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The aim of this cross-sectional study was to compare plasma C-peptide presence and levels in people without diabetes (CON) and with Type 1 diabetes and relate C-peptide status to clinical factors. In a subset we evaluated 50 microRNAs (miRs) previously implicated in beta-cell death and associations with clinical status and C-peptide levels. Diabetes age of onset was stratified as adult (≥ 18 y.o) or childhood (< 18 y.o.), and diabetes duration was stratified as ≤ 10 years, 10–20 years and > 20 years. Plasma C-peptide was measured by ultrasensitive ELISA. Plasma miRs were quantified using TaqMan probe-primer mix on an OpenArray platform. C-peptide was detectable in 55.3% of (n = 349) people with diabetes, including 64.1% of adults and 34.0% of youth with diabetes, p  < 0.0001 and in all (n = 253) participants without diabetes (CON). C-peptide levels, when detectable, were lower in the individuals with diabetes than in the CON group [median lower quartile (LQ)–upper quartile (UQ)] 5.0 (2.6–28.7) versus 650.9 (401.2–732.4) pmol/L respectively, p  < 0.0001 and lower in childhood versus adult-onset diabetes [median (LQ–UQ) 4.2 (2.6–12.2) pmol/L vs. 8.0 (2.3–80.5) pmol/L, p  = 0.02, respectively]. In the childhood-onset group more people with longer diabetes duration (> 20 years) had detectable C-peptide (60%) than in those with shorter diabetes duration (39%, p for trend < 0.05). Nine miRs significantly correlated with detectable C-peptide levels in people with diabetes and 16 miRs correlated with C-peptide levels in CON. Our cross-sectional study results are supportive of (a) greater beta-cell function loss in younger onset Type 1 diabetes; (b) persistent insulin secretion in adult-onset diabetes and possibly regenerative secretion in childhood-onset long diabetes duration; and (c) relationships of C-peptide levels with circulating miRs. Confirmatory clinical studies and related basic science studies are merited.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide–CoQ–respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold. For the body to work properly, cells must constantly ‘talk’ to each other using signalling molecules. Receiving a chemical signal triggers a series of molecular events in a cell, a so-called ‘signal transduction pathway’ that connects a signal with a precise outcome. Disturbing cell signalling can trigger disease, and strict control mechanisms are therefore in place to ensure that communication does not break down or become erratic. For instance, just as a thermostat turns off the heater once the right temperature is reached, negative feedback mechanisms in cells switch off signal transduction pathways when the desired outcome has been achieved. The hormone insulin is a signal for growth that increases in the body following a meal to promote the storage of excess blood glucose (sugar) in muscle and fat cells. The hormone binds to insulin receptors at the cell surface and switches on a signal transduction pathway that makes the cell take up glucose from the bloodstream. If the signal is not engaged diseases such as diabetes develop. Conversely, if the signal cannot be adequately switched of cancer can develop. Determining exactly how insulin works would help to understand these diseases better and to develop new treatments. Kearney et al. therefore set out to examine the biochemical ‘fail-safes’ that control insulin signalling. Experiments using computer simulations of the insulin signalling pathway revealed a potential new mechanism for negative feedback, which centred on a molecule known as Akt. The models predicted that if the negative feedback were removed, then Akt would become hyperactive and accumulate at the cell’s surface after stimulation with insulin. Further manipulation of the ‘virtual’ insulin signalling pathway and studies of live cells in culture confirmed that this was indeed the case. The cell biology experiments also showed how Akt, once at the cell surface, was able to engage the negative feedback and shut down further insulin signalling. Akt did this by inactivating a protein required to pass the signal from the insulin receptor to the rest of the cell. Overall, this work helps to understand cell communication by revealing a previously unknown, and critical component of the insulin signalling pathway.

To determine whether continuous subcutaneous insulin infusion (CSII) vs. multiple daily injections (MDI) therapy from near-diagnosis of type 1 diabetes is associated with reduced glycaemic variability (GV) and altered microRNA (miRNAs) expression. Adolescents (74% male) within 3-months of diabetes diagnosis (n = 27) were randomized to CSII (n = 12) or MDI. HbA1c, 1-5-Anhydroglucitol (1,5-AG), high sensitivity C-peptide and a custom TaqMan qPCR panel of 52 miRNAs were measured at baseline and follow-up (median (LQ-UQ); 535 (519–563) days). There were no significant differences between groups in baseline or follow-up HbA1c or C-peptide, nor baseline miRNAs. Mean ± SD 1,5-AG improved with CSII vs. MDI (3.1 ± 4.1 vs. − 2.2 ± − 7.0 mg/ml respectively, P  = 0.029). On follow-up 11 miRNAs associated with diabetes vascular complications had altered expression in CSII-users. Early CSII vs. MDI use is associated with lower GV and less adverse vascular-related miRNAs. Relationships with future complications are of interest.

Systems genetics has begun to tackle the complexity of insulin resistance by capitalising on computational advances to study high-diversity populations. ‘Diversity Outbred in Australia (DOz)’ is a population of genetically unique mice with profound metabolic heterogeneity. We leveraged this variance to explore skeletal muscle’s contribution to whole-body insulin action through metabolic phenotyping and skeletal muscle proteomics of 215 DOz mice. Linear modelling identified 553 proteins that associated with whole-body insulin sensitivity (Matsuda Index) including regulators of endocytosis and muscle proteostasis. To enrich for causality, we refined this network by focusing on negatively associated, genetically regulated proteins, resulting in a 76-protein fingerprint of insulin resistance. We sought to perturb this network and restore insulin action with small molecules by integrating the Broad Institute Connectivity Map platform and in vitro assays of insulin action using the Prestwick chemical library. These complementary approaches identified the antibiotic thiostrepton as an insulin resistance reversal agent. Subsequent validation in ex vivo insulin-resistant mouse muscle and palmitate-induced insulin-resistant myotubes demonstrated potent insulin action restoration, potentially via upregulation of glycolysis. This work demonstrates the value of a drug-centric framework to validate systems-level analysis by identifying potential therapeutics for insulin resistance.

Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects the motor neurons, causing progressive muscle weakness and paralysis. While research has focused on understanding pathological mechanisms in the motor cortex and spinal cord, there is growing evidence that extra-motor brain regions may also play a role in the pathogenesis or progression of ALS. Methods We generated 165 sample-matched post-mortem brain transcriptomes from 22 sporadic ALS patients with pTDP-43 pathological staging and 11 non-neurological controls. For each individual, five brain regions underwent mRNA sequencing: motor cortex (pTDP-43 inclusions always present), prefrontal cortex and hippocampus (pTDP-43 inclusions sometimes present), and occipital cortex and cerebellum (pTDP-43 inclusions rarely present). We examined gene expression, cell-type composition, transcript usage (% contribution of a transcript to total gene expression) and alternative splicing, comparing ALS-specific changes between brain regions. We also considered whether post-mortem pTDP-43 pathological stage classification defined ALS subgroups with distinct gene expression profiles. Results Significant gene expression changes were observed in ALS cases for all five brain regions, with the cerebellum demonstrating the largest number of total (> 3,000) and unique (60%) differentially expressed genes. Pathway enrichment and predicted activity were largely concordant across brain regions, suggesting that ALS-linked mechanisms, including inflammation, mitochondrial dysfunction and oxidative stress, are also dysregulated in non-motor brain regions. Switches in transcript usage were identified for a small set of genes including increased usage of a POLDIP3 transcript, associated with TDP-43 loss-of-function, in the cerebellum and a XBP1 transcript, indicative of unfolded protein response activity, in the motor cortex. Extensive variation in RNA splicing was identified in the ALS brain, with 26–41% of alternatively spliced genes unique to a given brain region. This included detection of TDP-43-associated cryptic splicing events such as the STMN2 cryptic exon which was shown to have a pTDP-43 pathology-specific expression pattern. Finally, ALS patients with stage 4 pTDP-43 pathology demonstrated distinct gene and protein expression changes in the cerebellum. Conclusions Together our findings highlighted widespread transcriptome alterations in ALS post-mortem brain and showed that, despite the absence of pTDP-43 pathology in the cerebellum, extensive and pTDP-43 pathological stage-specific RNA changes are evident in this brain region.

Mitochondria play an important role in both normal heart function and disease etiology. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteome study in the HMDP (72 strains, n=2-3 mice) and retrieved 848 mitochondrial proteins (quantified in ≥50 strains). High-resolution association mapping on their relative abundance levels revealed three trans -acting genetic loci on chromosomes (chr) 7, 13 and 17 that regulate distinct classes of mitochondrial proteins as well as cardiac hypertrophy. DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P =2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P =3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P =6.9E-05). Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Furthermore, a large cohort of Diversity Outbred mice was used to corroborate Lrpprc gene as a driver of mitochondrial DNA (mtDNA)-encoded gene regulation, and to show that the chr17 locus is specific to heart. Variations in all three loci were associated with heart mass in at least one of two independent heart stress models, namely, isoproterenol-induced heart failure and diet-induced obesity. These findings suggest that common variations in certain mitochondrial proteins can act in trans to influence tissue-specific mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.

Metastasis remains the major cause of death in breast cancer (BrCa) and lacks specific treatment strategies. The kynurenine pathway (KP) has been suggested as a key mechanism facilitating progression of BrCa. While KP activity has been explored in primary BrCa, its role in metastasis remains unclear. To better understand this, we examined changes in the KP of BrCa with no metastasis compared to BCa that produced local or distant metastases. Given that the cancer cell secretome plays a role in metastasis, we also investigated the relationship between changes in KP activity and serum proteins of patients with local or distant metastases. To investigate changes in the KP in BrCa, with and without metastasis, we quantified KP metabolites in blood sera collected from patients with stage 1 BrCa (n = 34), BrCa with local metastases (n = 46), BrCa with distant metastases (n = 20) and healthy controls (n = 39). The serum protein profile of the BrCa patients with local or distant metastasis was determined before correlation analyses were carried out to examine the relationship between changes in the KP and cancer serum proteins using SPSS. We found that the KP was elevated in BrCa patients with local and distant metastasis compared to healthy controls and stage 1 BrCa patients. The activity of kynurenine monooxygenase (KMO) and kynureninase (KYNU) A was positively associated with disease stage and was higher compared to healthy controls. Proteome analysis in patients with local or distant metastasis revealed the dysregulation of 14 proteins, 9 of which were up-regulated and 5 down-regulated at the distant metastasis stage. Importantly, three of these proteins have not been previously linked to BrCa metastasis. In the correlation studies between the KP profile, cancer serum proteins and metastasis status, KYNU A had the greatest number of significant associations with cancer serum protein, followed by KMO. Our findings reveal that the KP was regulated differently at various stages of BrCa and was more dysregulated in patients with local or distant metastasis. These KP activity changes showed a significant association with cancer serum proteins in BrCa patients with local or distant metastasis, highlighting the potential role of KP in BrCa metastasis.

Introduction Metastasis remains the major cause of death in breast cancer (BrCa) and lacks specific treatment strategies. The kynurenine pathway (KP) has been suggested as a key mechanism facilitating progression of BrCa. While KP activity has been explored in primary BrCa, its role in metastasis remains unclear. To better understand this, we examined changes in the KP of BrCa with no metastasis compared to BCa that produced local or distant metastases. Given that the cancer cell secretome plays a role in metastasis, we also investigated the relationship between changes in KP activity and serum proteins of patients with local or distant metastases. Methods To investigate changes in the KP in BrCa, with and without metastasis, we quantified KP metabolites in blood sera collected from patients with stage 1 BrCa ( n  = 34), BrCa with local metastases ( n  = 46), BrCa with distant metastases ( n  = 20) and healthy controls ( n  = 39). The serum protein profile of the BrCa patients with local or distant metastasis was determined before correlation analyses were carried out to examine the relationship between changes in the KP and cancer serum proteins using SPSS. Results We found that the KP was elevated in BrCa patients with local and distant metastasis compared to healthy controls and stage 1 BrCa patients. The activity of kynurenine monooxygenase (KMO) and kynureninase (KYNU) A was positively associated with disease stage and was higher compared to healthy controls. Proteome analysis in patients with local or distant metastasis revealed the dysregulation of 14 proteins, 9 of which were up-regulated and 5 down-regulated at the distant metastasis stage. Importantly, three of these proteins have not been previously linked to BrCa metastasis. In the correlation studies between the KP profile, cancer serum proteins and metastasis status, KYNU A had the greatest number of significant associations with cancer serum protein, followed by KMO. Conclusion Our findings reveal that the KP was regulated differently at various stages of BrCa and was more dysregulated in patients with local or distant metastasis. These KP activity changes showed a significant association with cancer serum proteins in BrCa patients with local or distant metastasis, highlighting the potential role of KP in BrCa metastasis.

ObjectiveTo identify blood proteins differentially expressed in children with and without delayed recovery from concussion.DesignA single centre, prospective, longitudinal cohort study.SettingThe Royal Children’s Hospital (RCH), a paediatric tertiary care centre in Victoria, Australia.ParticipantsChildren aged 5 to 18 years who presented to the RCH Emergency Department within 48 hours of a concussion (n=40). Controls included children with upper body orthopaedic injury (n=7), and otherwise healthy children undergoing elective day surgery at the RCH (n=33).Interventions (or Assessment of Risk Factors)Delayed recovery from concussion, determined using the Post-Concussion Symptoms Inventory at two weeks post-injury.Outcome MeasuresRelative abundance, obtained using single window acquisition of all theoretical mass spectra (SWATH-MS), and absolute concentration, obtained using multiple reaction monitoring (MRM) and enzyme linked immunosorbent assay (ELISA), of differentially abundant proteins in children with and without delayed recovery from concussion.Main ResultsSix of the proteins differentially expressed in children with and without delayed recovery from concussion were selected for validation using a protocol comprising of a statistical and non-statistical component. Most notably, a support vector machine including one protein, TC1, (temporary codename pending patent protection) differentiated between children with and without delayed recovery from concussion with an accuracy of 81% [95% confidence interval 65 – 96%].ConclusionsTC1 has been identified as a marker of delayed recovery from concussion in children, which has the capacity to transform the way that delayed recovery is assessed and treated in a clinical setting.