Hepatic glycogen content is important for glucose homeostasis and exhibits robust circadian rhythms that peak at the end of the active phase in mammals. The activities of the rate-limiting enzymes for glycogenesis and glycogenolysis also show circadian rhythms, and the balance between them forms the circadian rhythm of the hepatic glycogen content. However, no direct evidence has yet implicated the circadian clock in the regulation of glycogen metabolism at the molecular level. We show here that a Clock gene mutation damps the circadian rhythm of the hepatic glycogen content, as well as the circadian mRNA and protein expression of Gys2 (glycogen synthase 2), which is the rate-limiting enzyme of glycogenesis in the liver. Transient reporter assays revealed that CLOCK drives the transcriptional activation of Gys2 via two tandemly located E-boxes. Chromatin immunoprecipitation assays of liver tissues revealed that CLOCK binds to these E-box elements in vivo, and real time reporter assays showed that these elements are sufficient for circadian Gys2 expression in vitro. Thus, CLOCK regulates the circadian rhythms of hepatic glycogen synthesis through transcriptional activation of Gys2.

SummaryThe Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of bIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, which causes numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia or frailty include inflammation, muscle stem cell depletion, mitochondrial dysfunction, and loss of motor neurons, but whether there are key drivers of sarcopenia are not yet known. To gain deeper insights into age-related muscle loss, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, elderly, and frail human subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). This combined approach revealed changes in gene expression that occur with age and frailty in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in aged muscle. We validated several key genes changes in fixed human muscle tissue using digital spatial profiling. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with p21 cip1 -driven senescence in this subpopulation. Overall, our findings identify unique cellular subpopulations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related frailty.