Influence of inflammatory mechanisms in obese adipose tissue on pathomechanisms of allergic airway inflammation in mice

Asthma and obesity are non-communicable diseases with high and increasing prevalences. Both are characterized by the presence of local and systemic inflammatory processes, whereby their mutual pathomechanistic relationship within obesity-associated asthma are not fully elucidated. This research work hypothesized that inflammatory mechanisms in obese adipose tissue modify the inflammatory phenotype of an induced allergic airway inflammation (AAI) and that altered activation processes in CD4+ T cells are crucially involved. To test this hypothesis, BALB/c and C57BL/6 mice were fed for several weeks with a high-fat diet (HFD) in order to induce obesity-like metabolic changes and a mixed inflammatory phenotype of AAI was induced in the animals by exposure to house dust mite (HDM) extracts. In both strains, HFD resulted in comparable metabolic changes, such as higher plasma levels of a variety of metabolic parameters and features of type 2 diabetes. However, strain-dependent differences were observed with regard to excessive weight gain, which occurred only in C57BL/6 mice, but not in BALB/c mice. Weight gain in HFD-fed C57BL/6 mice was associated with pronounced adipocyte hypertrophy and increased inflammatory processes in the obese adipose tissue. As intended, HDM-exposure of control diet (ND)-fed mice resulted in a mixed inflammatory phenotype characterized by an influx of eosinophils and neutrophils in the bronchoalveolar lavage (BAL) and in lung tissue in both mouse strains. While per se no differences in the induced AAI between the HFD/HDM group and the ND/HDM group were detected in BALB/c mice, a more eosinophilic inflammation was observed in HFD-fed HDM-exposed C57BL/6 mice in comparison to their lean counterparts. Further investigations revealed increased levels of inflammatory cytokines in the BAL and higher numbers of inflammatory immune cells in the lung tissue of HFD/HDM C57BL/6 mice. This suggested that weight gain and associated inflammatory processes in obese adipose tissue are crucial for modifying characteristic features of the induced AAI. Single cell analysis of CD45+ lung cells was conducted to further analyze the cellular and molecular changes of the induced inflammatory phenotype in the airways of C57BL/6 mice. Interestingly, a CD4+ T cell subcluster was identified to be exclusively present in the lungs of lean, but not in obese HDM-exposed mice. In this subcluster, expression of markers associated with typical characteristics of Th2, Th1, and Treg cells and in addition, co-inhibitory receptors such as PD-1, CTLA-4, and LAG-3 was detected. Subsequent flow cytometric analyses confirmed the presence of Th2 and Treg cells expressing the coinhibitory receptors identified in lungs of HDM-exposed mice. To further investigate whether hypertrophic activated adipocytes release factors that might be involved in the regulation of co-inhibitory receptor expression of CD4+ T cells, supernatants from in vitro cultured adipocytes of HFD-fed mice were applied to differentiated Th2 and Treg cells. In line with the previous results, during the activation process lower numbers of Th2 cells were observed expressing CTLA-4 or LAG-3, while in Treg cells a reduction of CTLA-4+ cells was seen in response to the adipocyte supernatant. Overall, HFD changes the inflammatory phenotype of a HDM-induced AAI only in association with weight gain and related inflammatory processes in the obese adipose tissue. Further, the expression of co-inhibitory receptors PD-1, CTLA-4, and LAG-3 on CD4+ T cells, which may represent a putative disease-limiting mechanism, was observed in response to the induction of AAI only in lean mice. However, the absence of CD4+ T cells expressing such receptors might in turn underlie altered and potentially aggravated and/or prolonged inflammatory processes in obese HDM-exposed mice. By demonstrating obesity-mediated alterations of the AAI phenotype and the identification of putative immune mechanisms behind this observation, these findings provide significant deeper insights into the specific pathophysiology of obesity-associated asthma with implications for the development of stratified therapy concepts for this disease condition

Skeletal muscle tissue from the Goto-Kakizaki rat model of type-2 diabetes exhibits increased levels of the small heat shock protein Hsp27

In order to increase our understanding of diabetes-related muscle weakness, we carried out a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the Goto-Kakizaki rat model of type-2 diabetes. Fluorescence difference in-gel electrophoresis was performed to determine potential differences in the global protein expression profile of muscle extracts. Besides changes in contractile proteins and metabolic enzymes, the abundance of the small stress proteins αB-crystallin and Hsp27 was significantly increased. The up-regulation of the low-molecular-mass heat shock protein Hsp27 was confirmed by an alternative fluorescent staining method of two-dimensional gels and immunoblotting. The observed protein alterations in the cellular stress response, distinct metabolic pathways, regulatory mechanisms and the contractile apparatus might be directly or indirectly associated with peripheral resistance to insulin signalling, making these newly identified muscle proteins potential biomarkers of type-2 diabetes. Increased levels of molecular chaperones suggest considerably enhanced cellular stress levels in diabetic muscle fibres

Plasma Protein and MicroRNA Biomarkers of Insulin Resistance: A Network-Based Integrative -Omics Analysis

Although insulin resistance (IR) is a key pathophysiologic condition underlying various metabolic disorders, impaired cellular glucose uptake is one of many manifestations of metabolic derangements in the human body. To study the systems-wide molecular changes associated with obesity-dependent IR, we integrated information on plasma proteins and microRNAs in eight obese insulin-resistant (OIR, HOMA-IR > 2.5) and nine lean insulin-sensitive (LIS, HOMA-IR < 1.0) normoglycemic males. Of 374 circulating miRNAs we profiled, 65 species increased and 73 species decreased in the OIR compared to the LIS subjects, suggesting that the overall balance of the miRNA secretome is shifted in the OIR subjects. We also observed that 40 plasma proteins increased and 4 plasma proteins decreased in the OIR subjects compared to the LIS subjects, and most proteins are involved in metabolic and endocytic functions. We used an integrative -omics analysis framework called iOmicsPASS to link differentially regulated miRNAs with their target genes on the TargetScan map and the human protein interactome. Combined with tissue of origin information, the integrative analysis allowed us to nominate obesity-dependent and obesity-independent protein markers, along with potential sites of post-transcriptional regulation by some of the miRNAs. We also observed the changes in each -omics platform that are not linked by the TargetScan map, suggesting that proteins and microRNAs provide orthogonal information for the progression of OIR. In summary, our integrative analysis provides a network of elevated plasma markers of OIR and a global shift of microRNA secretome composition in the blood plasma

Skeletal muscle tissue from the Goto-Kakizaki rat model of type-2 diabetes exhibits increased levels of the small heat shock protein Hsp27

In order to increase our understanding of diabetes-related muscle weakness, we carried out a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the Goto-Kakizaki rat model of type-2 diabetes. Fluorescence difference in-gel electrophoresis was performed to determine potential differences in the global protein expression profile of muscle extracts. Besides changes in contractile proteins and metabolic enzymes, the abundance of the small stress proteins αB-crystallin and Hsp27 was significantly increased. The up-regulation of the low-molecular-mass heat shock protein Hsp27 was confirmed by an alternative fluorescent staining method of two-dimensional gels and immunoblotting. The observed protein alterations in the cellular stress response, distinct metabolic pathways, regulatory mechanisms and the contractile apparatus might be directly or indirectly associated with peripheral resistance to insulin signalling, making these newly identified muscle proteins potential biomarkers of type-2 diabetes. Increased levels of molecular chaperones suggest considerably enhanced cellular stress levels in diabetic muscle fibres

Deficiency of the bone mineralization inhibitor NPP1 protects against obesity and diabetes

The emergence of bone as an endocrine regulator has prompted a re-evaluation of the role of bone mineralization factors in the development of metabolic disease. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) controls bone mineralization through the generation of pyrophosphate, and levels of NPP1 are elevated both in dermal fibroblast cultures and muscle of individuals with insulin resistance. We investigated the metabolic phenotype associated with impaired bone metabolism in mice lacking the gene that encodes NPP1 (Enpp1−/− mice). Enpp1−/− mice exhibited mildly improved glucose homeostasis on a normal diet but showed a pronounced resistance to obesity and insulin resistance in response to chronic high-fat feeding. Enpp1−/− mice had increased levels of the insulin-sensitizing bone-derived hormone osteocalcin but unchanged insulin signalling within osteoblasts. A fuller understanding of the pathways of NPP1 could inform the development of novel therapeutic strategies for treating insulin resistance

ENPP1 Affects Insulin Action and Secretion: Evidences from In Vitro Studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser473, ERK1/2-Thr202/Tyr204 and GSK3-beta Ser9 phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser473, ERK1/2-Thr202/Tyr204 and GSK3-beta Ser9 in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities

IDENTIFYING THE MOLECULAR MECHANISMS OF EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING IN MUSCLE AND FAT BIOPSIES FROM CANCER PATIENTS

Cancer cachexia is responsible for one third of cancer–related deaths and contributes to the death of many others. More than 80% of cancer patients are cachectic towards the end of life. Despite intensive research, the mechanisms of cancer cachexia are still poorly understood. It is our hypothesis that identification of early changes in gene expression in cachexia will lead to an improved understanding of the mechanism that trigger this important problem in cancer patients. Thus, to shed light on the mechanisms involved in the major cachexia target tissues, we investigated the entire transcriptome in muscle and fat to identify altered expression of genes in cancer patients with and without cachexia. Samples of rectus abdominis muscle and visceral fat were collected at surgery from patients exhibiting 5-10% weight loss prior to surgery, compared with stable-weight patients. Analysis of all expressed genes was carried out using next generation sequencing (Illumina HiSeq 2500). Also, selected differentially expressed genes were confirmed using real time RT-PCR. In muscle, 30 genes showed highly significant changes in expression (25 downregulated and 5 upregulated: P In visceral fat, expression of 6 genes were downregulated and 10 upregulated with high statistical significance (

Circular RNAs: New players in Ageing and Age-related Chronic Disease

Circular RNAs (circRNAs) are an emerging class of non-coding RNA that may regulate expression during normal and disease states. Although circRNAs accumulate in in vivo models of ageing, their role in this process and its physiological consequences remains largely unanswered. In the course of this thesis, I assessed dysregulation of circRNA expression in RNA samples from ageing human peripheral blood and examined associations of their expression with various ageing outcomes in human, mammalian longevity and senescence in human cell types of various lineages, and in blood and islet samples from patients with type 2 diabetes; an exemplar disease of ageing. Of the 15 circRNAs validated in this study, I identified 4 (circDEF6, circEP300, circFOXO3 and circFNDC3B) that were associated with ageing outcomes (parental longevity or hand grip strength) in the InCHIANTI population study of ageing. CircFOXO3 and circEP300 also demonstrated differential expression in one or more human senescent cell types. 4 ageing outcomes associated circRNAs appeared to be conserved in mouse of which circPlekhm1 nominally correlated with median strain lifespan. As type 2 diabetes is an exemplar chronic disease of ageing, I also aimed to examine the role of circRNA in this disorder. I first defined the circRNA repertoire in human pancreatic islets and assessed their differential expression in conjunction with type 2 diabetes status and genotype at T2D risk loci. Following this, I determined their responsiveness to diabetomimetic stimuli in the human EndoC-βH1 beta cell line, and the potential for use as biomarkers of T2D in human peripheral blood. 4 of the five most abundant circRNAs expressed in human pancreatic islets circCIRBP, circZKSCAN, circRPH3AL and circCAMSAP1, were associated with diabetes status in islets. CircCIRBP and circRPH3AL were also differentially expressed in β-cells in response to elevated fatty acid. Despite this, no associations with T2D diabetes risk loci was identified. Cumulatively, the data generated from my work suggest that circRNAs have potential as regulators of gene expression during ageing and age-related disease, raising the possibility that they may have future utility as biomarkers or therapeutic targets for the management of age-related chronic disease outcomes