Circulating SFRP5 levels are elevated in drug-naïve recently diagnosed type 2 diabetic patients as compared with prediabetic subjects and controls.

BACKGROUND: Secreted frizzled-related protein 5 (SFRP5) has been linked to obesity. Results are conflicting regarding its association with type 2 diabetes (T2D) in humans. We aimed to investigate circulating SFRP5 in prediabetes and T2D and its potential association with parameters of insulin resistance and beta-cell function. METHODS: We studied 70 drug-naïve T2D patients, 70 prediabetic subjects and 70 controls. All subjects were body mass index matched to the T2D patients and overweight or obese. SFRP5, hormones and cytokines levels were measured by ELISA. RESULTS: Serum SFRP5 levels were elevated in T2D patients as compared with prediabetic subjects (median 15.6, interquartile range [9-24.5] ng/mL vs 9.8 [5-14.2] ng/mL, p < 0.001, respectively) and controls (15.6 [9-24.5] ng/mL vs 10.4 [6.7-16.6] ng/mL, P < 0.001, respectively). No differences were found in serum SFRP5 levels between prediabetic subjects and controls (9.8 [5-14.2] ng/mL vs 10.4 [6.7-16.6] ng/mL, p = 0.472, respectively). After adjusting for potential confounders (age, gender, body mass index, triglycerides, high-density lipoprotein cholesterol and blood pressure), T2D was still associated with higher values of SFRP5 as compared with prediabetes in multinomial logistic regression analysis (fully adjusted odds ratio 3.50, 95% confidence interval 1.40-8.79, p = 0.008). The association was more subtle when comparing T2D with normal glucose tolerance state (fully adjusted odds ratio 2.18, 95% confidence interval 0.91-5.21, p = 0.078). CONCLUSIONS: Circulating SFRP5 levels were independently associated with T2D as compared with prediabetes and normal glucose tolerance state.

Integrative analysis reveals novel pathways mediating the interaction between adipose tissue and pancreatic islets in obesity in rats.

AIMS/HYPOTHESIS: Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis. METHODS: Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis. RESULTS: The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells. CONCLUSIONS/INTERPRETATION: The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Tungstate promotes ß-cell survival in Irs2-/- mice.

Pancreatic ß-cells play a central role in type 2 diabetes (T2D) development, which is characterized by the progressive decline of the functional ß-cell mass that is associated mainly with increased ß-cell apoptosis. Thus, understanding how to enhance survival of ß-cells is key for the management of T2D. The insulin receptor substrate-2 (IRS-2) protein is pivotal in mediating the insulin/IGF signaling pathway in ß-cells. In fact, IRS-2 is critically required for ß-cell compensation in conditions of increased insulin demand and for ß-cell survival. Tungstate is a powerful antidiabetic agent that has been shown to promote ß-cell recovery in toxin-induced diabetic rodent models. In this study, we investigated whether tungstate could prevent the onset of diabetes in a scenario of dysregulated insulin/IGF signaling and massive ß-cell death. To this end, we treated mice deficient in IRS2 (Irs2(-/-)), which exhibit severe ß-cell loss, with tungstate for 3 wk. Tungstate normalized glucose tolerance in Irs2(-/-) mice in correlation with increased ß-cell mass, increased ß-cell replication, and a striking threefold reduction in ß-cell apoptosis. Islets from treated Irs2(-/-) exhibited increased phosphorylated Erk1/2. Interestingly, tungstate repressed apoptosis-related genes in Irs2(-/-) islets in vitro, and ERK1/2 blockade abolished some of these effects. Gene expression profiling showed evidence of a broad impact of tungstate on cell death pathways in islets from Irs2(-/-) mice, consistent with reduced apoptotic rates. Our results support the finding that ß-cell death can be arrested in the absence of IRS2 and that therapies aimed at reversing ß-cell mass decline are potential strategies to prevent the progression to T2D.

Targeting type 2 diabetes: lessons from a knockout model of insulin receptor substrate 2.

Insulin receptor substrate 2 (IRS2) is a widely expressed protein that regulates crucial biological processes including glucose metabolism, protein synthesis, and cell survival. IRS2 is part of the insulin - insulin-like growth factor (IGF) signaling pathway and mediates the activation of the phosphotidylinositol 3-kinase (PI3K)-Akt and the Ras-mitogen-activated protein kinase (MAPK) cascades in insulin target tissues and in the pancreas. The best evidence of this is that systemic elimination of the Irs2 in mice (Irs2(-/-)) recapitulates the pathogenesis of type 2 diabetes (T2D), in that diabetes arises as a consequence of combined insulin resistance and beta-cell failure. Indeed, work using this knockout mouse has confirmed the importance of IRS2 in the control of glucose homeostasis and especially in the survival and function of pancreatic beta-cells. These studies have shown that IRS2 is critically required for beta-cell compensation in conditions of increased insulin demand. Importantly, islets isolated from T2D patients exhibit reduced IRS2 expression, which supports the likely contribution of altered IRS2-dependent signaling to beta-cell failure in human T2D. For all these reasons, the Irs2(-/-) mouse has been and will be essential for elucidating the inter-relationship between beta-cell function and insulin resistance, as well as to delineate therapeutic strategies to protect beta-cells during T2D progression.

Downregulation of Sfrp5 promotes beta cell proliferation during obesity in the rat.

AIMS/HYPOTHESIS: During obesity, the increment in beta cell mass in response to the rising demand for insulin is essential to maintain normal glucose homeostasis. However, the precise cellular and molecular mechanisms involved in beta cell mass plasticity remain poorly understood. The Wnt signalling pathway has been suggested as one possible modulator of beta cell proliferation, which represents the principal process involved in beta cell mass expansion. Here, we sought to determine the mechanisms involved in beta cell mass proliferation using diet-induced obese rats. METHODS: Wistar rats aged 8 weeks old were fed a standard or cafeteria diet. Global transcriptomic analysis of pancreatic rat islets was performed using microarray analysis. Genetic loss-of-function approaches were performed in dispersed primary rat islets and the beta cell line INS1E. Gene expression was measured by real-time PCR, protein levels by immunoblot analysis, proliferation rates by ELISA and apoptosis by flow cytometry. RESULTS: Sfrp5, coding for secreted frizzled-related protein 5, is downregulated in the pancreatic islets of cafeteria-diet-fed rats as well as in the pancreatic islets of human obese patients. We demonstrate that silencing Sfrp5 increases beta cell proliferation, which correlates with activation of Wnt signalling and enhanced levels of proliferation markers. In addition, we show that expression of Sfrp5 in beta cells is modulated by IGF binding protein 3 (IGFBP3) secreted from visceral adipose tissue. CONCLUSIONS/INTERPRETATION: Together, these findings reveal an important role for SFRP5 and Wnt signalling in the regulation of beta cell proliferation in obesity.

Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.

BACKGROUND: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. MATERIALS AND METHODS: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. RESULTS: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. CONCLUSION: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

The triglycerides and glucose (TyG) index: A new marker associated with nonalcoholic steatohepatitis (NASH) in obese patients.

AIM: Diagnosis of nonalcoholic steatohepatitis (NASH) relies on liver biopsy. Noninvasive tools would be useful to target patients to refer for a biopsy. We aimed to determine the diagnostic value of the triglycerides and glucose (TyG) index, an insulin-resistance indicator, to predict NASH. METHODS: Our study included grade II-III obese patients aged 18-65 years undergoing bariatric surgery and included in the COMET (COllection of MEtabolic Tissues) biobank (NCT02861781). Liver biopsies performed during bariatric surgery were collected from the biobank along with blood derivatives. Biopsies were analysed according to the steatosis, activity and fibrosis (SAF) scoring system to diagnose NASH, nonalcoholic fatty liver disease (NAFLD), and fibrosis. Logistic regression models were performed to identify factors predicting NASH, NAFLD, and fibrosis. RESULTS: Of 238 analysed subjects (mean age 43±12 years, 33.6% men), 29% had type 2 diabetes. Steatosis was present in 67.2%, while NASH and advanced fibrosis (stage F3) were diagnosed in 18.1% and 2.9% respectively. TyG index was independently associated with NASH (odds ratio (OR): 4.7 [95% confidence interval: 2.3;9.5] P < 0.0001), NAFLD (OR: 2.0 [1.1;3.7] P = 0.03) and stages 2-3 fibrosis (OR: 4.0 [1.5;10.8] P = 0.007). NASH was also predicted by gamma-glutamyl transferase (GGT) with an area under the ROC curve: 0.79 [0.71;0.87 P = 0.04] for GGT and TyG index combined. CONCLUSION: In our cohort of severely obese patients, TyG index, when associated with GGT level, exhibited high diagnostic performance to predict NASH. Although validation in larger populations is needed, this result may be of considerable clinical value to predict need for liver biopsy.

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

Absence of anti-pendrin auto-antibodies in the sera of Tunisian patients with autoimmune thyroid diseases.

BACKGROUND: We previously demonstrated that the PDS gene is involved in the genetic susceptibility to autoimmune thyroid diseases (AITD) in Tunisia. In the same population, we now investigated the presence of anti-pendrin auto-antibodies (aAbs) in AITD patients' sera. METHODS: Thirty seven Tunisian AITD patients and 19 healthy subjects from families previously linked to the PDS gene, 75 unrelated patients and 20 healthy unrelated subjects were included in our study. The detection of anti-pendrin aAbs in patients' sera was performed by ELISA using membrane protein extracts of CHO cells expressing pendrin (CHO-hPDS) and by immunofluorescence using transient COS-7 cells expressing a GFP tagged pendrin. CHO cells transfected with human TPO in the same ELISA conditions were used as positive control. RESULTS: The majority of AITD patients' sera were positive for the presence of anti-TPO aAbs. In contrast, no reactivity was detected with CHO-hPDS membrane protein extracts. Likewise, no significant immunostaining was found on transfected COS-7cells upon exposure to patients' and controls' sera. CONCLUSIONS: Our data point to the absence of anti-pendrin aAbs in Tunisian AITD patients' sera.

Antithyroperoxidase antibody-dependent cytotoxicity in autoimmune thyroid disease.

CONTEXT: Thyroid antibody-dependent cytotoxicity has been reported in autoimmune thyroid disease (AITD). Indeed, the role of thyroperoxidase (TPO) autoantibodies (aAbs) in complement-mediated damage by binding to TPO expressed on the surface of human thyroid cells was demonstrated, whereas their activity in antibody-dependent cell cytotoxicity (ADCC) is not well established. OBJECTIVE: The aim of this study was to define the partners involved in antibody and complement-dependent cytotoxicity (CDC) in AITD and characterize which effector cells are involved in cytotoxicity mediated by anti-TPO aAbs using a chromium release assay. RESULTS: The relative capability of anti-TPO aAbs to mediate ADCC using human thyroid cells in culture varies from 11 to 74.5%, depending on the effectors cells used. The human monocyte cell line HL60 gives a better lysis than the THP-1 cell line as effector cells. It seems obvious that the mechanism of ADCC is mediated quite exclusively by FcgammaRI. Indeed, the two effector cell lines differ by the level of the FcgammaRI expression (91.83% for HL-60 cells and 22.55%t for the THP-1). In addition to ADCC, the anti-TPO aAbs mediate the destruction of thyrocytes by CDC (56%). CONCLUSIONS: These results demonstrate that anti-TPO aAbs can damage cultured thyroid cells by ADCC and CDC mechanisms. The monocytes, via their FcgammaRI, are important effector cells in ADCC mediated by anti-TPO aAbs and may contribute with T cells to the destruction of thyroid gland in AITD.