Metabolic dysfunction in human skin: Restoration of mitochondrial integrity and metabolic output by nicotinamide (niacinamide) in primary dermal fibroblasts from older aged donors

Abstract

Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in-depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20-70 years of age that encode for all subunits comprising complexes I-V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. "Oxidative phosphorylation" was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I-V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt-Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells.

Figure 1

Transcriptomics profiling of dermal sections from female facial cheek biopsies collected across age groups 20–70 years old. (a) Top 10 Canonical pathways in Caucasian face dermis of subject age groups 70 vs 20 (by log p) show inhibited oxidative phosphorylation as the top associated pathway (Fisher's p‐value = 7.28 × 10⁻¹⁵; z‐score = −5.92). Graded bar color indicates the magnitude of a pathway's z‐score, with predicted activated pathways (z‐score >0) shaded in orange and predicted inhibited pathways (z‐score <0) shaded in blue. White bars indicate neutral pathways (neither activated nor inhibited) and gray bars indicate pathways in which an activation state cannot be predicted. (b) Oxidative phosphorylation canonical pathway map shows reduced gene expression across all mitochondrial complexes as indicated by blue color. (c) Heatmap of 239 probe sets encoding for subunits of complexes I‐V face dermis from Caucasian subjects across multiple decades (20 s–70 s). Color range reflects normalized intensity values for each age group; blue (lower expression), red (higher expression)

Figure 2

Transcriptomics profiling comparing fibroblasts from younger and older aged donors: (a) hierarchical clustering of the statistically significant probe sets and individual samples in the old (purple, n = 5) vs young (green, n = 5) fibroblast comparison at day 0. Color range reflects normalized intensity values; blue (lower expression), red (higher expression). (b) Top 10 canonical pathways associated with the old vs. young fibroblast signature at day 0 show inhibited oxidative phosphorylation among top associated pathway (Fisher's p‐value = 2.86 × 10⁻⁶, z‐score = 3.58). (c) Heatmap comparing the average relative expression profiles of canonical oxidative phosphorylation genes in dermis biopsy sections from old (70–74 years of age, n = 25) and young (20–29 years of age, n = 30) subjects (left panel) with old (n = 5) vs. young (n = 5) fibroblasts (right panel). Color range reflects average normalized intensity values for each group; blue (lower expression), red (higher expression). (d) Trace profiles of probe sets encoding select subunits across age groups from dermal biopsies that were highly downregulated in old fibroblasts compared to young cells. Significance indicates comparisons to 20‐year‐old dermal biopsy group (***p < 0.001, *p < 0.10)

Figure 3

Select transcript expression levels of mitochondrial and nuclear‐encoded subunits following Nam treatment. (a) Following 7 days of Nam treatment, there was a significant increase in gene expression of the mitochondrial‐encoded genes MtND1 (subunit of complex I), MtCO1 (subunit of complex IV), MtATP8 (subunit of Complex V), and the ribosomal gene MtRNR1. As complex II is entirely nuclear‐encoded, no mitochondrial gene expression was able to be measured for this complex. (b) After 7 days of Nam treatment, there was a significant increase in gene expression of the nuclear‐encoded genes NDUFS1 (subunit of complex I), COX4 (subunit of complex IV), and ATP5A1 (subunit of complex V). There was no significant increase in gene expression of SDHA (subunit of complex II). Select complex activity following Nam treatment. (c) Mitochondrial complexes I and IV showed significant increases in activity when measured spectrophotometrically following 7 days of Nam treatment. Measurements of complex II activity showed no change over the 7 days of Nam treatment. The activity of all complexes was normalized against citrate synthase activity, which is used to determine complex activity per unit of mitochondrial content. Experiments were performed using dermal skin fibroblasts obtained from foreskin samples. (*p < 0.05, **p < 0.01, n = 13–15 donors ±SEM). Nam, nicotinamide; ns, not significant

Figure 4

Quantitation of cellular metabolism in primary dermal fibroblasts (ATCC) from females isolated from normal tissue from breast, abdomen, face, or unspecified sites as function of age and effect of Nam treatment on basal metabolism and OCR under stress conditions. (a) Basal measurements of OCR (blue circles) and ECAR (yellow circles) in fibroblasts spanning a range of young to aged donors were quantitated in the Seahorse XF Flux Analyzer. OCR, oxygen consumption rate; ECAR, extracellular acidification rate. (b) Basal measurements of OCR and ECAR in fibroblasts from young (18–20 years of age) and old (64–66 years of age) donors were quantitated. There was a significant difference in basal OCR (blue bars) between young (n = 10) and old (n = 15). Nam showed a significant increase in basal OCR in old donor cells after incubation for 7 days (n = 4). There were no significant differences in basal ECAR (yellow bars) between age groups or after Nam incubation. (c) Fibroblasts from a younger (20 years of age, red circles) and an older aged (65 years of age, open circles) donor were analyzed in the flux analyser and tested with sequential addition of oligomycin, FCCP, and rotenone +antimycin. 7 days of 1 mM Nam treatment of older aged donor cells showed a restoration of OCR to nearly levels in younger aged cells (blue triangles). (d) Quantitation of metabolic output from young (red bars, 18–20 years of age, average of n = 2 donors, 3 replicates, ±SEM) and older aged (white bars, 64–66 years of age, average of n = 3 donors, 5 replicates, ±SEM) donor fibroblasts as well as after treatment with Nam for 7 days (blue bars, 64–66 years of age; n = 3, ±SEM) showed a significant restoration of maximal respiration, ATP turnover and spare respiratory capacity to levels similar to younger aged cells (*p < 0.05, **p < 0.01, ***p < 0.001, ±SEM). ECAR, extracellular acidification rate; Nam, nicotinamide; OCR, oxygen consumption rate

Figure 5

Alteration in mitochondrial integrity and content by Nam in dermal fibroblasts from young and older aged donors. (a) Flow cytometry traces of primary dermal fibroblasts from young and old aged donors incubated with 1 mM Nam and stained with TMRM and MTG at days 3 and 7. Red dotted lines provide visual orientation of shifts across timepoints. (b) Quantification of TMRM and MTG staining intensity of cells from flow cytometry traces show lower mitochondrial integrity and content in older aged fibroblasts compared to young. Treatment with Nam caused significantly higher staining content in both younger (red bars) and older (blue bars) aged donor cells. (c) Fluorescence confocal photomicrographs of older aged donor fibroblasts for MTG (red fluorescence), DAPI for nuclear detection (blue fluorescence), and actin (green fluorescence) after treatment with 1 mM Nam for 2 and 5 days. White bar represents 10 µm. (d) Quantitation of MTO staining in older aged donor fibroblasts after Nam incubation showed a 70% and 132% increase in mitochondrial content area at days 2 and 5, respectively. (*p < 0.05, **p < 0.01, ***p < 0.001 ±SEM). MTG, MitoTrackerGreen; MTO, MitoTrackerOrange; Nam, nicotinamide; TMRM, tetramethylrhodamine, methyl ester

Figure 6

Mitophagy levels following Nam treatment. (a) Example images of human skin fibroblast cells transfected with mt‐Keima showing the mitochondrial network in green and the mitophagic vesicles in red. Cells from a young donor (26 years) and old donor (84 years) showing a control cell, a cell treated with Nam for 3 days, and a cell treated with 0.4 µM bafilomycin. White bar represents 10 µm. (b) Approximately 100–200 cells per category were analyzed (4 donors per category), from either young (17, 26, 28, and 33 years) or aged (75, 80, 83, and 84 years) donors, using dermal skin fibroblasts obtained from foreskin samples. Cells from young donors showed a 23% higher level of mitophagy than aged cells. Cells treated with Nam for 3 days showed a 19% and 32% increase in mitophagy in both younger and older donors, respectively. The bafilomycin controls all showed higher levels of mitophagy. (c) Increased levels of autophagy marker LC3 in dermal fibroblasts with Nam treatment. Fluorescence confocal photomicrographs of older aged donor fibroblasts for LC3 (red fluorescence), DAPI for nuclear detection (blue fluorescence), and tubulin (green fluorescence) after treatment with Nam for 3 days (left panel). Select area from control and Nam‐treated cells at high magnification (right panel) showing the internal distribution of LC3 in puncta form. (d) Number of puncta per cell was quantitated (n > 30 cells per treatment group) via image analysis and showed a 73% increase with Nam treatment compared to control cells (***p < 0.001, **p < 0.01, *p < 0.05). Nam, nicotinamide