Aberrant liver insulin receptor isoform a expression normalises with remission of type 2 diabetes after gastric bypass surgery.

Type 2 diabetes mellitus (T2DM) results from a combination of progressive insulin resistance and loss of pancreatic beta cell function and/or mass. Insulin signalling occurs through the insulin receptor, (INSR) which is alternatively spliced into two isoforms: INSRA (-exon 11) and INSRB (+exon 11). Because the INSR isoforms have different functional characteristics, their relative expression ratio has been implicated in the pathogenesis of insulin resistance and T2DM. We studied levels of INSR isoform mRNA in liver samples taken from 46 individuals with or without T2DM at Roux-en-Y (RYGB) surgery, and on average 17 (± 5.6) months later in 16 of the same individuals (8 diabetic and non-diabetic patients). INSRA or INSRB was also overexpressed in HepG2 cells to ascertain their effect on AKT phosphorylation and PCK1 expression as markers of insulin-mediated metabolic signalling. We found the INSRB:A isoform ratio was reduced in individuals with T2DM in comparison to those with normal glucose tolerance and normalised with remission of diabetes. The INSRB:A ratio increased due to a reduction in the alternatively spliced INSRA isoform following remission of diabetes. Overexpressing INSRA isoform in HepG2 hepatoma cells reduced inhibition of PCK1 transcription and did not increase AKT phosphorylation in response to insulin load compared to the effect of overexpressing the B isoform. Data presented here revitalizes the role of the INSR isoforms in the pathogenesis of T2DM, and suggests that an abrogated INSRB:A ratio that favours the INSRA isoform may negatively impact insulin-mediated metabolic signalling.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss.

BACKGROUND: Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. RESULTS: Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3' untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. CONCLUSIONS: This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Liver ENPP1 protein increases with remission of type 2 diabetes after gastric bypass surgery.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a progressive disease resulting from increasing insulin resistance and reduced pancreatic ß-cell insulin secretion. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibits insulin signalling and may contribute to the pathogenesis of T2DM. Others have found elevated ENPP1 levels in muscle, fat, and skin tissues from insulin resistant individuals, but similar data on liver ENPP1 is lacking. The purpose of this study was to compare expression and protein concentrations of ENPP1 in liver between patients with and without T2DM. METHODS: Roux-en-Y gastric bypass surgery (RYGB) results in remission of insulin resistance and T2DM thus presenting an opportunity to examine some critical aspects of these conditions. We measured liver ENPP1 gene and protein expression in individuals with or without T2DM at RYGB and on average 17 (±5.6) months later. RESULTS: We found liver ENPP1 protein abundance was lower in individuals with T2DM than in those with normal glucose tolerance, and increased after RYGB surgery in those individuals who had remission of T2DM. ENPP1 positively correlated with insulin sensitivity at the liver (as measured by HOMA-IR), which is contrary to what others have reported in other insulin target tissues. CONCLUSIONS: Liver ENPP1 expression in T2DM is the reverse of that expected based on expression in other tissues and is likely due to the unique role the liver has in insulin clearance. The work presented here adds another dimension to the role of ENPP1, and supports the hypothesis that ENPP1 may act as a natural modulator of insulin signalling in the liver.

Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance.

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor-insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real-time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR-A). We used a scaffold approach in which anti-insulin receptor monoclonal antibody 83-7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR-A capture. The multiplex SPR system (ProteOn XPR36™, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two-site binding pattern, consistent with the literature, with a high-affinity site (dissociation constant K(D1) = 38.1 ± 0.9 nM) and a low-affinity site (K(D2) = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K(D1) = 73.2 ± 1.8 nM and K(D2) = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high-affinity site was converted to the low-affinity site. The new SPR assay enables insulin-eIR-A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling.

Overexpression of aminoacylase 1 is associated with colorectal cancer progression.

Aminoacylase 1 (ACY1) is a cytosolic enzyme responsible for amino acid deacylation during intracellular protein degradation. ACY1 has been implicated in a number of human tumor types. However, the exact role of ACY1 in tumor development remains elusive because it was found to be lost in small cell lung cancer and renal cell carcinoma but overexpressed in colorectal cancer (CRC). The present study aims to further clarify the relationship of ACY1 with CRC progression. Immunohistochemical staining was performed in tissue microarrays composed of 120 cases of CRC using a monoclonal anti-ACY1 antibody. Immunoreactivity was analyzed in association with patients' clinicopathologic parameters and survival time. The role of ACY1 in cell proliferation and apoptosis was assessed by silencing its expression in HCT116 cells using a small interfering RNA. Strong expression of ACY1 was found to be significantly associated with more advanced TNM stage, lymph node metastasis, positive vascular invasion, and shorter cancer-specific survival. ACY1 knockdown significantly inhibited cell proliferation and induced apoptosis. We concluded that ACY1 expression in CRC varies with stage and appears to play a role in cell proliferation and apoptosis. Further evaluation of ACY1 as a clinically useful prognostic marker and a potential drug target for CRC would seem worthwhile.

Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis.

The emergence of laser capture microdissection (LCM) and two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to greatly improve the accuracy and sensitivity of global protein expression analysis. However, their combined use in profiling tumour proteome has rarely been reported. In this study, we applied these techniques to profile the protein expression changes of the late stage colorectal cancer (CRC) and its liver metastases. The study revealed that both the primary and secondary tumours showed a distinct protein expression profile compared to normal tissues, but were indistinguishable from each other. Differential analysis between the primary tumour and patient-matched normal colon mucosa identified a total of 71 proteins to be altered in CRC. Over 40% of these proteins have been previously reported as CRC-related proteins, validating the accuracy of the current analysis. We have also identified many previously unknown changes including overexpression of ACY1, HSC70, HnRNP I, HnRNP A3, SET, ANP32A and TUFM in CRC, which have been further verified by western blotting and immunohistochemistry. This study demonstrated that LCM in combination with 2D-DIGE is a powerful tool to analyse the proteome of tumour tissues and may lead to the identification of potential novel protein markers and therapeutic targets for cancer.

A randomised controlled trial of amniotic membrane in the treatment of a standardised burn injury in the merino lamb.

Burn injury is associated with disabling scar formation which impacts on many aspects of the patient's life. Previously we have shown that the fetus heals a deep dermal burn in a scarless fashion. Amniotic membrane (AM) is the outermost fetal tisue and has beeen used as a dressing in thermal injuries, though there is little data to support this use. To assess the efficacy of AM in scar minimisation after deep dermal burn wound, we conducted a randomised controlled study in the 1-month lamb. Lambs were delivered by caesarian section and the amniotic membranes stored after which lambs were returned to their mothers post-operatively. At 1 month, a standardised deep dermal burn was created under general anaesthesia on both flanks of the lamb. One flank was covered with unmatched AM, the other with paraffin gauze. Animals were sequentially euthanased from Day 3-60 after injury and tissue analysed for histopathology and immunohistochemically for alpha-smooth muscle actin (alphaSMA) content. AM resulted in reduced scar tissue as assessed histopathologically and reduced alphaSMA content. This study provides the first laboratory evidence that AM may reduce scar formation after burn injury.

A porcine deep dermal partial thickness burn model with hypertrophic scarring.

We developed a reproducible model of deep dermal partial thickness burn injury in juvenile Large White pigs. The contact burn is created using water at 92 degrees C for 15s in a bottle with the bottom replaced with plastic wrap. The depth of injury was determined by a histopathologist who examined tissue sections 2 and 6 days after injury in a blinded manner. Upon creation, the circular wound area developed white eschar and a hyperaemic zone around the wound border. Animals were kept for 6 weeks or 99 days to examine the wound healing process. The wounds took between 3 and 5 weeks for complete re-epithelialisation. Most wounds developed contracted, purple, hypertrophic scars. On measurement, the thickness of the burned skin was approximately 1.8 times that of the control skin at week 6 and approximately 2.2 times thicker than control skin at 99 days after injury. We have developed various methods to assess healing wounds, including digital photographic analysis, depth of organising granulation tissue, immunohistochemistry, electron microscopy and tensiometry. Immunohistochemistry and electron microscopy showed that our porcine hypertrophic scar appears similar to human hypertrophic scarring. The development of this model allows us to test and compare different treatments on burn wounds.

Collagen in the scarless fetal skin wound: detection with picrosirius-polarization.

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.

Deep dermal burn injury results in scarless wound healing in the ovine fetus.

Early to mid-term fetuses heal cutaneous incisional wounds without scars; however, fetal response to burn injury has not been ascertained. We present a fetal model of thermal injury and subsequent analysis of fetal and lamb response to burn injury. A reproducible deep dermal burn injury was created in the fetus by application of water at 66 degrees C for 7 seconds, and at 82 degrees C for 10 seconds to the lamb. Macroscopically, the area of fetal scald was undetectable from day 7 post injury, while all lamb scalds were readily identified and eventually healed with scarring. Using a five-point histopathology scoring system for alteration in tissue morphology, differences were detected between control and scalded skin at all stages in lamb postburn, but no difference was detected in the fetal model after day 7. There were also large differences in content of alpha-smooth muscle actin and transforming growth factor-beta1 between control and scalded lamb and these differences were statistically significant at day 14 (P < 0.01). This novel model of fetal and lamb response to deep dermal injury indicates that the fetus heals a deep burn injury in a scarless fashion. Further elucidation of this specific fetal process of burn injury repair may lead to improved outcome for patients with burn injury.