Nicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.

Glycogen is the storage form of carbohydrates in mammals. In humans the majority of glycogen is stored in skeletal muscles (∼500 g) and the liver (∼100 g). Food is supplied in larger meals, but the blood glucose concentration has to be kept within narrow limits to survive and stay healthy. Therefore, the body has to cope with periods of excess carbohydrates and periods without supplementation. Healthy persons remove blood glucose rapidly when glucose is in excess, but insulin-stimulated glucose disposal is reduced in insulin resistant and type 2 diabetic subjects. During a hyperinsulinemic euglycemic clamp, 70–90% of glucose disposal will be stored as muscle glycogen in healthy subjects. The glycogen stores in skeletal muscles are limited because an efficient feedback-mediated inhibition of glycogen synthase prevents accumulation. De novo lipid synthesis can contribute to glucose disposal when glycogen stores are filled. Exercise physiologists normally consider glycogen’s main function as energy substrate. Glycogen is the main energy substrate during exercise intensity above 70% of maximal oxygen uptake () and fatigue develops when the glycogen stores are depleted in the active muscles. After exercise, the rate of glycogen synthesis is increased to replete glycogen stores, and blood glucose is the substrate. Indeed insulin-stimulated glucose uptake and glycogen synthesis is elevated after exercise, which, from an evolutional point of view, will favor glycogen repletion and preparation for new “fight or flight” events. In the modern society, the reduced glycogen stores in skeletal muscles after exercise allows carbohydrates to be stored as muscle glycogen and prevents that glucose is channeled to de novo lipid synthesis, which over time will causes ectopic fat accumulation and insulin resistance. The reduction of skeletal muscle glycogen after exercise allows a healthy storage of carbohydrates after meals and prevents development of type 2 diabetes.

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser ⁶⁵ ) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser ¹¹¹ ) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser ¹¹¹ of each of the Rabs, we demonstrate that Rab Ser ¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser ¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser ¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser ⁶⁵ . We further show mechanistically that phosphorylation at Ser ¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser ¹¹¹ may represent novel biomarkers of PINK1 activity in vivo . Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non‐native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry‐green fluorescence protein‐mtFIS1 _101-152 ). Here, we report that both carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read‐out of PTEN‐induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1‐Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK‐binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non‐muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK‐dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1‐Parkin independent manner.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the V_max of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

Probes of E3 ligase transthiolation activity reveal the hierarchy of phosphorylation events leading to cellular parkin activation and show that disease-linked parkin mutants have impaired transthiolation activity. E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.