2022 Volume 45 Issue 7 Pages 934-939
Various diabetic drugs have been developed as the number of patients with type 2 diabetes has increased. Sodium-glucose cotransporter (SGLT)-2 inhibitors have been developed as novel therapeutic agents. However, SGLT-2 inhibitors cause skin dryness. The mechanism through which SGLT-2 inhibitors cause skin dryness is unknown. The purpose of this study was to investigate the mechanism through which dapagliflozin, a SGLT-2 inhibitor, induces skin dryness. Specific pathogen-free KK-Ay/TaJcl (type 2 diabetes model) mice were orally administered with SGLT-2 inhibitor (dapagliflozin) daily for 4 weeks at a dose of 1 mg/kg/d. Skin dryness induced in KK-Ay/TaJcl mice became severe after dapagliflozin administration. Dapagliflozin treatment decreased collagen type I and hyaluronic acid levels in mice; additionally, it affected the transforming growth factor (TGF)-β/hyaluronan synthase pathway, further reducing hyaluronic acid levels. The results indicate that the reduction in hyaluronic acid levels plays an important role in the occurrence of dry skin in diabetes.
In recent years, the number of diabetic patients has rapidly increased due to the westernization of food habits and alteration of social environment. In patients with type 2 diabetes, a lifestyle-induced disease, life improvement and pharmacotherapy are important.1) Progression of type 2 diabetes can cause complications such as nephropathy, retinopathy, neuropathy, myocardial infarction, and cerebral infarction.2) The purpose of medication is to prevent the onset and evolution of these symptoms via glycemic control. In 2014, a sodium-glucose cotransporter (SGLT)-2 inhibitor, which could inhibit glucose re-absorption in the kidney, was launched in the market. This SGLT-2 inhibitor can control blood glucose via an insulin-independent mechanism. As a result, the SGLT-2 inhibitor eliminates glucose toxicity and reduces burden on pancreatic beta cells. However, systemic tetter has been reported by the patients, and skin lesions have been observed.
The skin, which covers the entire body, acts as a physical and immunological barrier. Since an allergen or organism can easily invade dry skin,3) dry skin can lead to infectious diseases and allergies. Furthermore, itching occurs due to dryness, and dryness becomes severe due to excochleation.4)
In a previous study, an increase in the amount of moisture transpirations of the skin and a decrease in the amount of moisture retention as well as dry skin were observed in the KK-Ay/TaJcl type 2 diabetes model mice (KK-Ay/TaJcl mice).5) Furthermore, in KK-Ay/TaJcl mice, a decrease in the contents of hyaluronic acid and collagen type 1 of the skin was observed.6) A previous study showed that skin dryness is induced by reduction in collagens.7) We also found that skin dryness was induced in various diseases, collagen loss was also induced.8–11) This decrease in collagen was thought to be due to the destruction of the dermis and the barrier function of the skin. KK-Ay/TaJcl mice are obese, and inflammatory cytokines and reactive oxygen species are secreted from fat cells.12) These secretions indicated that skin dryness is induced by the effects of fibroblasts and mast cells.13) However, the influence of SGLT-2 inhibitors on skin dryness in KK-Ay/TaJcl mice has not been reported till date.
In this study, we aimed to examine the influence of dapagliflozin, a SGLT-2 inhibitor, on skin dryness in a type 2 diabetes mice model.
Ten-week-old specific-pathogen-free (SPF) C57BL/6N and KK-Ay/TaJcl male mice (CLEA Japan Inc., Tokyo, Japan) were used in the study. Mice were individually maintained in cages in an air-conditioned room at 23 ±1 °C under SPF conditions with a 12 h light–12 h dark cycle, with free access to drinking water and a pelleted basal diet. After 4 weeks (28 d), body weight (g), water intake (mL/d), and urine production (g/d) were measured. Additionally, small blood samples were collected from the tail vein of each mouse to measure blood glucose levels. Mice with a blood glucose level of 300 mg/dL or higher were considered to be diabetic. All experimental protocols were approved by the ethics committee of the Suzuka University of Medical Science. The study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Suzuka University of Medical Science (Approval No. 34) and carried out according to ARRIVE guidelines. All surgeries were performed under pentobarbital anesthesia, and efforts were made to minimize animal suffering.
Dapagliflozin TreatmentApproximately 1 mg/kg/d of dapagliflozin (Carbosynth, Compton, Berkshire, U.K.) in distilled water was orally administered for 4 weeks daily, and distilled water was administered to control animals.14)
Measurement of Transept Water Loss (TEWL) and Capacitance on Dorsal SkinTEWL and skin hydration were measured as an index of the skin barrier function. After 4 weeks, the dorsal skin of TEWL and skin hydration were evaluated by Tewameter TM300 (Courage + Khazaka Electronic GmbH, Cologen, Germany) and Corneometer CM825 (Courage + Khazaka Electronic GmbH), as in previous studies.15)
Preparation and Staining of the Dorsal SkinThe dorsal skin samples were collected from mice sacrificed and were fixed with 4% paraformaldehyde in phosphate buffer. After fixation, sample was frozen in Tissue Tec OCT compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Frozen sections were cut at 5 µm and was used for histological studies. For establishment of method to enable histological analysis, sections were hematoxylin–eosin stained. Collagen expression was evaluated by the Masson trichrome technique (trichrome stain kit [modified Masson’s]; ScyTec Laboratories, Inc., Logan, UT, U.S.A.).16) Toluidine blue staining was used for identifying mast cells. In addition, the stained sections of skin were observed by microscope and assessed according to conventional methods. Collagen type I was calculated from five random visual fields with constant area using Image J software (National Institutes of Health, Bethesda, MD, U.S.A.).
Enzyme-Linked Immunosorbent Assay (ELISA) Analysis of Cytokines and Matrix Metalloproteinase (MMP)-1 in Plasma, and Hyaluronic Acid, Hyaluronidase, and Hyaluronan Synthase (HAS)2 in SkinPlasma concentrations of interleukin (IL)-1, IL-6, and IL-10, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, MMP-1, hyaluronic acid, hyaluronidase, and HAS2 were measured using appropriate ELISA kits (IL-1 and IL-10, Proteintech, Rosemont, IL, U.S.A.; IL-6, Enzo Life Sciences, Farmingdale, NY, U.S.A.; TNF-α, TGF-β, and hyaluronic acid, R&D Systems, Minneapolis, MN, U.S.A.; MMP-1, MyBioSource, San Diego, CA, U.S.A.; hyaluronidase, AB Clonal Inc., Tokyo, Japan; and HAS2, LSBio, Seattle, WA, U.S.A.) according to manufacturer’s instructions. Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A.).
Statistical AnalysisEach data are expressed as the mean ± standard deviation (S.D.). More than two groups were compared by Tukey’s post-hoc test or Steel–Dwass test. Statistically significant was assumed at p < 0.05.
Figure 1 displays the body weight (Fig. 1A), water intake (Fig. 1B), urine production (Fig. 1C), and blood glucose (Fig. 1D) levels in KK-Ay/TaJcl mice. Body weight of KK-Ay/TaJcl mice was higher than that of control mice. Additionally, water intake, urine production, and glucose levels were also higher in KK-Ay/TaJcl mice than in control mice. In contrast, the body weight, water intake, and urine production in dapagliflozin-treated KK-Ay/TaJcl mice were similar to those in KK-Ay/TaJcl mice. However, the blood glucose level was ameliorated following dapagliflozin administration.
Values are expressed as the mean ± S.D. of five animals. * p < 0.05, ** p < 0.01. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
To examine skin dryness, we measured both TEWL (Fig. 2A) and skin hydration (Fig. 2B) levels of the stratum corneum in KK-Ay/TaJcl and control mice. No significant difference was observed in TEWL levels between the four groups. On the other hand, skin hydration levels were lower in KK-Ay/TaJcl mice than in control mice, and the skin hydration levels were lower in dapagliflozin-treated KK-Ay/TaJcl mice than in KK-Ay/TaJcl mice.
Values are expressed as the mean ± S.D. of five animals. ** p < 0.01. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
The dermal expression of collagen in KK-Ay/TaJcl mice was lower than that in control mice (Fig. 3). In KK-Ay/TaJcl mice, the dapagliflozin-treated group showed higher values than the vehicle-treated group. There was no difference in control mice between the dapagliflozin-treated group and the vehicle-treated group.
Histological analysis of skin sections, performed using hematoxylin–eosin staining (A) and Masson-trichrome staining (B). The data show one representative experiment performed on five animals. Values are expressed as the mean ± S.D. derived from five animals. * p < 0.05, ** p < 0.01. Scale bar = 100 μm. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
The plasma levels of TNF-α (Fig. 4A), IL-6 (Fig. 4B), and IL-10 (Fig. 4C) increased in KK-Ay/TaJcl mice. In dapagliflozin-treated KK-Ay/TaJcl mice, although TNF-α level was not different from that of KK-Ay/TaJcl mice, IL-6 and IL-10 levels increased as compared to those in KK-Ay/TaJcl mice. On the other hand, plasma TGF-β levels (Fig. 4D) were lower in KK-Ay/TaJcl mice than in the control mice. Furthermore, TGF-β levels were lower in dapagliflozin-treated KK-Ay/TaJcl mice than in KK-Ay/TaJcl mice.
We measured the plasma levels of TNF-α, IL-6, IL-10, and TGF-β in mice using ELISA. Values are expressed as the mean ± S.D. derived from five animals. * p < 0.05, ** p < 0.01. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
HAS2 level in the skin of KK-Ay/TaJcl mice decreased compared with that of the control group (Fig. 5A). Additionally, HAS2 levels in the skin of dapagliflozin-treated KK-Ay/TaJcl mice were lower than those in KK-Ay/TaJcl mice. Conversely, the dermal level of hyaluronidase increased in KK-Ay/TaJcl mice, and it increased more in dapagliflozin-treated KK-Ay/TaJcl mice (Fig. 5B). The dermal level of hyaluronic acid decreased in KK-Ay/TaJcl mice than in control mice. Further, dermal hyaluronic acid levels were lower in dapagliflozin-treated KK-Ay/TaJcl mice than in KK-Ay/TaJcl mice (Fig. 5C).
We measured the levels of HAS2, hyaluronidase, and hyaluronic acid in the skin of mice using ELISA. Values are expressed as the mean ± S.D. derived from five animals. * p < 0.05, ** p < 0.01. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
Plasma IL-1 (Fig. 6A) and MMP-1 (Fig. 6B) levels and expression of mast cells in the skin (Fig. 6C) were the highest in KK-Ay/TaJcl mice, and they decreased following dapagliflozin treatment.
Arrows indicate mast cells. The expression of mast cells in skin sections was determined using toluidine blue staining. The data show one representative experiment performed on five animals. Scale bar = 100 μm. The plasma levels of IL-1 and MMP-1 in mice were measured using an ELISA kit. Values are expressed as mean ± S.D. derived from five animals. * p < 0.05, ** p < 0.01. Dapa: Dapagliflozin. DM: KK-Ay/TaJcl mice. Control: C57BL/6j mice.
In this study, skin dryness was induced in KK-Ay/TaJcl mice, and it became severe after dapagliflozin administration. The total collagen expression decreased in KK-Ay/TaJcl mice; however, the decrease in collagen expression was suppressed following dapagliflozin administration. Additionally, plasma levels of TNF-α, IL-6, and IL-10 were higher in KK-Ay/TaJcl mice than in control mice. Furthermore, IL-6 and IL-10 levels increased with dapagliflozin administration. Conversely, TGF-β levels decreased in KK-Ay/TaJcl mice compared with that in control mice, and it further decreased after dapagliflozin administration. HAS2 and hyaluronic acid levels significantly decreased and hyaluronidase level significantly increased in dapagliflozin-treated KK-Ay/TaJcl mice compared with that in KK-Ay/TaJcl mice. Additionally, the levels of IL-1 and MMP-1 and number of mast cells significantly increased in KK-Ay/TaJcl mice compared with those in control mice but decreased in dapagliflozin-administered KK-Ay/TaJcl mice compared with those in KK-Ay/TaJcl mice.
Skin dryness occurred in KK-Ay/TaJcl mice, and a decrease in the contents of hyaluronic acid and collagen was suggested as the cause. This mouse model of diabetes is associated with obesity. Therefore, it was considered that the cause of decrease in collagen content was due to inflammation induced by diabetes caused by obesity. An increase in the levels of IL-1, an inflammatory cytokine, activates mast cells.17) Activated mast cells increase the secretion of MMP-1, a collagenolytic enzyme, and it leads to a reduction in collagen levels.18) With the decrease in hyaluronic acid levels due to a high amount of moisture retention, the increase in the levels of inflammatory cytokines, such as IL-6, is a concern. It has been reported that IL-6 synthesizes hyaluronidase, a hyaluronic acid degrading enzyme, and it decomposes hyaluronic acid.19,20)
On the other hand, when dapagliflozin was administered to KK-Ay/TaJcl mice, a depression in TGF-β levels and increase in IL-10 levels were induced. IL-10 inhibits inflammatory cytokines and suppresses collagen degradation.21,22) Therefore, it was considered that in dapagliflozin administered KK-Ay/TaJcl mice, the drug suppressed skin dryness compared with that in KK-Ay/TaJcl mice. However, in this study, skin dryness of dapagliflozin-administered KK-Ay/TaJcl mice deteriorated as compared to that of KK-Ay/TaJcl mice. A depression in TGF-β could be one of the reasons. Although TGF-β promotes the synthesis of hyaluronic acid,23) TGF-β production is inhibited via dapagliflozin administration, HAS decreases, and as a result, hyaluronic acid levels decrease. Although collagen levels increased via dapagliflozin administration, the decrease in hyaluronic acid was strongly inclined to skin dryness. Additionally, amelioration of obesity was not observed, although an amelioration of blood sugar level was observed after dapagliflozin administration. Therefore, there was a possibility of no decrease in the levels of the inflammatory cytokine from adipose cell origin and decomposition of hyaluronic acid.
Furthermore, skin dryness was induced by administering dapagliflozin to control mice. Dapagliflozin treatment increased the levels of inflammatory cytokines, such as IL-1, IL-6, and TNF-α, in the control group, and this inflammatory cytokine was considered to have disassembled hyaluronic acid. However, it is not known how dapagliflozin affects these cytokines. In particular, there are no reports on the effects of dapagliflozin on the skin. Therefore, further studies are needed on the effects of dapagliflozin on each organ.
In this study, we observed skin dryness in KK-Ay/TaJcl (type 2 diabetes model) mice. Furthermore, skin dryness deteriorated more after administration of dapagliflozin, a anti-diabetic medicine. Skin dryness in KK-Ay/TaJcl mice was considered to be a result of the decrease in collagen type I via the IL-1/mast cell/MMP-1 system and hyaluronic acid via the IL-6/hyaluronidase system. Additionally, the TGF-β/HAS pathway was suppressed after dapagliflozin administration, and a decrease in hyaluronic acid levels was induced (Fig. 7). In the current study, we used a mouse model, and our observations are not based on human examination. Therefore, future studies should focus on clinical tests on humans to validate our findings.
This study was supported by JSPS KAKENHI (Grant No. 18K06082).
The authors declare no conflict of interest.
