Defining the underlying defect in insulin action in type 2 diabetes.

Insulin resistance is one of the earliest defects in the pathogenesis of type 2 diabetes. Over the past 50 years, elucidation of the insulin signalling network has provided important mechanistic insights into the abnormalities of glucose, lipid and protein metabolism that underlie insulin resistance. In classical target tissues (liver, muscle and adipose tissue), insulin binding to its receptor initiates a broad signalling cascade mediated by changes in phosphorylation, gene expression and vesicular trafficking that result in increased nutrient utilisation and storage, and suppression of catabolic processes. Insulin receptors are also expressed in non-classical targets, such as the brain and endothelial cells, where it helps regulate appetite, energy expenditure, reproductive hormones, mood/behaviour and vascular function. Recent progress in cell biology and unbiased molecular profiling by mass spectrometry and DNA/RNA-sequencing has provided a unique opportunity to dissect the determinants of insulin resistance in type 2 diabetes and the metabolic syndrome; best studied are extrinsic factors, such as circulating lipids, amino acids and other metabolites and exosomal microRNAs. More challenging has been defining the cell-intrinsic factors programmed by genetics and epigenetics that underlie insulin resistance. In this regard, studies using human induced pluripotent stem cells and tissues point to cell-autonomous alterations in signalling super-networks, involving changes in phosphorylation and gene expression both inside and outside the canonical insulin signalling pathway. Understanding how these multi-layered molecular networks modulate insulin action and metabolism in different tissues will open new avenues for therapy and prevention of type 2 diabetes and its associated pathologies.

Activation of the PDGFRα-Nrf2 pathway mediates impaired adipocyte differentiation in bone marrow mesenchymal stem cells lacking Nck1.

BACKGROUND: The limited options to treat obesity and its complications result from an incomplete understanding of the underlying molecular mechanisms regulating white adipose tissue development, including adipocyte hypertrophy (increase in size) and hyperplasia (increase in number through adipogenesis). We recently demonstrated that lack of the adaptor protein Nck1 in mice is associated with reduced adiposity and impaired adipocyte differentiation. In agreement, Nck1 depletion in 3 T3-L1 cells also attenuates adipocyte differentiation by enhancing PDGFRα activation and signaling. This is accompanied by higher expression of PDGF-A, a specific PDGFRα ligand, that may contribute to enhanced activation of PDGFRα signaling in the absence of Nck1 in white adipose tissue. However, whether Nck1 deficiency also impairs adipogenic differentiation in bone marrow still remains to be determined. METHODS: To address this point, Nck1-deficient derived bone marrow mesenchymal stem/stromal cells (BM-MSCs) and C3H10T1/2 mesenchymal stem cells were differentiated into adipocytes in vitro. Genes and proteins expression in these cellular models were determined using qPCR and western blotting respectively. Pharmacological approaches were used to assess a role for Nrf2 in mediating Nck1 deficiency effect on mesenchymal stem cells adipocyte differentiation. RESULTS: Nck1 deficiency in both BM-MSCs and C3H10T1/2 results in impaired adipocyte differentiation, accompanied by increased activation of the transcription factor Nrf2, as shown by increased mRNA levels of Nrf2 target genes, including PDGF-A. Using pharmacological activator and inhibitor of Nrf2, we further provide evidence that Nrf2 is an important player in PDGFRα signaling that mediates expression of PDGF-A and impaired adipogenesis in Nck1-deficient BM-MSCs and C3H10T1/2 cells. CONCLUSION: This study demonstrates that Nck1 deficiency in mesenchymal stem cells impairs adipogenesis through activation of the PDGFRα-Nrf2 anti-adipogenic signaling pathway. Video Abstract.

Interactions among insulin resistance, epigenetics, and donor sex in gene expression regulation of iPSC-derived myoblasts.

About 25% of people in the general population are insulin resistant, increasing the risk for type 2 diabetes (T2D) and metabolic disease. Transcriptomic analysis of induced pluripotent stem cells differentiated into myoblasts (iMyos) from insulin-resistant (I-Res) versus insulin-sensitive (I-Sen) nondiabetic individuals revealed that 306 genes increased and 271 genes decreased in expression in iMyos from I-Res donors with differences of 2-fold or more. Over 30 of the genes changed in I-Res iMyos were associated with T2D by SNPs and were functionally linked to insulin action and control of metabolism. Interestingly, we also identified more than 1,500 differences in gene expression that were dependent on the sex of the cell donor, some of which modified the insulin resistance effects. Many of these sex differences were associated with increased DNA methylation in cells from female donors and were reversed by 5-azacytidine. By contrast, the insulin sensitivity differences were not reversed and thus appear to reflect genetic or methylation-independent epigenetic effects.

Effect of Preoperative Duloxetine Hydrochloride on Reducing Postoperative Morphine Requirement after Open Radical Cholecystectomy in Cancer Patients: A Randomized Controlled Study.

Background: Recently, opoids are linked with cancer recurrence. Duloxetine hydrochloride (DH), an anxiolytic may reduce total opoid requirement after cancer surgery. Aims: We assessed the efficacy of a single dose of DH in reducing the total morphine requirement after open radical cholecystectomy. We also calculated the Visual Analog Scale (VAS) score, patient satisfaction score (PSS), and time taken to the use of the first rescue analgesic. Setting and Designes: This is a prospective, randomized, double blind, controlled study conducted in the patients aged 20-70 years (American Society of Anaesthesiologists classes I-III) undergoing open radical cholecystectomy under general anesthesia for carcinoma gall bladder. Materials and Methods: The patients were divided into two groups of 32 patients each by computer-generated randomization. Group A received oral DH (60 mg); Group B received identical placebo capsules 2 h before surgery with a sip of water. Postoperatively, intravenous morphine was given using a patient-controlled analgesia pump. After 24 h, total morphine consumption, the VAS score, time to the first rescue analgesia, and PSS were recorded. Statistical Analysis: Statistical Package for the Social Sciences software (SPSS version 22.0, IBM Corp., Chicago, IL, USA 2013). P value < 0.05 or 0.001 was considered statistically significant. Results: The total morphine consumption and VAS score were significantly lower in Group A. No significant effects was observed on PSS. Conclusion: A single 60 mg dose of DH administered 2 h before open radical cholecystectomy reduced total morphine consumption and improved VAS score postoperatively with no effect on PSS.

Signaling defects associated with insulin resistance in nondiabetic and diabetic individuals and modification by sex.

Insulin resistance is present in one-quarter of the general population, predisposing these people to a wide range of diseases. Our aim was to identify cell-intrinsic determinants of insulin resistance in this population using induced pluripotent stem cell-derived (iPSC-derived) myoblasts (iMyos). We found that these cells exhibited a large network of altered protein phosphorylation in vitro. Integrating these data with data from type 2 diabetic iMyos revealed critical sites of conserved altered phosphorylation in IRS-1, AKT, mTOR, and TBC1D1 in addition to changes in protein phosphorylation involved in Rho/Rac signaling, chromatin organization, and RNA processing. There were also striking differences in the phosphoproteome in cells from men versus women. These sex-specific and insulin-resistance defects were linked to functional differences in downstream actions. Thus, there are cell-autonomous signaling alterations associated with insulin resistance within the general population and important differences between men and women, many of which also occur in diabetes, that contribute to differences in physiology and disease.

Disconnect between the developing eye and craniofacial prominences in the avian embryo.

In the amniote embryo, the upper jaw and nasal cavities form through coordinated outgrowth and fusion of craniofacial prominences. Adjacent to the embryonic prominences are the developing eyes, which abut the maxillary and lateral nasal prominences. The embryos of extant sauropsids (birds and nonavian reptiles) develop particularly large eyes in comparison to mammals, leading researchers to propose that the developing eye may facilitate outgrowth of prominences towards the midline in order to aid prominence fusion. To test this hypothesis, we performed unilateral and bilateral ablation of the developing eyes in chicken embryos, with the aim of evaluating subsequent prominence formation and fusion. Our analyses revealed minor interaction between the developing craniofacial prominences and the eyes, inconsequential to the fusion of the upper beak. At later developmental stages, the skull exhibited only localized effects from missing eyes, while geometric morphometrics revealed minimal effect on overall shape of the upper jaw when it develops without eyes. Our results indicate that the substantial size of the developing eyes in the chicken embryo exert little influence over the fusion of the craniofacial prominences, despite their effect on the size and shape of maxillary prominences and components of the skull.

Harnessing adipogenesis to prevent obesity.

Obesity and associated metabolic complications, including diabetes, cardiovascular and hepatic diseases, and certain types of cancers, create a major socioeconomic burden. Obesity is characterized by excessive expansion of white adipose tissue resulting from increased adipocyte size, and enhanced adipocyte precursor cells proliferation and differentiation into mature adipocytes, a process well-defined as adipogenesis. Efforts to develop therapeutically potent strategies to circumvent obesity are impacted by our limited understanding of molecular mechanisms regulating adipogenesis. In this review, we discuss recently discovered molecular mechanisms restraining adipogenesis. In this perspective, the discoveries of white adipose tissue endogenous adipogenesis-regulatory cells (Aregs) that negatively regulate adipocyte differentiation, platelet-derived growth factor receptor isoform α (PDGFRα) activation and downstream signaling that hinder adipocyte precursors differentiation, and a group of obesity-associated non-coding RNAs (ncRNAs) that regulate adipogenesis open up promising therapeutic avenues to prevent and/or treat obesity.

Comparative evaluation of effect of chlorhexidine, Azadirachta indica (neem), and Aloe barbadensis miller (Aloe vera) on resin-dentin bond stabilization using shear bond testing: An in vitro study.

INTRODUCTION: Cavity disinfectants help to remove the microbial remnants; hence, its use prior to any restoration is valuable, and a search for alternative to chlorhexidine (CHX) is required which may be more efficacious and can overcome the drawbacks of CHX. OBJECTIVE: The aim of this study was to evaluate the effect of application of three different cavity disinfectants in a clinically relevant time period on the immediate and delayed shear bond strengths (SBSs) of an etch-and-rinse adhesive system to dentin. MATERIALS AND METHODS: For SBS testing, flat coronal dentin surfaces were prepared in two hundred extracted human molars. Specimens were randomly assigned to four groups according to the disinfectant used: Group I: Control (no disinfectant), Group II: 2% CHX solution, Group III: Aloe barbadensis miller (Aloe vera) solution, and Group IV: Azadirachta indica (neem) solution. Specimens were bonded using Prime and Bond NT adhesive which was employed according to the manufacturer's instructions. Resin composite cylinder buildups were done in all the samples. The modes of failure were noted after visual examination using a binocular stereomicroscope. Samples were also analyzed under scanning electron microscope for observation of resin-dentin interface. SBS results were analyzed using one-way ANOVA followed by Tukey's post hoc test. RESULTS: The results showed that CHX, Aloe vera, and neem had improved bond strengths as compared to the control group for both immediate and delayed SBSs. CONCLUSION: From the results of the study, the authors concluded that Aloe vera and neem can be used as alternative cavity disinfectants to CHX.

Nck1 Deficiency Impairs Adipogenesis by Activation of PDGFRα in Preadipocytes.

Obesity results from an excessive expansion of white adipose tissue (WAT), which is still poorly understood from an etiologic-mechanistic perspective. Here, we report that Nck1, a Src homology domain-containing adaptor, is upregulated during WAT expansion and in vitro adipogenesis. In agreement, Nck1 mRNA correlates positively with peroxisome proliferator-activated receptor (PPAR) γ and adiponectin mRNAs in the WAT of obese humans, whereas Nck1-deficient mice display smaller WAT depots with reduced number of adipocyte precursors and accumulation of extracellular matrix. Furthermore, silencing Nck1 in 3T3-L1 preadipocytes increases the proliferation and expression of genes encoding collagen, whereas it decreases the expression of adipogenic markers and impairs adipogenesis. Silencing Nck1 in 3T3-L1 preadipocytes also promotes the expression of platelet-derived growth factor (PDGF)-A and platelet-derived growth factor receptor (PDGFR) α activation and signaling. Preventing PDGFRα activation using imatinib, or through PDGF-A or PDGFRα deficiency, inhibits collagen expression in Nck1-deficient preadipocytes. Finally, imatinib rescues differentiation of Nck1-deficient preadipocytes. Altogether, our findings reveal that Nck1 modulates WAT development through PDGFRα-dependent remodeling of preadipocytes.

Nck2 Deficiency in Mice Results in Increased Adiposity Associated With Adipocyte Hypertrophy and Enhanced Adipogenesis.

Obesity results from an excessive expansion of white adipose tissue (WAT) from hypertrophy of preexisting adipocytes and enhancement of precursor differentiation into mature adipocytes. We report that Nck2-deficient mice display progressive increased adiposity associated with adipocyte hypertrophy. A negative relationship between the expression of Nck2 and WAT expansion was recapitulated in humans such that reduced Nck2 protein and mRNA levels in human visceral WAT significantly correlate with the degree of obesity. Accordingly, Nck2 deficiency promotes an adipogenic program that not only enhances adipocyte differentiation and lipid droplet formation but also results in dysfunctional elevated lipogenesis and lipolysis activities in mouse WAT as well as in stromal vascular fraction and 3T3-L1 preadipocytes. We provide strong evidence to support that through a mechanism involving primed PERK activation and signaling, Nck2 deficiency in adipocyte precursors is associated with enhanced adipogenesis in vitro and adiposity in vivo. Finally, in agreement with elevated circulating lipids, Nck2-deficient mice develop glucose intolerance, insulin resistance, and hepatic steatosis. Taken together, these findings reveal that Nck2 is a novel regulator of adiposity and suggest that Nck2 is important in limiting WAT expansion and dysfunction in mice and humans.