Exploring mitochondrial metabolism of wild‐type and diabetic mice skin explants using the Seahorse technology (2024)

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Skin Res Technol
v.30(3); 2024 Mar
PMC10920985

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Skin Res Technol. 2024 Mar; 30(3): e13638.

Published online 2024 Mar 7. doi:10.1111/srt.13638

PMCID: PMC10920985

PMID: 38454567

Justine Dugrain,¹ Laurence Canaple,^1,² Nicolas Picard,¹ Dominique Sigaudo‐Roussel,¹ and Christelle Bonod¹

Author information Article notes Copyright and License information PMC Disclaimer

Associated Data

Data Availability Statement

Abstract

Background

Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate‐based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux.

Materials and methods

Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild‐type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile.

Results

Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non‐mitochondrial oxygen consumption than wild‐type or type I diabetic (T1D) skin.

Conclusion

Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

Keywords: diabetes, metabolism, mitochondria, oxidative phosphorylation, Seahorse technology, skin

Abbreviations

FCCP: carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone
OCR: oxygen consumption rate
STZ: Streptozotocin
T1D: Type I diabetic
T2D: Type II diabetic

1. INTRODUCTION

Skin is the largest organ in the human body representing the first barrier against external aggressions such as mechanical stress, radiation, pollution or microorganisms. Thus, injured skin must heal quickly to recover its barrier functions. Diabetes is a metabolic disease which impairs skin functions and structures. Two forms of diabetes prevail: type I and type II. Type I diabetes (T1D) affects 5–10% of the population and frequently appears during childhood or adolescence. It is characterized by the destruction of the β‐cells in the pancreas which prevents synthesis of insulin, necessary to stimulate uptake of glucose in adipocytes, hepatocytes and skeletal muscle cells. Type II diabetes (T2D) concerns more than 90% of diabetic patients. This form of diabetes consists of cellular insulin resistance combined with an imbalanced insulin secretory response.¹^,² Chronic hyperglycemia is the main shared feature of these two types of diabetes, which leads to skin complications. Several studies showed that 30%–82% of diabetic patients have cutaneous impairments.³ Among these complications, 6.3% of patients suffer from a substantial delay in wound healing, which results in chronic ulcers.⁴ Chronic ulcers occur because of reduced sensitivity due to neuropathy that prevents patient from detecting the lesion. It may also be coupled with a low capacity to fight infections. Consequences are limb amputation or even death. Lower limb amputation occurs every 20 s in the world because of diabetes complications.⁵ Thus, diabetic ulcers represent one of the big worldwide public health issues. Both T1D and T2D are associated with mitochondrial dysfunctions.⁶

Mitochondria are known to produce phosphocreatine using creatine kinase, an enzyme present in the mitochondrial inter‐membrane space.⁷ Creatine kinase has already been shown to be increased in leg ulcers, leading to a lower level of ATP.⁸^,⁹ This enzymatic process is preceded by mitochondrial respiration which is known to be essential for skin functions as it produces the main source of ATP through the oxidative phosphorylation process. Mitochondrial respiration first involves the oxidation of NADH or FADH2 by the four protein complexes of the respiratory chain to remove electrons it contains. These electrons are transported through the respiratory chain while releasing energy which is used by the four complexes to pump H+ protons from the matrix to the inner membrane of the mitochondria. Oxygen is the final electron acceptor in the electron transport chain. It is reduced to water to clear the chain from the remaining electron energy. The resulting electrochemical proton gradient provides energy to the ATP synthase which will thereby produce ATP. Studies already showed decreased mitochondrial respiration in diabetic skeletal muscle,¹⁰ diabetic heart¹¹ or pancreatic β‐cells.¹² However, little is known about mitochondrial respiration in diabetic skin.

The Seahorse bioanalyzer is an innovative technology to assess mitochondrial respiration by measuring oxygen consumption in real‐time. The skin mitochondrial metabolism has already been analyzed at cell level, in keratinocytes,¹³ fibroblasts¹⁴ and macrophages,¹⁵ but also in ex vivo epidermis¹⁶ and reconstructed epidermis.¹⁷ However, nothing is known about mitochondrial respiration on whole, healthy or diabetic skin.

The aim of this preliminary study was to characterized, for the first time, using the Seahorse technology on the whole tissue, the ex vivo mitochondrial respiration function of wild‐type and diabetic mice skin.

2. MATERIAL AND METHODS

2.1. Animals

The study was conducted in 6weeks‐old male C57BL/6 (T1D) or 11weeks‐old db/db (T2D) mice (Janvier labs, Saint‐Berthevin, France). The animals were kept under controlled conditions of temperature (22±2°C) and humidity (60±5%), on a 12‐hours light/dark cycle, with ad libitum access to water and food. All procedures were carried out in accordance with the guidelines for ethical care of experimental animals of the European Community and approved by the Ethic Committee C2EA015, n°26546.

2.2. Induction of diabetes

After an 8‐h fast, T1D was chemically induced by a single intraperitoneal injection of streptozotocin (STZ) (Zanosar®, Keocyt, Montrouge, France; at a dose of 180mg/kg). Mice were considered diabetic when blood glucose level was ≥300mg/dL, 3 days after injection. 2 UI of insulin (Caninsulin, MSD) were administered in diabetic animals every two days and body weight was monitored each week. Experiments were conducted 4weeks after the induction of diabetes. T2D mice blood glucose level was>400mg/dL the day of the experiment, which confirms their diabetic phenotype. Experiments were conducted at 4weeks of diabetes.

2.3. Punch biopsy

Mice were euthanized, dorsal skin was depilated and excised. Skin punches were taken using sterile biopsy punches (1mm, 1.5mm or 4.0mm diameter; kai medical) (Figure1A).

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FIGURE 1

Cell Mito Stress Test on mice skin. To perform the assay, mice's backs were depilated and the skin was removed to take punch biopsies. Each punch was placed, dermis up, in the Seahorse well‐plate which was then filled with Seahorse medium. The four drugs were added in the appropriate injection port of the Seahorse cartridge and the program started (A). The typical OCR curve obtained enables to calculate the different parameters of the mitochondrial respiration (B).

2.4. Seahorse cell mito stress test

Skin punches were individually placed into a Seahorse XF24 Islet Capture Microplate (103518‐100; Agilent Technologies) and 4 empty wells were used as controls. Each well was then covered with islet capture screens and filled with 500µL of XF DMEM medium (103575‐100) complemented with glucose (10mM), pyruvate (1mM) and glutamine (2mM). Oligomycin, carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP) and a mix of rotenone/antimycin A at 10X concentration (40µM or 60µM) were prepared in the XF DMEM medium and respectively loaded into the injection ports A, B and C of the Seahorse XFe24 sensor cartridge which was previously hydrated overnight with the XF Calibrant solution (100840‐000; Agilent) at 37°C in a CO₂‐free incubator. The sensor cartridge was then inserted into the Agilent Seahorse XFe24 Analyzer. Once the calibration was complete, the calibration plate was replaced by the microplate containing the tissue punches and a specific assay program of the “Cell Mito Stress Test” designed for murine skin was started (Figure1A and Table1). At the end of the assay, a typical graph of the test was generated (Figure1B). Data were normalized to the real surfaces of punches.

TABLE 1

Detailed parameters of the final assay protocol.

Final assay protocol parameters
Skin side	dermis
Punch size (diameter)	1.5mm
Program length	Baseline: 8 cycles 3min/2min/3min Oligomycin: 10 cycles 3min/2min/3min FCCP: 6 cycles 3min/2min/3min Rotenone/Antimycin A: 10 cycles 3min/2min/3min
Drugs concentration (µM)	4/4/4

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Note: Here is the optimized protocol used to run the final assay comparing wild‐type, T1D and T2D mice skin. 3min/2min/3min correspond to 3min mix, 2min wait and 3min measure.

2.5. Statistical analysis

The results are expressed as means±SEM. One‐way ANOVA and Kruskal‐Wallis test Statistical tests were performed using GraphPad Prism 7.00 (GraphPad Software, Inc).

3. RESULTS

3.1. Dermis side of wild‐type skin have a higher mitochondrial respiration than epidermis side

Since the objective of this study was to measure mitochondrial activities on the whole skin, the first parameter to determine was the most pertinent side of the tissue on which the test should be carried out. Thus, the oxygen consumption rate (OCR) of punches from wild‐type mice were compared either with their epidermis or dermis side up, in contact with the probe of the cartridge. Figure2A shows that, for 4mm skin punches, dermis side of skin (black curve) have a statistically higher basal respiration, around 500pmol/min than epidermis side (gray curve) whose basal OCR is around 380pmol/min. However, after the injection of oligomycin there is no response for the epidermis side while the OCR decreased for the dermis side, from 500 to 400pmol/min. Dermis side also responds to FCCP, a mitochondrial uncoupler that induces maximal uncoupled respiration rate, and rotenone/antimycin A, inhibitors of the mitochondrial respiration that act on complexes I and III of the electron transport chain, respectively. The measurement of each mitochondrial parameters showed an increase of the ATP‐linked respiration from the dermis side compared to the epidermis face (Figure2B). We conclude that dermis side is more suitable for OCR measurements than epidermis side.

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FIGURE 2

Optimization of the Cell Mito Stress Test on mice skin. OCR of dermis and epidermis sides of wild‐type mice's 4mm skin punches after sequential addition of oligomycin, FCCP and rotenone/antimycin A (A). Difference of ATP production capacity between both skin sides. Data are presented as mean ± SEM (n=5), unpaired t‐test (p<0.05) (B). Comparison of the basal respiration of 4mm, 1,5 and 1mm skin punches (C). Effect of oligomycin (D) and FCCP (E) on the OCR of the three skin punch sizes, Kruskal‐Wallis test and one‐way ANOVA (*p<0.05 and ****p<0.0001).

3.2. Increased concentration of FCCP and rotenone/antimycin A showed no improvement of the OCR curve shape

In order to reach the optimal measurement of OCR, the concentrations of FCCP and rotenone/antimycin A were increased from 4µM to 6µM, while oligomycin was maintained at 4µM. The calculation of different parameters linked to these drugs showed that neither maximal respiration, nor spare respiratory capacity and non‐mitochondrial oxygen consumption was significantly different between 4/4/4µM and 4/6/6µM (Data not shown). Overall, increasing FCCP and rotenone/antimycin A concentrations is unnecessary to improve their effect on skin mitochondrial respiration.

3.3. Punch size influences mitochondria response to FCCP addition

As suggested by Underwood etal., 2020 on the brain, a reduction of the punch size could increase the drug responses, by improving the drug diffusion and so the global respiration profile. To test this question, we performed a Cell Mito Stress Test on wild‐type skin with three different punch sizes: 4, 1.5 or 1mm.

The basal OCR is proportional to the size of the punch with a very low OCR for the 1 mm² punch (Figure2C). Moreover, oligomycin effect does not depend on the punch size as there are no significant variations of OCR between each punch sizes (Figure2D). However, the response to FCCP treatment is dependent on the punch size with a better efficiency for the smallest punches. For instance, the increase of the OCR due to FCCP is 40.7% for 1.5mm versus 16.8% for 4mm punch (Figure2E). In order to get a high OCR and a good sensitivity to FCCP, we used 1.5mm punches to test the difference between wild‐type and diabetic skin (see standardized protocol in Table1).

3.4. Only type II diabetic mice skin presents a diminished mitochondrial metabolism

The mitochondrial metabolism of wild‐type, T1D and T2D mice skin has been measured using the standardized protocol shown in Table1. For that, experiments were conducted on 10 punches from each mouse (n=10), from wild‐type (n=3), T1D mice induced after streptozotocin injection (n=3) and T2D mice (db/db) (n=4).

Figure3A showed that both wild‐type and T1D skin mitochondrial respiration have a similar pattern, unlike T2D skin which especially has a less pronounced response to oligomycin, but the same FCCP‐induced maximal uncoupling respiration. Hence, basal respiration (Figure3B) was slightly but significantly lower in T2D skin than in wild‐type skin. Interestingly, the rates of phosphorylating oxygen consumption (Figure3C) and non‐mitochondrial oxygen consumption (Figure3D) in T2D skin were significantly lower and higher compared to the two other skin types, respectively. On the contrary, no significant differences in these three parameters were measured between wild‐type and T1D skin (Figure3B–D).

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FIGURE 3

OCR of wild‐type, type I and type II diabetic mice's skin (A). Difference of basal respiration (B) (one‐way ANOVA, *p<0.05), phosphorylation oxygen consumption (C) (one‐way ANOVA, ****p<0.0001) and non‐mitochondrial respiration (D) (Kruskal‐Wallis test, ****p<0.0001) between each skin types. Data are presented as mean±SEM.

4. CONCLUSIONS AND PERSPECTIVES

With the important public health problem that represents diabetes,¹⁸ a greater understanding of diabetic skin metabolism is required. The substantial energy rate provided by mitochondria is essential for an effective skin wound healing process. Mitochondrial respiration is known to be impaired, together with a decrease of the organelle ATP production capacities in some types of diabetic cells.¹⁰^,¹¹^,¹²

The aim of this study thus was to develop a reliable method for assessing mitochondrial respiration function of whole skin from wild‐type and diabetic mice through the Seahorse XFe extracellular flux analyzer, to measure the OCR in real time using a common Mito Stress assay. This would be of great interest as it allows to directly measure the OCR in fresh tissues without any required isolation step of cells or mitochondria. This optimization will be a precious timesaver and results will also reflect the real mitochondrial respiration profile of the skin cells kept in their complex cellular ecosystems. Moreover, the OCR of 20 skin explants can be measured simultaneously in less than a day, which brings even more quickness and robustness to the method. This technology has already been used to study mitochondrial bioenergetics in whole tissues such as retina,¹⁹^,²⁰ brain²¹ or even epidermis.¹⁶ However, no experiment has been conducted on the whole skin.

In the present study, we provide a reliable protocol to assay mitochondrial function on the whole skin.

Firstly, we report that, compared with epidermis side, dermis is the best side of the skin which would be exposed to the sensor in order to perform mitochondrial assay. This result is not surprising in that epidermis upper layer is composed of dead corneocytes, which have no energy needs. In addition, this dead layer of cells can limit the diffusion of mitochondrial inhibitor and uncoupler, thus impairing their action on the living cells within the tissue. One solution could have been to separate the epidermis from the dermis and measure the mitochondrial respiration of the “vital” side of the epidermis called the basal layer. This method has been developed by Schniertshauer etal.¹⁶ and the results were convincing. However, this method entails a supplementary step which alter the integrity of the tissue and can also be complicated to settle on mice skin.

Secondly, we show that skin punch sizes matter to increase the sensitivity of mitochondrial assay because the mitochondrial response to uncoupler is significantly better with the smallest punches. As previously found for brain sample sizes,¹⁵ this better mitochondrial response is probably due to a betterdrugs diffusion in the smaller tissue size. In the present study, the best compromise was obtained with 1.5mm diameter punch.

In order to test our protocol, we conducted a preliminary study to compare the metabolic activity of skin from wild‐type and diabetic mice. Diabetic skin was obtained from mice induced after streptozotocin injection to mimic T1D or from db/db mice for T2D. Wild‐type and T1D skin have similar metabolic profile suggesting that T1D murine skin has no mitochondrial dysfunction despite the risk of skin healing delay. In contrast, T2D skin exhibited a significant decreased basal and phosphorylating respiration, suggesting a lower ability to sustained ATP synthesis compared to wild‐type skin.

Our data suggest that mitochondrial function is altered in the skin of type 2 diabetes. These observations are supported by a previous study that showed a severe alteration of skin wound healing in db/db mice compared to wild‐type and STZ‐induced diabetic mice, which healed about 10 and 7 days sooner, respectively.²² These observations may be explained by the fact that the spontaneous mutation of the leptin receptor gene in db/db mice leads to a progressive diabetes development, unlike STZ mice.²³ db/db mice diabetes may thus have more time to impact energetic metabolism. A prospective observational study conducted on diabetic patients also highlighted that cutaneous lesions were significantly more observed in T2D patients (98.2%) compared with T1D patients (34.3%).³ These observations were supported by a meta‐analysis study reporting that the global prevalence of diabetic foot ulceration was higher in T2D (6.4%) than in T1D (5.5%).⁴

Whether such mitochondrial dysfunction drives or is correlated with the development of chronic wounds observed in T2D patients remains to be furtherly investigated.

In conclusion, we developed the first method to measure mitochondrial metabolism of mice skin and showed that it can be used to detect mitochondrial alteration in diabetic model. This method could be used in the future, not only on mice skin, but also on human or other species skin in healthy and pathological conditions. The interest will be to analyze whether a specific skin pathology could have a consequence on the skin metabolism and function.

CONFLICT OF INTEREST STATEMENT

The authors declar no conflicts of interest.

ACKNOWLEDGMENTS

We acknowledge the contribution of the SFR Biosciences (UAR3444/CNRS, US8/Inserm, ENS de Lyon, UCBL) especially the AniRA ImmOs‐seahorse facility. We thank Damien Roussel for his precious help. SFR Biosciences (UAR3444/US8) supported the study.

Notes

Dugrain J, Canaple L, Picard N, Sigaudo‐Roussel D, Bonod C. Exploring mitochondrial metabolism of wild‐type and diabetic mice skin explants using the Seahorse technology. Skin Res Technol. 2024;30:e13638. 10.1111/srt.13638 [PubMed] [CrossRef] [Google Scholar]

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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