Ann-Christin Nyström

A PROTEOME ANALYSIS OF LIVERS FROM OBESE (OB/OB) MICE TREATED WITH THE PEROXISOME PROLIFERATOR WY14,643

The PPAR (peroxisome proliferator activated receptor) transcription factors are ligand‐activated receptors which regulate genes involved in lipid metabolism and homeostasis. PPARα is preferentially expressed in the liver and PPARγ preferentially in adipose tissue. Activation of PPARα leads to peroxisome proliferation in rodents and increased β‐oxidation of fatty acids. PPARγ‐activation leads to adipocyte differentiation and improved insulin signaling of mature adipocytes. Both of these PPAR receptors are potential targets for treatment of dyslipidemia in man. Studies by others using a proteomics approach have characterized the effects of PPARα agonists in livers from lean healthy mice. However, we wanted to map the effects of a therapeutic dose of a PPARα agonist in a disease model of insulin resistance and diabetes, the obese diabetic ob/ob mouse, by proteomics. Therefore, ob/ob mice, which have highly elevated levels of plasma triglycerides, glucose and insulin, were treated for one week with WY14,643 (180 μmol/kg/day), a well‐characterized selective PPARα agonist. Plasma triglycerides, glucose and insulin levels were determined and we found significant therapeutic effects on triglycerides and glucose levels. The liver protein compositions were investigated by high‐resolution two‐dimensional gel electrophoresis which showed that WY14,643 produced up‐regulation of at least 16 spots. These were identified by mass spectrometry and 14 spots were found to be components of the peroxisomal fatty acid metabolism. Thus, WY14,643 at a therapeutic dose, caused induction of peroxisomal fatty acid β‐oxidation in obese diabetic mice.

Thiourea enhances mapping of the proteome from murine white adipose tissue

Adipose tissue imposes problems in two‐dimensional (2‐D) analysis due to its extremely high content of fat. To improve protein separation detergents and chaotropes were varied in the IEF step. The most important factor for obtaining distinct spots in the 2‐D gel was whether thiourea was included or not. Many high molecular weight spots became resolved by using thiourea, while no spots disappeared or showed inferior characteristics, thus approximately twice as many spots were possible to quantify. Hydrophobic indices were compared for a set of proteins that gave rise to sharper spots with proteins that were not improved on the use of thiourea. The comparison did not give any statistically significant difference between the two groups of proteins. One of the effects obtained by inclusion of thiourea was that the dominating protein, serum albumin, appeared as more condensed spots allowing other minor proteins to be detected. This work resulted in a protocol which greatly enhances the resolution of proteins in adipose tissue. A 2‐D map of mouse white adipose tissue from epididymal fat pads was constructed in which 140 spots were identified by mass spectrometry. This work lays the ground for our further studies on white adipose tissue in metabolic diseases such as obesity and dyslipidemia.

Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes

Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients, marking the existence of an insulin-resistant signature that was retained in cultured myotubes. In conclusion, BZ treatment further exacerbates insulin resistance and unmasks intrinsic features of metabolic disease in myotubes derived from type 2 diabetic patients. Our results highlight the existence of a confounding inherent abnormality in cellular protein dynamics in metabolic disease, which is uncovered through concurrent inhibition of the proteasome system.

Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies.

Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an in situ hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.

The interleukin-like epithelial-mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer

Production and secretion of pro-metastatic proteins is a feature of many tumor cells. The FAM3C interleukin-like epithelial-to-mesenchymal-transition (EMT) inducer (ILEI) has been shown to be strongly up-regulated in several cancers and to be essential for tumor formation and metastasis in epithelial cells, correlating with a significant decrease in overall survival in colon and breast cancer patients. ILEI has been seen to interact with the γ-secretase presenilin 1 subunit (PS1). However, not much is known about the mechanism-of-action or the detailed ILEI structure. We present here the crystal structures of FAM3C ILEI and show that it exists as monomers but also as covalent dimers. The observed ILEI β-β-α fold confirmed previous indications that the FAM3C proteins do not form classical four-helix-bundle structures as was initially predicted. This provides the first experimental evidence that the interleukin-like EMT inducers are not evolutionarily related to the interleukins. However, more surprisingly, the ILEI dimer structure was found to feature a trans-linked domain swap, converting an intramolecular disulfide to intermolecular. Interestingly, dimeric but not monomeric ILEI was subsequently found to cause a dose-dependent increase in EpRas cell invasiveness comparable with TGF-β, indicating that the dimer might be the active ILEI species. This is in line with a parallel study showing that covalent oligomerization of ILEI is essential for EMT and tumor progression in vivo. The structures and the activity data give some first insight into the relationship between dimerization and ILEI function as well as indicate an intriguing link between ILEI, the PS1-protease, TGF-β, and the TGF-β receptor 1.

Transient expression, purification and characterisation of human full-length PPARγ2 in HEK293 cells.

Effective anti-diabetic drugs known as thiazolidinediones (e.g. rosiglitazone, pioglitazone) exert their therapeutic effects through their agonistic activity at the peroxisome proliferator-activated receptor gamma (PPARγ). As a multidomain transcription factor, PPARγ forms heterodimers with different retinoid X receptors (RXRs) to modulate target gene expression at the transcriptional level in response to natural or synthetic ligands. Difficulties in producing either of the two major human PPARγ isoforms (PPARγ1 and PPARγ2) as pure full-length proteins in adequate quantity has hindered detailed mechanistic studies of PPARγ and its ancillary protein partners. Here we report an efficient transient expression system to produce recombinant human full-length PPARγ2 protein. The DNA encoding the human full-length PPARγ2 was cloned into a mammalian episomal vector and transiently expressed in human embryonic kidney 293 (HEK293-6E) cells with an expression level of 10mg/L culture. Identity of the purified recombinant PPARγ2 protein was confirmed by mass spectrometry analysis. The purified PPARγ2 protein was active in ligand binding and could be phosphorylated in vitro by Cdk5/p25 at Ser 273. Further studies showed that selected PPARγ modulators inhibited Cdk5-mediated PPARγ2 Ser 273 phosphorylation in vitro. Our results demonstrate the feasibility of producing large quantities of pure and functional human full-length PPARγ2 suitable for drug discovery applications.

The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1

The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated in vitro. The acute glucose lowering effect was studied in lean wild-type and GPR120 null mice following oral or intravenous glucose tolerance tests. In vitro, in GPR120 overexpressing cells, both agonists signaled through Gαq, Gαs and the β-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both in vitro in STC-1 cells and in vivo following oral administration. In vivo GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 null mice. Exendin 9–39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.