Targeting postsynaptic glutamate receptor scaffolding proteins PSD-95 and PICK1 for obesity treatment.

Human genome-wide association studies (GWAS) suggest a functional role for central glutamate receptor signaling and plasticity in body weight regulation. Here, we use UK Biobank GWAS summary statistics of body mass index (BMI) and body fat percentage (BF%) to identify genes encoding proteins known to interact with postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors. Loci in/near discs large homolog 4 (DLG4) and protein interacting with C kinase 1 (PICK1) reached genome-wide significance (P < 5 × 10-8) for BF% and/or BMI. To further evaluate the functional role of postsynaptic density protein-95 (PSD-95; gene name: DLG4) and PICK1 in energy homeostasis, we used dimeric PSD-95/disc large/ZO-1 (PDZ) domain-targeting peptides of PSD-95 and PICK1 to demonstrate that pharmacological inhibition of PSD-95 and PICK1 induces prolonged weight-lowering effects in obese mice. Collectively, these data demonstrate that the glutamate receptor scaffolding proteins, PICK1 and PSD-95, are genetically linked to obesity and that pharmacological targeting of their PDZ domains represents a promising therapeutic avenue for sustained weight loss.

Harnessing the power of proteomics in precision diabetes medicine.

Precision diabetes medicine (PDM) aims to reduce errors in prevention programmes, diagnosis thresholds, prognosis prediction and treatment strategies. However, its advancement and implementation are difficult due to the heterogeneity of complex molecular processes and environmental exposures that influence an individual's disease trajectory. To address this challenge, it is imperative to develop robust screening methods for all areas of PDM. Innovative proteomic technologies, alongside genomics, have proven effective in precision cancer medicine and are showing promise in diabetes research for potential translation. This narrative review highlights how proteomics is well-positioned to help improve PDM. Specifically, a critical assessment of widely adopted affinity-based proteomic technologies in large-scale clinical studies and evidence of the benefits and feasibility of using MS-based plasma proteomics is presented. We also present a case for the use of proteomics to identify predictive protein panels for type 2 diabetes subtyping and the development of clinical prediction models for prevention, diagnosis, prognosis and treatment strategies. Lastly, we discuss the importance of plasma and tissue proteomics and its integration with genomics (proteogenomics) for identifying unique type 2 diabetes intra- and inter-subtype aetiology. We conclude with a call for action formed on advancing proteomics technologies, benchmarking their performance and standardisation across sites, with an emphasis on data sharing and the inclusion of diverse ancestries in large cohort studies. These efforts should foster collaboration with key stakeholders and align with ongoing academic programmes such as the Precision Medicine in Diabetes Initiative consortium.

Mass spectrometry-based proteomics approaches to interrogate skeletal muscle adaptations to exercise.

Acute exercise and chronic exercise training elicit beneficial whole-body changes in physiology that ultimately depend on profound alterations to the dynamics of tissue-specific proteins. Since the work accomplished during exercise owes predominantly to skeletal muscle, it has received the majority of interest from exercise scientists that attempt to unravel adaptive mechanisms accounting for salutary metabolic effects and performance improvements that arise from training. Contemporary scientists are also beginning to use mass spectrometry-based proteomics, which is emerging as a powerful approach to interrogate the muscle protein signature in a more comprehensive manner. Collectively, these technologies facilitate the analysis of skeletal muscle protein dynamics from several viewpoints, including changes to intracellular proteins (expression proteomics), secreted proteins (secretomics), post-translational modifications as well as fiber-, cell-, and organelle-specific changes. This review aims to highlight recent literature that has leveraged new workflows and advances in mass spectrometry-based proteomics to further our understanding of training-related changes in skeletal muscle. We call attention to untapped areas in skeletal muscle proteomics research relating to exercise training and metabolism, as well as basic points of contention when applying mass spectrometry-based analyses, particularly in the study of human biology. We further encourage researchers to couple the hypothesis-generating and descriptive nature of omics data with functional analyses that propel our understanding of the complex adaptive responses in skeletal muscle that occur with acute and chronic exercise.

Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M.

The discovery of exercise-regulated circulatory factors has fueled interest in organ crosstalk, especially between skeletal muscle and adipose tissue, and the role in mediating beneficial effects of exercise. We studied the adipose tissue transcriptome in men and women with normal glucose tolerance or type 2 diabetes following an acute exercise bout, revealing substantial exercise- and time-dependent changes, with sustained increase in inflammatory genes in type 2 diabetes. We identify oncostatin-M as one of the most upregulated adipose-tissue-secreted factors post-exercise. In cultured human adipocytes, oncostatin-M enhances MAPK signaling and regulates lipolysis. Oncostatin-M expression arises predominantly from adipose tissue immune cell fractions, while the corresponding receptors are expressed in adipocytes. Oncostatin-M expression increases in cultured human Thp1 macrophages following exercise-like stimuli. Our results suggest that immune cells, via secreted factors such as oncostatin-M, mediate a crosstalk between skeletal muscle and adipose tissue during exercise to regulate adipocyte metabolism and adaptation.

High-throughput proteomics uncovers exercise training and type 2 diabetes-induced changes in human white adipose tissue.

White adipose tissue (WAT) is important for metabolic homeostasis. We established the differential proteomic signatures of WAT in glucose-tolerant lean and obese individuals and patients with type 2 diabetes (T2D) and the response to 8 weeks of high-intensity interval training (HIIT). Using a high-throughput and reproducible mass spectrometry-based proteomics pipeline, we identified 3773 proteins and found that most regulated proteins displayed progression in markers of dysfunctional WAT from lean to obese to T2D individuals and were highly associated with clinical measures such as insulin sensitivity and HbA1c. We propose that these distinct markers could serve as potential clinical biomarkers. HIIT induced only minor changes in the WAT proteome. This included an increase in WAT ferritin levels independent of obesity and T2D, and WAT ferritin levels were strongly correlated with individual insulin sensitivity. Together, we report a proteomic signature of WAT related to obesity and T2D and highlight an unrecognized role of human WAT iron metabolism in exercise training adaptations.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Organ-specific metabolic pathways distinguish prediabetes, type 2 diabetes, and normal tissues.

Environmental and genetic factors cause defects in pancreatic islets driving type 2 diabetes (T2D) together with the progression of multi-tissue insulin resistance. Mass spectrometry proteomics on samples from five key metabolic tissues of a cross-sectional cohort of 43 multi-organ donors provides deep coverage of their proteomes. Enrichment analysis of Gene Ontology terms provides a tissue-specific map of altered biological processes across healthy, prediabetes (PD), and T2D subjects. We find widespread alterations in several relevant biological pathways, including increase in hemostasis in pancreatic islets of PD, increase in the complement cascade in liver and pancreatic islets of PD, and elevation in cholesterol biosynthesis in liver of T2D. Our findings point to inflammatory, immune, and vascular alterations in pancreatic islets in PD that are hypotheses to be tested for potential contributions to hormonal perturbations such as impaired insulin and increased glucagon production. This multi-tissue proteomic map suggests tissue-specific metabolic dysregulations in T2D.

Exercise timing influences multi-tissue metabolome and skeletal muscle proteome profiles in type 2 diabetic patients - A randomized crossover trial.

AIMS/HYPOTHESIS: Metabolic effects of exercise may partly depend on the time-of-day when exercise is performed. We tested the hypothesis that exercise timing affects the adaptations in multi-tissue metabolome and skeletal muscle proteome profiles in men with type 2 diabetes. METHODS: Men fitting the inclusion (type 2 diabetes, age 45-68 years and body mass index 23-33 kg/m2) and exclusion criteria (insulin treatment, smoking, concurrent systemic disease, and regular exercise training) were included in a randomized crossover trial (n = 15). Participants included in this metabolomics and proteomics analysis fully completed all exercise sessions (n = 8). The trial consisted of two weeks of high-intensity interval training (HIT) (three sessions/week) either in the morning (08:00, n = 5) or afternoon (16:45, n = 3), a two-week wash-out period, and an additional two weeks of HIT at the opposing time. Participants and researchers were not blinded to group allocation. Blood, skeletal muscle and subcutaneous adipose tissue were obtained before the first, and after each training period. Broad-spectrum, untargeted proteomic analysis was performed on skeletal muscle, and metabolomic analysis was performed on all biosamples. Differential content was assessed by linear regression and pathway set enrichment analyses were performed. Coordinated metabolic changes across tissues were identified by Spearman correlation analysis. RESULTS: Metabolic and proteomic profiles remained stable after two weeks of HIT, and individual metabolites and proteins were not altered, irrespective of the time of day at which the training was performed. However, coordinated changes in relevant metabolic pathways and protein categories were identified. Morning and afternoon HIT similarly increased plasma diacylglycerols, skeletal muscle acyl-carnitines, and subcutaneous adipose tissue sphingomyelins and lysophospholipids. Acyl-carnitines were central to training-induced metabolic cross-talk across tissues. Plasma carbohydrates, via the penthose phosphate pathway, were increased and skeletal muscle lipids were decreased after morning compared to afternoon HIT. Skeletal muscle lipoproteins were higher, and mitochondrial complex III abundance was lower after morning compared to afternoon HIT. CONCLUSIONS/INTERPRETATION: We provide a comprehensive analysis of a multi-tissue metabolomic and skeletal muscle proteomic responses to training at different times of the day in men with type 2 diabetes. Increased circulating lipids and changes in adipose tissue lipid composition were common between morning and afternoon HIT. However, afternoon HIT increased skeletal muscle lipids and mitochondrial content to a greater degree than morning training. Thus, there is a diurnal component in the metabolomic and proteomic response to exercise in men with type 2 diabetes. The clinical relevance of this response warrants further investigation.

Exercise suppresses tumor growth independent of high fat food intake and associated immune dysfunction.

Epidemiological data suggest that exercise training protects from cancer independent of BMI. Here, we aimed to elucidate mechanisms involved in voluntary wheel running-dependent control of tumor growth across chow and high-fat diets. Access to running wheels decreased tumor growth in B16F10 tumor-bearing on chow (- 50%) or high-fat diets (- 75%, p < 0.001), however, tumor growth was augmented in high-fat fed mice (+ 53%, p < 0.001). Tumor growth correlated with serum glucose (p < 0.01), leptin (p < 0.01), and ghrelin levels (p < 0.01), but not with serum insulin levels. Voluntary wheel running increased immune recognition of tumors as determined by microarray analysis and gene expression analysis of markers of macrophages, NK and T cells, but the induction of markers of macrophages and NK cells was attenuated with high-fat feeding. Moreover, we found that the regulator of innate immunity, ZBP1, was induced by wheel running, attenuated by high-fat feeding and associated with innate immune recognition in the B16F10 tumors. We observed no effects of ZBP1 on cell cycle arrest, or exercise-regulated necrosis in the tumors of running mice. Taken together, our data support epidemiological findings showing that exercise suppresses tumor growth independent of BMI, however, our data suggest that high-fat feeding attenuates exercise-mediated immune recognition of tumors.

The proteomic profile of the human myotendinous junction.

Proteomics analysis of skeletal muscle has recently progressed from whole muscle tissue to single myofibers. Here, we further focus on a specific myofiber domain crucial for force transmission from muscle to tendon, the myotendinous junction (MTJ). To overcome the anatomical constraints preventing the isolation of pure MTJs, we performed in-depth analysis of the MTJ by progressive removal of the muscle component in semitendinosus muscle-tendon samples. Using detergents with increasing stringency, we quantified >3000 proteins across all samples, and identified 112 significantly enriched MTJ proteins, including 24 known MTJ-enriched proteins. Of the 88 novel MTJ markers, immunofluorescence analysis confirmed the presence of tetraspanin-24 (CD151), kindlin-2 (FERMT2), cartilage intermediate layer protein 1 (CILP), and integrin-alpha10 (ITGA10), at the human MTJ. Together, these human data constitute the first detailed MTJ proteomics resource that will contribute to advance understanding of the biology of the MTJ and its failure in pathological conditions.

Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle.

Insulin-stimulated muscle glucose uptake is a key process in glycemic control. This process depends on the redistribution of glucose transporters to the surface membrane, a process that involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with an increased risk of type 2 diabetes, and observations from carriers of a TBC1D1 variant associate this protein to a severe obesity phenotype. Here, we identified interactors of the endogenous TBC1D4 protein in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors was regulated by insulin, including proteins known to be involved in glucose metabolism (e.g., 14-3-3 proteins and α-actinin-4 [ACTN4]). TBC1D1 also coprecipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin or exercise in young, healthy, lean individuals. Similarly, the exercise- and insulin-regulated phosphorylation of the TBC1D1-TBC1D4 complex was intact. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phosphoregulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with type 2 diabetes. Altogether, we provide a repository of TBC1D4 interactors in human and mouse skeletal muscle that serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.

Protocol to characterize mitochondrial supercomplexes from mouse tissues by combining BN-PAGE and MS-based proteomics.

The assembly of mitochondrial respiratory complexes into supercomplexes has significant implications for mitochondrial function. This protocol details mitochondrial isolation from mouse tissues and the use of blue native gel electrophoresis (BN-PAGE) to separate pre-identified mitochondrial supercomplexes into different gel bands. We then describe the excision of the individual bands, followed by in-gel protein digestion and peptide desalting for mass spectrometry (MS)-based proteomics. This protocol provides a time-efficient measurement of the abundance and distribution of proteins within known supercomplexes. For complete details on the use and execution of this profile, please refer to Gonzalez-Franquesa et al. (2021).

Integrated Liver and Plasma Proteomics in Obese Mice Reveals Complex Metabolic Regulation.

Obesity leads to the development of nonalcoholic fatty liver disease (NAFLD) and associated alterations to the plasma proteome. To elucidate the underlying changes associated with obesity, we performed liquid chromatography-tandem mass spectrometry in the liver and plasma of obese leptin-deficient ob/ob mice and integrated these data with publicly available transcriptomic and proteomic datasets of obesity and metabolic diseases in preclinical and clinical cohorts. We quantified 7173 and 555 proteins in the liver and plasma proteomes, respectively. The abundance of proteins related to fatty acid metabolism were increased, alongside peroxisomal proliferation in ob/ob liver. Putatively secreted proteins and the secretory machinery were also dysregulated in the liver, which was mirrored by a substantial alteration of the plasma proteome. Greater than 50% of the plasma proteins were differentially regulated, including NAFLD biomarkers, lipoproteins, the 20S proteasome, and the complement and coagulation cascades of the immune system. Integration of the liver and plasma proteomes identified proteins that were concomitantly regulated in the liver and plasma in obesity, suggesting that the systemic abundance of these plasma proteins is regulated by secretion from the liver. Many of these proteins are systemically regulated during type 2 diabetes and/or NAFLD in humans, indicating the clinical importance of liver-plasma cross talk and the relevance of our investigations in ob/ob mice. Together, these analyses yield a comprehensive insight into obesity and provide an extensive resource for obesity research in a prevailing model organism.

Discovery of thymosin ß4 as a human exerkine and growth factor.

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in interorgan cross talk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin ß4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in the plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular cross talk.

Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome.

Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR-an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.

Mass-spectrometry-based proteomics reveals mitochondrial supercomplexome plasticity.

Mitochondrial respiratory complex subunits assemble in supercomplexes. Studies of supercomplexes have typically relied upon antibody-based quantification, often limited to a single subunit per respiratory complex. To provide a deeper insight into mitochondrial and supercomplex plasticity, we combine native electrophoresis and mass spectrometry to determine the supercomplexome of skeletal muscle from sedentary and exercise-trained mice. We quantify 422 mitochondrial proteins within 10 supercomplex bands in which we show the debated presence of complexes II and V. Exercise-induced mitochondrial biogenesis results in non-stoichiometric changes in subunits and incorporation into supercomplexes. We uncover the dynamics of supercomplex-related assembly proteins and mtDNA-encoded subunits after exercise. Furthermore, exercise affects the complexing of Lactb, an obesity-associated mitochondrial protein, and ubiquinone biosynthesis proteins. Knockdown of ubiquinone biosynthesis proteins leads to alterations in mitochondrial respiration. Our approach can be applied to broad biological systems. In this instance, comprehensively analyzing respiratory supercomplexes illuminates previously undetectable complexity in mitochondrial plasticity.

Human thermogenic adipocyte regulation by the long noncoding RNA LINC00473.

Human thermogenic adipose tissue mitigates metabolic disease, raising much interest in understanding its development and function. Here, we show that human thermogenic adipocytes specifically express a primate-specific long non-coding RNA, LINC00473 which is highly correlated with UCP1 expression and decreased in obesity and type-2 diabetes. LINC00473 is detected in progenitor cells, and increases upon differentiation and in response to cAMP. In contrast to other known adipocyte LincRNAs, LINC00473 shuttles out of the nucleus, colocalizes and can be crosslinked to mitochondrial and lipid droplet proteins. Up- or down- regulation of LINC00473 results in reciprocal alterations in lipolysis, respiration and transcription of genes associated with mitochondrial oxidative metabolism. Depletion of PLIN1 results in impaired cAMP-responsive LINC00473 expression and lipolysis, indicating bidirectional interactions between PLIN1, LINC00473 and mitochondrial oxidative functions. Thus, we suggest that LINC00473 is a key regulator of human thermogenic adipocyte function, and reveals a role for a LincRNA in inter-organelle communication and human energy metabolism.

Proteomics-Based Comparative Mapping of the Secretomes of Human Brown and White Adipocytes Reveals EPDR1 as a Novel Batokine.

Adipokines secreted from white adipose tissue play a role in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism.

Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation.

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.