The impact of mild hypoxia exposure on myokine secretion in human obesity.

BACKGROUND/OBJECTIVE: Compelling evidence indicates that myokines act in an autocrine, paracrine and endocrine manner to alter metabolic homeostasis. The mechanisms underlying exercise-induced changes in myokine secretion remain to be elucidated. Since exercise acutely decreases oxygen partial pressure (pO2) in skeletal muscle (SM), the present study was designed to test the hypothesis that (1) hypoxia exposure impacts myokine secretion in primary human myotubes and (2) exposure to mild hypoxia in vivo alters fasting and postprandial plasma myokine concentrations in humans. METHODS: Differentiated primary human myotubes were exposed to different physiological pO2 levels for 24 h, and cell culture medium was harvested to determine myokine secretion. Furthermore, we performed a randomized single-blind crossover trial to investigate the impact of mild intermittent hypoxia exposure (MIH: 7-day exposure to 15% O2, 3x2h/day vs. normoxia: 21% O2) on in vivo SM pO2 and plasma myokine concentrations in 12 individuals with overweight and obesity (body-mass index ≥ 28 kg/m2). RESULTS: Hypoxia exposure (1% O2) increased secreted protein acidic and rich in cysteine (SPARC, p = 0.043) and follistatin like 1 (FSTL1, p = 0.021), and reduced leukemia inhibitory factor (LIF) secretion (p = 0.009) compared to 3% O2 in primary human myotubes. In addition, 1% O2 exposure increased interleukin-6 (IL-6, p = 0.004) and SPARC secretion (p = 0.021), whilst reducing fatty acid binding protein 3 (FABP3) secretion (p = 0.021) compared to 21% O2. MIH exposure in vivo markedly decreased SM pO2 (≈40%, p = 0.002) but did not alter plasma myokine concentrations. CONCLUSIONS: Hypoxia exposure altered the secretion of several myokines in primary human myotubes, revealing hypoxia as a novel modulator of myokine secretion. However, both acute and 7-day MIH exposure did not induce alterations in plasma myokine concentrations in individuals with overweight and obesity. CLINICAL TRIALS IDENTIFIER: This study is registered at the Netherlands Trial Register (NL7120/NTR7325).

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase.

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis-Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 µM) and Thr649 (KM ∼25 µM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 µM), Ser711 (KM ∼79 µM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

Adaptation of Oxidative Phosphorylation Machinery Compensates for Hepatic Lipotoxicity in Early Stages of MAFLD.

Alterations in mitochondrial function are an important control variable in the progression of metabolic dysfunction-associated fatty liver disease (MAFLD), while also noted by increased de novo lipogenesis (DNL) and hepatic insulin resistance. We hypothesized that the organization and function of a mitochondrial electron transport chain (ETC) in this pathologic condition is a consequence of shifted substrate availability. We addressed this question using a transgenic mouse model with increased hepatic insulin resistance and DNL due to constitutively active human SREBP-1c. The abundance of ETC complex subunits and components of key metabolic pathways are regulated in the liver of these animals. Further omics approaches combined with functional assays in isolated liver mitochondria and primary hepatocytes revealed that the SREBP-1c-forced fatty liver induced a substrate limitation for oxidative phosphorylation, inducing enhanced complex II activity. The observed increased expression of mitochondrial genes may have indicated a counteraction. In conclusion, a shift of available substrates directed toward activated DNL results in increased electron flows, mainly through complex II, to compensate for the increased energy demand of the cell. The reorganization of key compounds in energy metabolism observed in the SREBP-1c animal model might explain the initial increase in mitochondrial function observed in the early stages of human MAFLD.

Dapagliflozin reduces thrombin generation and platelet activation: implications for cardiovascular risk reduction in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium-glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. METHODS: Male, 8-week-old LDL-receptor-deficient (Ldlr-/-) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. RESULTS: Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet-leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr-/- mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10-9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10-9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. CONCLUSIONS/INTERPRETATION: We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin-platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin's beneficial cardiovascular risk profile.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Oral Treatment with RD2RD2 Impedes Development of Motoric Phenotype and Delays Symptom Onset in SOD1G93A Transgenic Mice.

Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.

Abscisic Acid Receptors and Coreceptors Modulate Plant Water Use Efficiency and Water Productivity.

Improving the water use efficiency (WUE) of crop plants without trade-offs in growth and yield is considered a utopic goal. However, recent studies on model plants show that partial restriction of transpiration can occur without a reduction in CO2 uptake and photosynthesis. In this study, we analyzed the potentials and constraints of improving WUE in Arabidopsis (Arabidopsis thaliana) and in wheat (Triticum aestivum). We show that the analyzed Arabidopsis wild-type plants consume more water than is required for unrestricted growth. WUE was enhanced without a growth penalty by modulating abscisic acid (ABA) responses either by using overexpression of specific ABA receptors or deficiency of ABA coreceptors. Hence, the plants showed higher water productivity compared with the wild-type plants; that is, equal growth with less water. The high WUE trait was resilient to changes in light intensity and water availability, but it was sensitive to the ambient temperature. ABA application to plants generated a partial phenocopy of the water-productivity trait. ABA application, however, was never as effective as genetic modification in enhancing water productivity, probably because ABA indiscriminately targets all ABA receptors. ABA agonists selective for individual ABA receptors might offer an approach to phenocopy the water-productivity trait of the high WUE lines. ABA application to wheat grown under near-field conditions improved WUE without detectable growth trade-offs. Wheat yields are heavily impacted by water deficit, and our identification of this crop as a promising target for WUE improvement may help contribute to greater food security.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP.

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n = 15), early-stage CRCs (n = 15), late-stage CRCs (n = 15)]. Two proteins-clusterin and glutathione synthetase (GSH-S)-featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.

Adipokinome Signatures in Obese Mouse Models Reflect Adipose Tissue Health and Are Associated with Serum Lipid Composition.

Adipocyte and hepatic lipid metabolism govern whole-body metabolic homeostasis, whereas a disbalance of de novo lipogenesis (DNL) in fat and liver might lead to obesity, with severe co-morbidities. Nevertheless, some obese people are metabolically healthy, but the "protective" mechanisms are not yet known in detail. Especially, the adipocyte-derived molecular mediators that indicate adipose functionality are poorly understood. We studied transgenic mice (alb-SREBP-1c) with a "healthy" obese phenotype, and obob mice with hyperphagia-induced "sick" obesity to analyze the impact of the tissue-specific DNL on the secreted proteins, i.e., the adipokinome, of the primary adipose cells by label-free proteomics. Compared to the control mice, adipose DNL is reduced in both obese mouse models. In contrast, the hepatic DNL is reduced in obob but elevated in alb-SREBP-1c mice. To investigate the relationship between lipid metabolism and adipokinomes, we formulated the "liver-to-adipose-tissue DNL" ratio. Knowledge-based analyses of these results revealed adipocyte functionality with proteins, which was involved in tissue remodeling or metabolism in the alb-SREBP-1c mice and in the control mice, but mainly in fibrosis in the obob mice. The adipokinome in "healthy" obesity is similar to that in a normal condition, but it differs from that in "sick" obesity, whereas the serum lipid patterns reflect the "liver-to-adipose-tissue DNL" ratio and are associated with the adipokinome signature.

CDH13 abundance interferes with adipocyte differentiation and is a novel biomarker for adipose tissue health.

BACKGROUND: CDH13, an atypical member of the cadherin superfamily, has been identified in adipocyte secretomes of lean mouse models. CDH13 abundance differs in mouse models according to their susceptibility to develop metabolic disorders, but the role of CDH13 in adipose tissue is unknown. METHODS: Secreted CDH13 protein levels and mRNA levels in visceral adipose tissue were determined in lean and obese mouse models. In vitro studies were performed in 3T3-L1 adipocytes to determine the role of CDH13 in adipocyte differentiation. The pathophysiological impact of visceral adipose tissue CDH13 mRNA and circulating CDH13 levels were determined in humans (normal-weight men n = 37, obese men n = 109 including n = 51 type 2 diabetes patients) and in obese patients (n = 14) pre- and post-metabolic surgery. RESULTS: This study shows that in visceral adipose tissue CDH13 protein secretion and mRNA levels were decreased in obese mouse models. Mechanistically, CDH13 affects lipid metabolism during adipogenesis but not in mature adipocytes. CDH13 knockdown during adipogenesis reduced fatty acid uptake and lipid content in developing adipocytes. Furthermore, CDH13 depletion during adipogenesis lowered the induction of PPARγ and C/EBPα expression. These observations are of pathophysiological impact since visceral adipose tissue CDH13 mRNA and circulating CDH13 levels were decreased in obese men compared to normal-weight controls. Weight loss induced by bariatric surgery restored circulating CDH13 to levels found in normal-weight controls. CONCLUSIONS: CDH13 levels in adipose tissue and the circulation are affected by obesity in mouse models and humans and are restored by weight loss in humans. CDH13 interferes with the differentiation potential of adipocytes and therefore is a marker for plasticity of fat tissue that might reflect the health status of adipose tissue.

Inactivation of SREBP-1a Phosphorylation Prevents Fatty Liver Disease in Mice: Identification of Related Signaling Pathways by Gene Expression Profiles in Liver and Proteomes of Peroxisomes.

The key lipid metabolism transcription factor sterol regulatory element-binding protein (SREBP)-1a integrates gene regulatory effects of hormones, cytokines, nutrition and metabolites as lipids, glucose, or cholesterol via phosphorylation by different mitogen activated protein kinase (MAPK) cascades. We have previously reported the impact of SREBP-1a phosphorylation on the phenotype in transgenic mouse models with liver-specific overexpression of the N-terminal transcriptional active domain of SREBP-1a (alb-SREBP-1a) or a MAPK phosphorylation site-deficient variant (alb-SREBP-1a∆P; (S63A, S117A, T426V)), respectively. In this report, we investigated the molecular basis of the systemic observations by holistic analyses of gene expression in liver and of proteome patterns in lipid-degrading organelles involved in the pathogenesis of metabolic syndrome, i.e., peroxisomes, using 2D-DIGE and mass spectrometry. The differences in hepatic gene expression and peroxisomal protein patterns were surprisingly small between the control and alb-SREBP-1a mice, although the latter develop a severe phenotype with visceral obesity and fatty liver. In contrast, phosphorylation site-deficient alb-SREBP-1a∆P mice, which are protected from fatty liver disease, showed marked differences in hepatic gene expression and peroxisomal proteome patterns. Further knowledge-based analyses revealed that disruption of SREBP-1a phosphorylation resulted in massive alteration of cellular processes, including signs for loss of targeting lipid pathways.

Deficiency in lymphotoxin ß receptor protects from atherosclerosis in apoE-deficient mice.

RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice.

OBJECTIVE: Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. APPROACH AND RESULTS: Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. CONCLUSIONS: HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.

Association between copy-number variation on metabolic phenotypes and HDL-C levels in patients with polycystic ovary syndrome.

Polygenic diseases with a broad phenotypic spectrum, such as polycystic ovary syndrome (PCOS), present a particular challenge in terms of identifying the underlying genetic mechanisms, nevertheless genetic variants have impact on the individual phenotype. We aimed to determine if next to genetic variations like SNPs further mechanisms might play a role in the pathogenesis of PCOS. We examined the effect of copy-number variations (CNVs) on metabolic phenotypes in PCOS. The intragenic rs1244979, rs2815752 in NEGR1 gene, and rs780094 in GCKR gene were genotyped and CNVs were determined by droplet digital polymerase chain reaction (ddPCR) in PCOS patients (n = 153) and controls without metabolic syndrome (n = 142). The study indicated that SNPs are not associated with the pathogenesis of PCOS but affect metabolic phenotypes. The CNVs investigated show a lower variability in PCOS than in CON. Furthermore, we provided direct evidence that the copy number, but not the genotype of the CNV in the genomic regions of rs780094(GCKR) is associated with low level of high-density lipoprotein cholesterol in PCOS. This study supports the hypothesis that not only genetic variants, but also CNVs in metabolically relevant genes, have an effect on metabolic phenotypes in our group of PCOS patients.

Novel Insights into the Adipokinome of Obese and Obese/Diabetic Mouse Models.

The group of adipokines comprises hundreds of biological active proteins and peptides released from adipose tissue. Alterations of those complex protein signatures are suggested to play a crucial role in the pathophysiology of multifactorial, metabolic diseases. We hypothesized that also the pathophysiology of type-2-diabetes is linked to the dysregulation of the adipocyte secretome. To test this, we investigated mouse models with monogenic defects in leptin signaling which are susceptible to adipositas (C57BL/6 Cg-Lepob (obob)) or adipositas with diabetes (C57BL/KS Cg-Leprdb (dbdb)) according to their genetic background. At the age of 17 weeks, visceral fat was obtained and primary murine adipocytes were isolated to harvest secretomes. Quantitative proteome analyses (LC-ESI-MS/MS) identified more than 800 potential secreted proteins. The secretome patterns revealed significant differences connected to the pathophysiology of obese mice. Pathway analyses indicated that these differences focus on exosome modelling, but failed to provide more precise specifications. To investigate the relationship of secretome data to insulin sensitivity, we examined the content of diabetogenic lipids, i.e., diacylglycerols (DAGs), identified as key players in lipid-induced insulin resistance. In contrast to obob mice, fat tissue of dbdb mice showed elevated DAG content, especially of DAG species with saturated fatty acid C16:0 and C18:0, while unsaturated fatty acid C16:1 were only changed in obob. Furthermore, DAG signatures of the models specifically correlate to secreted regulated adipokines indicating specific pathways. In conclusion, our data further support the concept that the fat tissue is an endocrine organ that releases bioactive factors corresponding to adipose tissue health status.

Peroxisomes compensate hepatic lipid overflow in mice with fatty liver.

UNLABELLED: Major causes of lipid accumulation in liver are increased import or synthesis or decreased catabolism of fatty acids. The latter is caused by dysfunction of cellular organelles controlling energy homeostasis, i.e., mitochondria. Peroxisomes also appear to be an important organelle in lipid metabolism of hepatocytes, but little is known about their role in the development of non-alcoholic fatty liver disease (NAFLD). To investigate the role of peroxisomes alongside mitochondria in excessive hepatic lipid accumulation, we used leptin-resistant db/db mice on C57BLKS background, a mouse model that develops hyperphagia-induced diabetes with obesity and NAFLD. Proteome and gene expression analyses along with lipid analyses in the liver revealed differential expression of genes related to lipid metabolism and ß-oxidation, whereas genes for peroxisomal proteins were predominantly regulated. CONCLUSION: Our investigations show that in fatty liver disease in combination with obesity and diabetes, the hepatocyte-protecting organelle peroxisome is altered. Hence, peroxisomes might indicate a stage of pre-NAFLD, play a role in the early development of NAFLD and appear to be a potential target for treatment and prevention of NAFLD.

Lipidomics-Reshaping the Analysis and Perception of Type 2 Diabetes.

As a consequence of a sedentary lifestyle as well as changed nutritional behavior, today's societies are challenged by the rapid propagation of metabolic disorders. A common feature of diseases, such as obesity and type 2 diabetes (T2D), is the dysregulation of lipid metabolism. Our understanding of the mechanisms underlying these diseases is hampered by the complexity of lipid metabolic pathways on a cellular level. Furthermore, overall lipid homeostasis in higher eukaryotic organisms needs to be maintained by a highly regulated interplay between tissues, such as adipose tissue, liver and muscle. Unraveling pathological mechanisms underlying metabolic disorders therefore requires a diversified approach, integrating basic cellular research with clinical research, ultimately relying on the analytical power of mass spectrometry-based techniques. Here, we discuss recent progress in the development of lipidomics approaches to resolve the pathological mechanisms of metabolic diseases and to identify suitable biomarkers for clinical application. Due to its growing impact worldwide, we focus on T2D to highlight the key role of lipidomics in our current understanding of this disease, discuss remaining questions and suggest future strategies to address them.

Secretome profiling of primary human skeletal muscle cells.

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Platelet proteome analysis reveals integrin-dependent aggregation defects in patients with myelodysplastic syndromes.

Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/µl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbß3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbß3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's ß3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbß3 signaling that may contribute to the hemorrhagic diathesis observed in these patients.