Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative, label-free fluorescence imaging.

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.