Dasari, Surendra

Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon’s catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino acid metabolites (amino-metabolites) across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or saline infusion alone. Despite a twofold or greater increase in the regional exchange of amino-metabolites by glucagon, whole-body kinetics and concentrations of amino acids (AA) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential (EAA) AA while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methylhistidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched-chain AA and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.

Obesity is associated with several skeletal muscle impairments which can be improved through an aerobic exercise prescription. The possibility that exercise responsiveness is diminished in people with obesity has been suggested but not well‐studied. PURPOSE The purpose of this study was to investigate how obesity influences acute exercise responsiveness within skeletal muscle. METHODS Non‐obese (NO; n=19; 10F/9M; BMI=25.1 ± 2.8 kg/m 2 ) and Obese (O; n=21; 14F/7M; BMI=37.3 ± 4.6 kg/m 2 ) adults performed 30 minutes of single‐leg cycling at 70% of VO 2 peak. Serial muscle biopsies (vastus lateralis) were collected before exercise and 3 and 6 hours post‐exercise to measure protein synthesis and gene expression. RESULTS The exercise‐induced fold change in mixed muscle protein synthesis trended (p=0.058) higher in NO (1.28 ± 0.54‐fold) compared to O (0.95 ± 0.42‐fold) and was inversely related to BMI (r=‐.374, p=0.027). RNA sequencing revealed 331 and 280 genes that were up or downregulated after exercise in NO and O, respectively. Gene set enrichment analysis showed O had blunted post‐exercise pathways related to MAPK signaling, chemokine‐mediated signaling, FC receptor mediated stimulation signaling, cell proliferation, apoptosis, and RNA polymerase II promotion. Quantitative polymerase chain reaction of select metabolic ( PGC1a, PGC1b, TFAM, PDK4, IRS, MAPK, and PRKAA1 ) and muscle growth ( SLC, TBC, MSTN, MyoD, TRIM32, FOX03, and FBXO3) genes were similar between groups 3 and 6 hours after exercise. Conclusion These data highlight several unique pathways in individuals with obesity that result in a blunted exercise response.

Background Immortalized cell lines are conventionally used to examine bacterial virulence and host responses. These models lack heterogeneity, complexity and do not allow interrogation of host specific characteristics. Human intestinal organoids provide a model of human epithelium and allow determination of whether bacterial infections elicit host dependent responses. Objective Determine how gender and stress influence responses to Campylobacter infection using human colonic organoids. Methods Colonic biopsies were collected from 20 volunteers (10/sex), with Hospital Anxiety and Depression Score of <4 (low stress) or >14 (high stress). Crypts were isolated from biopsies. Monolayers were generated, experimentally infected with 3 different clinical isolates of C. jejuni (OD 600 1) and averaged. Bacterial virulence and transepithelial resistance (TER) was measured throughout infection. Proteomics was done 24h post‐infection, with analysis via 1D‐LC‐MS using an Orbitrap Eclipse. Data was processed with MS2 ID pipeline MyriMatch‐IDPicker and proteins FDR<0.1 considered significant. Results Colonoid cultures were generated from 75% of volunteers. C. jejuni strains attachmed and invaded all colonoids. Greater bacterial attachment was seen on high stress volunteer colonoids compared low stress (0.08 ± 0.11 vs. 0.03 ± 0.08 CFU/starting inoculum for males, 0.015 ± 0.036 vs. 0.014 ± 0.014 for females, p<0.05 for both) and on high stress males compared to females (p<0.001). Colonoids from high stress volunteers had a significant drop in TER 24h post‐infection compared to non‐infected controls (42.1 ± 30.7 vs 125.2 ± 62.4, % baseline, p<0.05). Proteomic analysis showed minimal differences between high and low stress or between female and male colonoids at baseline (4 and 9 proteins respectively). Upon infection 195 proteins were differentially abundant with 161 increased in stressed organoids (FDR<0.1). Increased apoptotic pathway activation and cellular reorganization; pro‐apoptotic death‐associated protein‐kinase 3 (DAPK3), and DNA damage response serine/threonine‐protein kinase protein (TAOK3) were seen. Gene set enrichment analysis (GSEA) showed increased interferon alpha and beta signaling, IL‐1 and IL‐17 responses and MHC1/2 antigen processing in high stress infected monolayers (FDR<0.1). No differences between male high and low stress organoids post infection. In contrast, 1289 proteins were differentially abundant between high and low stress females with 84% up in stressed organoids. IL‐1 and IL‐12 signaling was up in high stress female organoids (FDR<0.1). Conclusions Colonoid monolayers can be developed from human volunteers and used to evaluate host pathogen interactions. Colonoid cultures highlight variability in C. jejuni infectivity and host responses; with robust C. jejuni virulence seen with high stress male individuals and the greatest change in proteomic profile in response to infection in high stress females. These findings demonstrate utility of colonoids and provide a platform to study personalized host responses to infection.

AbstractObjectiveThe health benefits of exercise are well documented, but several exercise‐response parameters are attenuated in individuals with obesity. The goal of this pilot study was to identify molecular mechanisms that may influence exercise response with obesity.MethodsA multi‐omics comparison of the transcriptome, proteome, and phosphoproteome in muscle from a preliminary cohort of lean individuals (n = 4) and individuals with obesity (n = 4) was performed, before and after a single bout of 30 minutes of unilateral cycling at 70% maximal oxygen uptake (VO2 peak). Mass spectrometry and RNA sequencing were used to interrogate the proteome, phosphoproteome, and transcriptome from muscle biopsy tissue.ResultsThe main findings are that individuals with obesity exhibited transcriptional and proteomic signatures consistent with reduced mitochondrial function, protein synthesis, and glycogen synthesis. Furthermore, individuals with obesity demonstrated markedly different transcriptional, proteomic, and phosphoproteomic responses to exercise, particularly biosynthetic pathways of glycogen synthesis and protein synthesis. Casein kinase II subunit alpha and glycogen synthase kinase‐3β signaling was identified as exercise‐response pathways that were notably altered by obesity.ConclusionsOpportunities to enhance exercise responsiveness by targeting specific molecular pathways that are disrupted in skeletal muscle from individuals with obesity await a better understanding of the precise molecular mechanisms that may limit exercise‐response pathways in obesity.

AbstractObjectiveTo define the proteomic profile of sporadic late‐onset nemaline myopathy (SLONM) and explore its pathogenesis.MethodsWe performed mass spectrometry on laser‐dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL).ResultsThere were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T‐cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers.ConclusionOur findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

AbstractBackgroundOral lichen planus confers a 1% risk of transformation to oral squamous cell carcinoma. While prior exome sequencing studies have identified multiple genetic mutations in oral squamous cell carcinoma, mutational analyses of lichen planus‐derived OSCC are lacking. We sought to clarify genomic events associated with oral lichen planus transformation.MethodsUsing rigorous diagnostic criteria, we retrospectively identified patients with non‐transforming oral lichen planus (i.e., known to be non‐transforming with 5 years of clinical follow‐up; n = 17), transforming oral lichen planus (tissue marginal to oral squamous cell carcinoma, n = 9), or oral squamous cell carcinoma arising in lichen planus (n = 17). Gene mutational profiles derived from whole‐exome sequencing on fixed mucosal specimens were compared among the groups.ResultsThe four most frequently mutated genes in transforming oral lichen planus and oral squamous cell carcinoma (TP53, CELSR1, CASP8, and KMT2D) identified 12/17 (71%) of oral squamous cell carcinomas and 5/9 (56%) of transforming oral lichen planus but were absent in non‐transforming oral lichen planus. We identified other known oral squamous cell carcinoma mutations (TRRAP, OBSCN, and LRP2) but also previously unreported mutations (TENM3 and ASH1L) in lichen planus‐associated oral squamous cell carcinomas.ConclusionsThese findings suggest alterations in DNA damage response and apoptosis pathways underlie lichen planus‐related oral squamous cell carcinoma transformation and are supported by mutational signatures indicative of DNA damage. This study characterized patterns of mutational events present in oral lichen planus associated with squamous cell carcinoma and in squamous cell carcinoma associated with oral lichen planus but not in non‐transforming oral lichen planus.

Abstract Aims In heart failure (HF), pulmonary venous hypertension (PVH) produces pulmonary hypertension (PH) with remodeling of pulmonary veins (PV) and arteries (PA). In a porcine PVH model, we performed proteomic-based bioinformatics to investigate unique pathophysiologic mechanisms mediating PA and PV remodeling. Methods and results Large PV were banded (PVH, n = 10) or not (Sham, n = 9) in piglets. At sacrifice, PV and PA were perfusion labelled for vessel-specific histology and proteomics. The PA and PV were separately sampled with laser-capture micro-dissection for mass spectrometry. Pulmonary vascular resistance [Wood Units; 8.6 (95% confidence interval: 6.3, 12.3) vs. 2.0 (1.7, 2.3)] and PA [19.9 (standard error of mean, 1.1) vs. 10.3 (1.1)] and PV [14.2 (1.2) vs. 7.6 (1.1)] wall thickness/external diameter (%) were increased in PVH (P &lt; 0.05 for all). Similar numbers of proteins were identified in PA (2093) and PV (2085) with 94% overlap, but biological processes differed. There were more differentially expressed proteins (287 vs. 161), altered canonical pathways (17 vs. 3), and predicted upstream regulators (PUSR; 22 vs. 6) in PV than PA. In PA and PV, bioinformatics indicated activation of the integrated stress response and mammalian target of rapamycin signalling with dysregulated growth. In PV, there was also activation of Rho/Rho-kinase signalling with decreased actin cytoskeletal signalling and altered tight and adherens junctions, ephrin B, and caveolae-mediated endocytosis signalling; all indicating disrupted endothelial barrier function. Indeed, protein biomarkers and the top PUSR in PV (transforming growth factor-beta) suggested endothelial to mesenchymal transition in PV. Findings were similar in human autopsy specimens. Conclusion These findings provide new therapeutic targets to oppose pulmonary vascular remodeling in HF-related PH.