid oxidation active (cool conditions), skin specific activity was reduced by 30%: a, comparison of skin from mice housed at TMN versus cool, p=0.0001. D. Skin from BALB/c female and males 2 and 24 hours after radiolabel administration showed a progressive increase in specific activity; n≥3. E-G. Skins from BALB/cJ females were separated into 3 fractions, sebome (extracted from hair), dWAT (separated from skin), and the skin remainder (epi). F. Specific activities of epi and dWAT fractions from BALBc/J females housed at TMN or 10°C (n=3) showed approximately equal distribution. G. Lipid classes were separated by thin layer chromatography (TLC), into triglyceride (TG), wax diester (WDE) and cholesterol ester (CE) and scraped for counting (a representative image is labeled with dpm/mg of tissue equivalent), from 3 adipose depots from BALB/cJ females, along with hair, skin and separated skins (SS). All radiolabel was retrieved from the triglyceride fraction.

Fig. 3.. Dietary fat persists in skin and adipose depots for several weeks.

A,B. Dilution and elimination of the radiolabel in BALB/cJ females is indicated for 2 weeks post-gavage, for serum and liver (see also Fig.S2, dpm/mg tissue, all data points; n≥3). Timepoints shown are 2h, 24h, 3 days (72h), 1 week (168h) and 2 weeks (336h) (n≥3 for each timepoint). C. A scheme of the potential fates of ³H-triolein (acyl group ³H-label): acyl group reshuffling, fatty acid oxidation (FAO), degradation of acyl groups and dilution into general metabolic pool, or uptake of the whole TG moiety. These are not discriminated by assay of dpm/mg tissue. D-G. Relative specific activity of adipose depots and skin, expressed with respect to time, for mice housed either at 29°C (approximately thermoneutral) or in cool housing (10°C). H. Statistical analysis of specific activity of tissues indicated for the 2-week timepoint.

Fig. 4.. Skins of calorie restricted mice show minimal lipid uptake, depleted dWAT and a reduced mitotic index in the interfollicular epithelium.

A. Representative H&E-stained sections from skins of C57BL/6J male and female mice fed ad libitum (AL) or calorie-restricted (30% for 3 weeks; CR; n=6), illustrate profound dWAT depletion (quantified in E). Scale bar=100μm. B-E. The relative effect of CR on body weight (B), and adipose depots (C, pgWAT; D, iWAT) for C57BL/6J males illustrates extreme lipodystrophy of dWAT depot (E). F. Uptake of ³H labeled radiotracer into total skin lipid, or triglyceride (TG) or phospholipid (PL) purified by TLC for C57BL/6J males (Fig.S9). G. Circulating levels of ³H labeled radiotracer. H. The mitotic index of keratinocytes was evaluated by Ki67 staining of paraffin-embedded skins, and the thickness of the epidermis quantified in I, J, for both C57BL/6J males and females. Scale bar =25μm.

Fig. 5.. Dietary acyl chains are taken up by skin-associated triglycerides.

A. Feeding of Western diet for 3 days induces typical changes of circulating lipid classes, shown here for C57BL/6J female mice (n=4; WD or chow-fed). Data is shown as an unsupervised heatmap of sera samples (EtherPE, ether phosphatidylethanolamines; PC, phosphatidylcholines; Cer, ceramides; CE cholesterol esters; PE, phosphatidylethanolamines; LPC, lysophosphatidylethanolamines; AcCar, acyl carnitines; FA, free fatty acids; n=4). Scale for heat map is shown as fold change. B. Volcano plots of the circulating lipidome and epidermis, showing significantly changed lipids (WD/chow), including TGs with medium chain fatty acids (MCFA), a signature of Western diet consumption. C. Fold change in specific acyl chains of serum and epidermis (epi) from 3d WD-fed mice is indicated, noting MCFA acyl chains. Total amounts of MCFA in serum, epidermis, sebome and dWAT of WD-fed mice is shown in Fig.S4. D. Fully specified MCFA-containing TGs were compared for serum and for skin, to test for a direct correlation of entire TG species (implicating direct delivery). Species marked with ○ or ○ are unique to either serum or skin (respectively) or are present only in serum (WD-fed only). E. Specific activity of adipose depots and skins from C57BL/6J female mice administered tetrahydrolipstatin (THL, lipoprotein lipase inhibitor) before radiotracer administration (n=3). See also MCFA content and lipid profiles of diets (Fig.S3).

Fig. 6.. Mice fed a low-isoleucine Western diet rapidly develop heat-permeable skins.

A,B. C57BL/6 female mice made obese by 16 weeks of WD feeding (WD DIO; TD.88137), were switched to low isoleucine (WDIL; TD.200692), low branched chain amino acid (WD 1/3xBCAA; TD.200691), or amino acid-defined WD (WD DIO; TD.200690). A control cohort were maintained on a regular fat content diet for the duration (no DIO; TD200693). B. Body composition was measured at 3 weeks post diet-switch (see also Fig. S5 for lean/fat body composition assay results; n≥4), illustrating the loss of nearly 15g of body fat within 21 days. C. To test the impact of a switch to WDIL diet feeding, female C57BL/6J mice (10–15 weeks old) were switched to Western diet (WD) or low-isoleucine Western diet (WDIL) for 3 days, and daily water and food consumption assessed (expressed as both grams/day or kcal/day/g body weight; C-E, n=7–8). F. Body weights were measured after 3 days. G,H. Changes to adipocyte depot lipid loads were calculated as the average area of pgWAT adipocytes, thickness of dWAT, or the lipid droplet area/cell for BAT, shown also as representative H&E-stained skin sections. I-K. TEWL and FLIR assays of thermal barrier function for mice fed chow, WD, or WDIL for 3 days (n=7–8, TEWL; n=5 FLIR).

Fig. 7.. Multi-modal lipid analysis of skins.

Skin tissue fractions produced as described in Fig.2 and Materials and Methods were analyzed for their lipid composition using LC/Q-TOF mass spectrometry. A. Using AUC as an approximation, the relative amounts of each detectable lipid class are described for each of sebome, epidermis and dWAT. The larger grey circle shown around the sebome indicates the neutral lipid wax esters (and potentially other classes) which are under-annotated by mass spectrometry. The larger pink circle around the epidermal lipids indicates the cross-linked proteolipid sheath called the stratum corneum, highly enriched with ceramides, which is not solubilized by these methods, therefore not detectable. B. The total number of reliably detected unique species identified for this analysis of epidermis or sebome from female mice fed HFD is shown. (Ceramide score includes both positive and negative modes). C. To interrogate the majority wax diester (WDE) class that predominates in sebome, WDE fractions are retrieved from preparative TLC plates and acyl chains derivatized to FAMEs, for analysis by GC/MS. Diacylglycerol O-acyltransferase (DGAT1) can serve as a wax synthase, and DGAT1 knockout mice have deficiencies in skin wax synthesis and function^,, therefore extracts from mice with a DGAT1 mutation were used to indicate which fractions are likely to be wax diesters. CE, cholesterol esters; WDE, wax diesters: b4 (band4), unknown species; TG, triglycerides; FFAs, free fatty acids; chol, cholesterol. D. Comparison of ceramide species present in serum, skin and sebome (see also Fig.S6, annotated species), to illustrate the specificity of each ceramide cohort. E. Quantitation of total amounts of ceramide species identified by LC/QTOF-MS in serum, skin and sebome per mg of tissue (taken from positive mode data). F. Diversity of ceramide species shown for sebome and skin (total number of species identified in negative mode). Annotation key: S=sphingosine, P=phytosphingosine, DS=dihydro-sphingosine, A=α-hydroxy, B=β-hydroxy, N=non-hydroxylated.

Fig. 8.. High fat diet consumption results in changes to components known to be thermally active in both sebome and skin lipidomes.

A,B. The relative abundance (HFD/chow) of each lipid class is shown for sebome and epidermis from both BALB/cJ males and females. This HFD/chow ratio is calculated for males and females separately, since their skins are so different. The numbers in red along the top row show the total number of species compared for each class (n=4; additional lipid abbreviations noted here are DGs, diacylglycerols; HexCers, hexaceramides; PIs, phosphoinositides; PSs, phosphoserines; SMs, sphingomyelins). C. Evaluation of efficiency of FAME derivatization of WDE fraction. Shown is a charred analytical TLC separation of lipid extracts from hair of mice fed control or HFD diets for 3 days (pooled from 3 mice, repeated twice on separate samples). The lipid mix directly extracted from hair is shown labeled in grey, the WDE fraction after base-esterification to derivatize acyl-fatty acid methyl esters (FAMEs) are shown labeled in black (no residual bands at WDE moblity). Standards are shown in blue. WDE, wax diester; b4, unidentified band 4; FFA (free fatty acids) and TGs are identified by comigration with standards. Tentative identification of alkanes and fatty dihydroxy alcohols (FAdiOH) is also indicated. D. Quantitation of the increase in TGs present in sebome from male and female BALBc/J mice fed HFD, together with the relative acyl chain composition of the TGs (E). F. Each of the top 5 ceramide classes in sebome of HFD-fed male BALB/cJ mice increases by approximately the same degree (n=4). G,H. Quantitation of total FAMEs (G), together with specific data on 35 FAME species derived from the wax diester fraction of sebome, observed in the resolution range of the GC column. Peaks are reported as retention times, chain lengths C18-C26. Nine FAME species were tentatively identified by comparison with FAMEs in the standard mix (see also Fig.S7 for abundance waterfall plot). I. Summary scheme of the changes of sebome and epidermis observed in response to HFD feeding.

Fig. 9.. Low isoleucine Western diet prevents dietary delivery of acyl chains and mobilization of pro-inflammatory oxylipids from the epidermal reservoir.

A. Relative appearance of Western-diet enriched MCFAs in epidermis of WDIL-fed C57BL/6J female mice compared to WD-fed mice (see also Fig.4; n=4). B. Heatmap of changes induced in circulating lipidome for WD- and WDIL-fed mice. C,D. The relative abundance of each lipid class is shown for C57BL/6J male sebome and epidermis, for control, WD and WDIL-fed mice. See also Fig.S8, inventory of total species interrogated. E. Oxylipid acyl chains, precursors of inflammatory cytokines (shown left hand side) were quantified for epidermal fractions from mice in all 3 diet conditions.

Fig. 10.. Summary of changes of heat-permeable skins induced by feeding low isoleucine diet.

A. Several classes of lipids show changes specific to low isoleucine-fed C57BL/6J female mice (A-D, n=4). Illustrated are the trends present for the entire class, followed by a specific example of each. E. Quantitation of WDEs and FAME derivatization by TLC for all 3 diet conditions (annotated as for Fig.8). F. FAME mixtures were analyzed by GC of WDE-derived FAMEs (prepared as for Fig.8H), illustrating the general suppression of WDE synthesis by WD consumption, and the reversal of this change when diet contains low isoleucine (see also Fig.S7, waterfall plot showing relative abundance). G. Summary scheme of the changes of sebome and epidermis observed in response to WD/WDIL feeding.