ObjectiveAugmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.MethodsWe exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization.ResultsSkeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability.ConclusionsThese results identify skeletal muscle cells as requiring NAMPT to maintain NAD+ availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD+ availability.

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in β cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although β cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key β cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed (“disallowed”) in β cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in βPax6KO versus control β cells, and the former displayed attenuated increases in cytosolic Ca2+. Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated β-cells, Pdx1 and Nkx6.1, were observed in islet “hub” cells. These results indicate that Pax6 is required for the functional identity of adult β cells. Furthermore, deficiencies in β cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of β-cell development and function. Consequently, the mis-expression of members of this group may contribute to the risk of type 2 diabetes (T2D). Here, we investigate roles for an antisense lncRNA expressed from the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in β-cell function. The transcription factor Pax6 is required for the development of pancreatic islets and maintenance of a fully differentiated β-cell phenotype. Pax6os1/PAX6-AS1 expression was increased in pancreatic islets and β-cell lines at high glucose concentrations, in islets from mice fed a high fat diet, and in those from patients with type 2 diabetes. Silencing or deletion of Pax6os1/PAX6-AS1 in MIN6 cells and EndoC-βH1cells, respectively, upregulated β-cell signature genes, including insulin. Moreover, shRNA-mediated silencing of PAX6-AS1 in human islets not only increased insulin mRNA, but also enhanced glucose-stimulated insulin secretion and calcium dynamics. In contrast, inactivation of Pax6os1 in mice was largely without effect on glucose homeostasis, though female Pax6os1 null mice on high fat diet (HFD) showed a tendency towards enhanced glucose clearance. Together, our results suggest that increased expression of PAX6-AS1 at high glucose levels may contribute to β-cell dedifferentiation and failure in some forms of type 2 diabetes. Thus, targeting PAX6-AS1 may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in type 2 diabetes.