
Patients with diabetes mellitus (DM) often suffer from diverse skin disorders, which might be attributable to skin barrier dysfunction. To explore the role of lipid alterations in the epidermis in DM skin disorders, we quantitated 49 lipids (34 ceramides, 14 free fatty acids (FFAs), and cholesterol) in the skin epidermis, liver, and kidneys of db/db mice, a Type 2 DM model, using UPLC-MS/MS. The expression of genes involved in lipid synthesis was also evaluated. With the full establishment of hyperglycemia at the age of 20 weeks, remarkable lipid enrichment was noted in the skin of the db/db mice, especially at the epidermis and subcutaneous fat bed. Prominent increases in the ceramides and FFAs (>3 fold) with short or medium chains (
Hundreds of lipid species occur in the skin epidermis, where they constitute structural components, maintain cutaneous physiology, and participate in the progression of diseases (Kendall
Alterations in ceramides and FFAs are closely associated with the pathophysiology of various dermatoses (Ishikawa
In diabetes mellitus (DM), which causes profound and broad changes in energy metabolism and dyslipidemia (Kumar
Here, we examine the epidermal lipids of db/db mice at 20 weeks of age, which represent Type 2 DM with long-standing hyperglycemia (Quondamatteo, 2014). We used sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to quantitate 34 ceramides (CER-NP (non-hydroxy fatty acid conjugated to phytosphingosine), CER-NDS (non-hydroxy fatty acid conjugated to dihydrosphingosine), CER-NS (non-hydroxy fatty acid conjugated to sphingosine), and CER-AP (α-hydroxy fatty acid conjugated to phytosphingosine) with varying carbon chain lengths), 14 FFAs (C12, C14, C16, C16:1, C18, C18:1, C18:2, C20, C22, C22:1, C24, C24:1, C26, and C28) and cholesterol (CHOL) in the skin epidermis of db/db mice compared to their normal littermates, db/m mice. In addition, to explore the relationship between the skin and body lipidomes, we examined liver and kidney lipid profiles, the organs most affected by DM. We used principal component analysis (PCA), a metabolomics tool widely used to identify novel biomarkers (Joo
An AccQ•Tag Ultra derivatization kit (borate buffer and reagent) and AccQ•Tag Ultra eluents A and B were obtained from Waters (Milford, MA, USA). Ammonium formate, formic acid, and potassium hydroxide were acquired from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid solution and sodium hydroxide were purchased from Daejung (Seoul, Korea). HPLC-grade methanol, ethyl acetate, isopropyl alcohol, and chloroform were purchased from Burdick & Jackson (Muskegon, MI, USA). CHOL and the FFAs (C12, C14, C16, C16:1, C18, C18:1, C18:2, C20, C22, C22:1, C24, C24:1, C26, and C28) were obtained from Sigma-Aldrich. Deuterated FFAs C16-D3, C20-D3, and C18:1-D2 were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Synthetic CER-NP, CER-NDS, and CER-NS were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Synthetic CER-AP was kindly provided by Evonik (Essen, Germany). 2,2′-dithiodipyridin, 2-picolylamine, and triphenylphosphine were purchased from Sigma-Aldrich. All other reagents used were of the highest grade available. The water used was ultra-pure deionized water (18.2 MΩ•cm) produced using a Milli-Q Gradient system (Millipore, Bedford, MA, USA).
C57BLKS/J-db/db and age-matched control C57BLKS/J-m+/db mice (five-week-old males, Japan SLC Inc., Hamamatsu, Japan) were housed in a room maintained at 22 ± 2°C with a 12 h dark/12 h light cycle. All animal experiments were conducted according to the Institutional Animal Care and Use Committee of the Ewha Laboratory Animal Genomics Center (IACUC-14-109). Mice were given free access to standard rodent pellets (Purina, Seongnam, Korea). Body weight, fasting blood glucose concentration, and HbA1c level were measured at 8, 14, and 20 weeks. Blood was withdrawn from the cervical vein at the indicated time points; the blood hemoglobin A1c (HbA1c) level was determined using a DCA2000 HbA1c reagent kit (SIEMENS Healthcare Diagnostics, Inc., Tarrytown, NY, USA), and blood glucose was measured using a glucometer (OneTouch Ultra, Johnson & Johnson Co., CA, USA). The moisture and trans-epidermal water loss (TEWL) of each group was measured at the end of the experiments (20 weeks) with a MoistureMeterSC and Vapometer (Delfin Technology, Kuopio, Finland), respectively, after removing hair with an electrical shaver. Liver and skin samples were collected after sacrifice with CO2.
For the histological examination of the skin, 7 mm×1 cm full-thickness skin was excised from the dorsum near the tail and stitched to filter paper before fixing in 10% formalin. For the oil red O staining, skin samples were freshly collected and immediately frozen in Optimal Cutting Temperature (OCT) compound (Tissue TEK®, Sakura Finetek, Tokyo, Japan). Frozen sections were made 8 μm in thickness using a cryostat. Slides were fixed in 70% ethyl-alcohol and then placed in propylene glycol for 5 min. After that, the slides were incubated in 0.7% oil red O (Sigma-Aldrich) solution for 7 min at 60°C. Slides were rinsed with 85% propylene glycol for 3 min and then rinsed with distilled water. Counterstaining was done with Mayer’s hematoxylin for 5 min, and then the slides were rinsed thoroughly in tap water. Slides were mounted in warmed glycerin jelly solution.
To examine the immunohistochemistry of ceramide, paraffin-embedded skin sections were de-paraffinized and sequentially rehydrated. Antigen retrieval was performed using ph6.0 Target Retrieval solution (DAKO, Glostrup, Denmark) in a pressure cooker for 15 min. After cooling on ice for at least 1 h, sections were incubated in 3% H2O2 for 30 min to block endogenous peroxidase. Sections were washed twice with PBS and blocked with a Mouse on Mouse (M.O.M.) kit (Vector Laboratories, Burlingame, CA, USA) overnight at 4°C followed by a protein block (DAKO) for 1 h at room temperature. Then, sections were incubated with anti-ceramide antibody (1:1000, Enzo, Farmingdale, NY, USA) overnight at 4°C. After 3 washes in PBS, sections were incubated in horseradish peroxidase (HRP)-conjugated secondary mouse antibody (DAKO) for 15 min at room temperature. For immunohistochemistry experiments, diaminobenzidine (DAB) (DAKO) was used for antibody development, and Mayer’s hematoxylin (DAKO) was used for counter staining.
To prepare the epidermal sheet for lipid sampling, the skin was disinfected with povidone and treated in 0.25% EDTA-free trypsin for digestion overnight. The dermis was removed with tweezers to collect the epidermis. To prepare the liver tissue sample, the liver was perfused with PBS, and 100 mg tissue was collected from the same lobe in each animal. For the kidney samples, about 50 mg of kidney cortex was collected.
Epidermal sheets or tissues were minced into small pieces in ice-cold 0.1% ammonium acetate followed by homogenization in a Precellys 24 (6500 rpm 1×30s, Bertin Instrument, Montigny-le-Bretonneux, France). Methanol (1.5 mL) was added to a 200 μL sample aliquot, which was placed into a glass tube with a Teflon-lined cap, and the tube was vortexed. Then, 5 mL of methyl-tertbutyl ether (MTBE) was added, and the mixture was incubated for 1 h at room temperature in a shaker. Phase separation was induced by adding 1.25 mL of MS-grade water. After 10 min of incubation at room temperature, the sample was centrifuged at 1,000 g for 10 min. The top 4 mL (organic phase) was collected. The solvent was volatilized using a Speedvac (EZ-2 Plus, Genevac, Ipswich, UK). Each 4 mL of the filtrate was united and evaporated to dryness using a Speedvac at 40°C. The residues (extractable lipids) were reconstituted in 500 μL of chloroform–methanol 2:1 (v/v) for analysis by UPLC-MS/MS. A standard stock solution of individual lipids (ca. 1 mg/mL) was prepared by dissolving specific amounts of authentic standards in chloroform-methanol (1:1, v/v) and then storing it at −20°C. The working standard solutions of the lipid mixtures were serially diluted with chloroform-methanol (2:1, v/v) to obtain the concentrations needed for calibration curve standards. FFAs were quantified through derivatization according to a method previously described (Joo
The chromatographic separation was carried out using an ACQUITY UPLC system (Waters, Manchester, UK) equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1×50 mm). The column temperature and autosampler tray temperature were maintained at 55°C and 4°C, respectively. The mobile phase for the CER (CER-NDS, CER-NS, CER-AP, CER-NP) and FFA analyses consisted of methanol: water=1:1 (v/v, 2 mM ammonium formate and 0.1% formic acid, solvent A) and methanol: isopropyl alcohol=6:4 (v/v, 2 mM ammonium formate and 0.1% formic acid, solvent B). Gradient elution was initiated with 20% B (50% B for the CER analysis) for 0.5 min and changed to 100% B in 2 min. The isocratic elution with 100% B was kept for 5 min and then returned to 20% B (50% B for CERs analysis) in 6.5 min. The total run time was 6.5 min. The flow rate was 0.3 mL/min, and the injection volume was 2 μL.
The UPLC-MS/MS analysis was performed using a Waters Micromass Quattro Premier XE triple quadrupole mass spectrometer. The ESI-MS spectra were acquired in the positive ion multiple reaction monitoring (MRM) mode with a capillary voltage of 3.5 kV, source temperature of 120°C, desolvation temperature of 350°C, desolvation gas flow rate of 750 L/h, and cone gas flow rate of 50 L/h. The optimum cone voltages for the FFAs and CERs were set to 20 and 25 eV, respectively. The collision energy for the MRM mode was optimized for each analyte.
The cholesterol analysis was performed with a gradient elution of water (solvent A) and acetonitrile (solvent B). The gradient elution was initiated with 85% B for 0.5 min and changed to 100% B in 1.5 min. The isocratic elution with 100% B was kept for 3.5 min and then returned to 85% B in 5.0 min. The flow rate was 0.4 mL/min, and the injection volume was 2 μL.
The APCI-MS spectra were acquired in positive ion mode with an APCI corona current of 4.0 mA, a source temperature of 120°C, desolvation temperature of 400°C, and desolvation gas flow rate of 500 L/h. The optimum cone voltage was 35 V. The collision energy for the MRM mode was optimized to 6 eV (
Liver and skin samples were lysed using Trizol (Invitrogen, CA, USA). After the addition of chloroform, samples were centrifuged at 12,000 rpm for 10 min. The aqueous phase was mixed with isopropanol, and RNA pellets were collected by centrifugation (12,000 rpm, 15 min, 4°C). The RNA pellets were washed with 70% ethanol and dissolved in RNase-free, DEPC (diethyl pyrocarbonate)-treated water (Waltham, MA, USA). The RNA yield was estimated by determining the optical density at 260 nm with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, INC., Wilmington, DE, USA).
The relative mRNA expression levels were measured using quantitative real-time PCR. cDNA was synthesized from 1250 ng of total RNA with oligo (dT) (Bioelpis, Seoul, Korea). SYBR Green PCR master mix and a StepOnePlusTM real-time PCR machine (Applied Biosystems, Warrington, UK) were used in each reaction. The sequence of primers was as follows: forward LXRα, 5′-ACT TTG CCA AAC AGC TCC CT-3′; reverse LXRα, 5′-AAG GTG ATG CTC TCA CTG CC-3′; forward LXRβ, 5′-TGG ACG ATG CAG AGT ATG CC-3′; reverse LXRβ, 5′-TCC TCG TGT AGG AGA GGA GC-3′; forward PPARγ, 5′-TGA ACG TGA AGC CCA TCG AG-3′; reverse PPARγ, 5′-CGA TCT GCC TGA GGT CTG TC-3′; forward Elovl6, 5′-CTG GAT GCA GCA TGA CAA CG-3′; reverse Elovl6, 5′-GCC GAT GTA GGC CTC AAA GA-3′; forward Elovl1, 5′-TAC CCC ATC ATC ATC CAC CT-3′; reverse Elovl1, 5′-GGA GCT CCA TTT TGC TGA AC-3′; forward Elovl4, 5′-GTC TCT CTA CAC CGA CTG CC-3′; reverse Elovl4, 5′-CCG GTT TTT GAC TGC TTC GG-3′; forward FAS, 5′-AGC TAC CGG GCA AAG ATG AC-3′; reverse FAS, 5′-CCC GAT CTT CCA GGC TCT TC-3′; forward SCD, 5′-AGC CTG TTC GTT AGC ACC TT-3′; reverse SCD, 5′-CCA GGA TAT TCT CCC GGG ATT G-3′. Cycling parameters were 51°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 51°C for 1 min.
Data are presented as the mean ± SD. PCA for the lipid profiles were done using SIMCA-P+ (v12.0 version, Umetrics, Umea, Sweden). Data were analyzed by Student’s t-test to identify statistically significant differences from the control group. Significance was acknowledged when
DM was well-established in the db/db mice from the age of 14 weeks, as determined by high body weights (Fig. 1A, 1B), evident hyperglycemia (Fig. 1C), and significantly increased levels of hemoglobin A1c (HbAc1) (Fig. 1D). At the age of 20 weeks, HbAc1 was 2.6 fold higher than in the db/m mice, and blood glucose exceeded the control by >3.0 fold, reflecting a chronic stage of severe hyperglycemia. At 20 weeks, skin moisture and TEWL were significantly lower in the db/db mice than in the db/m mice (Fig. 1E), suggesting the impairment of epidermal homeostasis. Histological examination revealed that the skin of the db/db mice was markedly thickened (Fig. 2A), which was largely attributable to an increased subcutaneous fat bed. In contrast, the epidermis and dermis of the db/db mice was rather compressed and had a flaky appearance. Lipid-specific oil-red O staining of the skin suggested a profound increase in the lipids in the epidermis and subcutis of the db/db mice (Fig. 2B). The immunohistochemical analysis of ceramide using ceramide-specific antibody revealed that the distribution and content of ceramides were also substantially altered in the skin epidermis of the db/db mice (Fig. 2C).
Ceramides, FFAs, and CHOL constitute the major skin epidermal lipid classes. Their composition significantly affects epidermal homeostasis, and their alteration is closely linked with skin diseases (Li
To investigate the mechanism underlying the alteration of epidermal and liver lipids in db/db mice, we evaluated the expression of genes associated with lipid synthesis reported previously (Rabionet
In this study, we have demonstrated that db/db mice at the age of 20 weeks (representing longstanding, chronic hyperglycemia) exhibited reduced skin moisture and TEWL (Fig. 1A–1E) compared to controls. Of note, ceramides and FFAs with medium chains were highly enriched in the skin epidermis of db/db mice, and those alteration patterns were repeated in the liver (Fig. 3A, 3B), whereas in the kidney, the difference from db/m mice was markedly attenuated (Fig. 3C), suggesting the existence of cross-talk in lipid synthesis and metabolism between the epidermis and the liver.
In this study, we demonstrated that db/db mice show significantly reduced skin hydration and TEWL compared to db/m mice (skin moisture, 3.6 ± 0.18 (n=10) vs. 13.6 ± 3.8 (n=5); TEWL 14.87 ± 0.5944 (n=10) vs. 30.82 ± 2.481 (n=5), t-test,
Ceramides and FFAs with medium chains were enriched in the skin epidermis (Fig. 3A) of db/db mice, and those patterns were similarly observed in the liver (Fig. 3B). Interestingly, the alteration pattern of lipids in DM was distinct from that observed in skin affected by atopic dermatitis (AD), a representative inflammatory dermatose with xerosis. In AD, asymmetric alteration of ceramides across chain lengths was observed, and ceramides with short chains (≤C22) increased in the epidermis, whereas ceramides with medium or long chains (≥C24) decreased significantly (Joo
In addition to hyperglycemia, DM causes dyslipidemia, although its extent and patterns vary across tissues and with disease severity (Sas
Interestingly, the lipid profiles of both db/db and db/m mice were similar in the skin and livers of individual animals, suggesting cross-talk between the skin and liver in lipid synthesis and metabolism. The PCA revealed a remarkable difference in the lipids of both the skin and the liver between db/db and db/m mice (Fig. 4A–4F). Of note, the lipids of marked distinction in both tissues largely overlapped, further supporting the close relationship between the skin and the liver in lipid synthesis. Indeed, the liver is known as a central organ for synthesizing and distributing lipids to peripheral tissues, and coordinated changes in certain lipids occur between the skin and the liver in disease states (Miyazaki
In addition, we demonstrated that the
In conclusion, we have demonstrated that remarkable changes occur in the lipid profiles of the skin epidermis of db/db mice, a Type 2 DM model, and that those changes coincide with changes in the liver and kidneys. The alteration of these lipids, which are critical to the normal homeostasis of the skin epidermis, could explain, at least in part, the abnormal skin barrier, hydration, and compromised defense against infection seen in DM patients.
None.
This work was supported by the National Research Foundation of Korea (Grant No. 2016R1A2B4006575 and 2017R1A6A3A11034070) and the Korea government (MSIT) (2018R1A5A2025286).
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