👤 John A Humphrey

Calorie restriction (CR; calorie intake reduced by ∼20%-40% below ad libitum, AL, intake) potentiates skeletal muscle insulin sensitivity during old age by incompletely understood mechanisms. We aimed to identify CR-induced changes in muscle insulin signaling that may explain this enhanced sensitivity. We examined how CR (65% of AL intake for 8-weeks) alters muscle insulin action and signaling in aged rats (24-month old) of both sexes. We assessed insulin-stimulated glucose uptake (ISGU) in muscle together with deep phosphoproteomic profiling. CR enhanced ISGU in both sexes, with higher ISGU in females regardless of diet. We identified 590 diet-responsive phosphosites, indicating extensive CR-induced remodelling of muscle phosphorylation, particularly within structural and contractile pathways. Strikingly, 70% of these sites were sex-specific. Numerous insulin-responsive sites were identified (193 in females; 107 in males) with 60 overlapping sites. The magnitude of the insulin-effects among all significantly regulated sites correlated between sexes. S1443 phosphorylation on EH domain-binding protein 1-like protein-1 (Ehbp1l1; a potential regulator of Rab proteins that control GLUT4 glucose transporter trafficking) was insulin-responsive in both sexes but only associated to ISGU in females. Personalized phosphoproteomic analysis also identified insulin-responsive sites on Leiomodin-1 (Lmod1) that correlated with ISGU across individuals. Both Lmod1 and Ehbp1l1 have strong genetic association with glycemic traits in humans, reinforcing their translational relevance. This study revealed sex-dependent and sex-independent phosphosignaling mechanisms that associate with muscle insulin responsiveness as well as hundreds of sex-specific, CR-responsive phosphosites. These findings provide a rich resource for future research on CR and insulin sensitivity. Show less

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder. Show less

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS. We have applied this device to the analysis of human plasma from a clinical trial of IF. In this longitudinal study employing 8-weeks IF, we identified significant abundance differences induced by the IF intervention, including increased apolipoprotein A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3). These changes correlated with a significant decrease in plasma triglycerides after the IF intervention. Given that these proteins have a role in regulating apolipoprotein particle metabolism, we propose that IF had a positive effect on lipid metabolism through modulation of HDL particle size and function. In addition, we applied a novel human protein variant database to detect common protein variants across the participants. We show that consistent detection of clinically relevant peptides derived from both alleles of many proteins is possible, including some that are associated with human metabolic phenotypes. Together, these findings illustrate the power of accessible workflows for proteomics analysis of clinical samples to yield significant biological insight. Show less

Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not been identified in birds, the objectives of this work were to clone, sequence, and characterize the genomic organization of ChREBP and Mlx genes and to determine the expression of ChREBP, Mlx, and several related genes including liver X receptor (LXR), SREBP-1 and thyroid hormone responsive Spot 14 (Spot 14) in chickens. Alternative splicing resulted in two ChREBP mRNA transcript variants that code for predicted proteins of 895 and 869 amino acids. The chicken Mlx gene produced a single mRNA transcript that codes for a predicted protein of 245 amino acids. Chicken ChREBP and Mlx predicted proteins shared high amino acid homology with select portions of corresponding mammalian proteins. In chickens, Mlx, SREBP-1, and LXR were expressed at comparable levels in all tissues examined. However, ChREBP demonstrated significant tissue-specific expression with the highest mRNA levels found in liver and duodenum and Spot 14 was expressed predominantly in liver and abdominal fat. Using Western blotting, the presence of ChREBP protein was detected in chicken liver tissue. Our findings add new insight into a potential role for specific transcription factors such as ChREBP and Mlx in the glucose-dependent regulation of lipogenesis in birds. Show less

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action. Show less