MiPsummer Programme pdf - Mitochondrial Physiology Society

27
Abstract # 1
Nicotinamide phosphoribosyltransferase knockdown impairs mitochondrial function in mouse myoblasts
Marianne A. Andersen 1 , Sara G. Vienberg 1 , Josef Brandauer 1,2 , Birgitte Holst 3 & Jonas T. Treebak 1 .
1
Section of Integrative Physiology, The Novo Nordisk Foundation Center for Basic Metabolic Research,
Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen.
2 Health Sciences Department, Gettysburg College, Gettysburg PA, USA.
3
Section of Metabolic Receptology and Enteroendocrinology, The Novo Nordisk Foundation Center for Basic
Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen.
In skeletal muscle proper mitochondrial biogenesis and function is critical for maintaining insulin sensitivity
as mitochondrial dysfunction is associated with development of metabolic disorders such as insulin
resistance and type 2 diabetes. A family of energy sensing enzymes called sirtuins deacetylates and thus
activates transcription factors involved in mitochondrial biogenesis. Nicotinamide adenine dinucleotide
(NAD) is a required substrate for sirtuin activation which results in conversion of NAD to nicotinamide
(NAM). NAM directly inhibits sirtuin activity, and resynthesis of NAD is likely essential for maintaining
sirtuin activity. Nicotinamide phosphoribosyltransferase (Nampt) is the ratelimiting enzyme in the
recycling pathway that converts NAM to NAD, but evidence for a role of Nampt in maintaining sirtuin
activity and mitochondrial function is lacking. The aim of the current study was to determine the role of
Nampt in maintaining mitochondrial function in response to metabolic stress such as the transition
between substrate utilization in skeletal muscle cells. We hypothesized that mitochondrial function would
be impaired in absence of Nampt.
We have generated a stable Nampt knockdown (KD) mouse myoblast (C2C12) cell line using a shRNA
lentiviral approach. Nampt mRNA expression and protein abundance was reduced by 80% in the KD cells.
Mitochondrial function was determined using a Seahorse X96 Extracellular Flux Analyzer. Basal
mitochondrial respiration was unaffected by Nampt KD compared to cells expressing nonsense shRNA
(control). However, maximal respiratory capacity induced by the oxidative phosphorylation uncoupler
Carbonyl cyanide 4(trifluoromethoxy) phenylhydrazone (FCCP) was significantly (p