Insulin and leptin oscillations license food-entrained browning and metabolic flexibility.

Timed feeding drives adipose browning, although the integrative mechanisms for the same remain unclear. Here, we show that twice-a-night (TAN) feeding generates biphasic oscillations of circulating insulin and leptin, representing their entrainment by timed feeding. Insulin and leptin surges lead to marked cellular, functional, and metabolic remodeling of subcutaneous white adipose tissue (sWAT), resulting in increased energy expenditure. Single-cell RNA-sequencing (scRNA-seq) analyses and flow cytometry demonstrate a role for insulin and leptin surges in innate lymphoid type 2 (ILC2) cell recruitment and sWAT browning, since sWAT depot denervation or loss of leptin or insulin receptor signaling or ILC2 recruitment each dampens TAN feeding-induced sWAT remodeling and energy expenditure. Consistently, recreating insulin and leptin oscillations via once-a-day timed co-injections is sufficient to favorably remodel innervated sWAT. Innervation is necessary for sWAT remodeling, since denervation of sWAT, but not brown adipose tissue (BAT), blocks TAN-induced sWAT remodeling and resolution of inflammation. In sum, reorganization of nutrient-sensitive pathways remodels sWAT and drives the metabolic benefits of timed feeding.

Functional characterization of Polr3a hypomyelinating leukodystrophy mutations in the S. cerevisiae homolog, RPC160.

Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast Rpc160 to single Pol III leukodystrophy mutations in the pore domain can be overcome by a second mutation in the domain.