Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Adiposity Measurements and Metabolic Syndrome Are Linked Through Circulating Neuregulin 4 and Adipsin Levels in Obese Adults

BackgroundAdiposity and adipokines are associated with metabolic disorders, but little is known regarding that whether adiposity measurements link metabolic syndrome (MetS) through circulating neuregulin 4 (Nrg4) and adipsin levels.Materials and MethodsA total of 1212 subjects with a waist circumference greater than 90 cm for men or 80 cm for women were enrolled from a Chinese community. Circulating Nrg4 and adipsin levels were measured using commercial kits. Mediation analyses of circulating Nrg4 and adipsin were performed in the study using linear and logistic regression.ResultsSubjects with MetS had higher waist circumference, visceral fat level, and circulating adipsin level, and lower levels of circulating Nrg4 and muscle mass to visceral fat (MVF) ratio (all P < 0.05). In multivariable logistic regression analyses, after adjusting for confounding variables, per standard deviation (SD) increase in waist circumference and visceral fat level were significantly associated with increased odds of MetS [OR (95% CI), 1.42 (1.22–1.64); 2.20 (1.62–2.99); respectively]; and per SD reduction in MVF ratio was significantly associated with reduced odds of MetS [OR (95% CI), 0.65 (0.55–0.77)]. In the mediation analyses, both circulating Nrg4 and adipsin levels mediated the association between waist circumference (8.31% and 18.35%, respectively), visceral fat level (7.50% and 9.98%, respectively), and MVF ratio (5.80% and 9.86%, respectively) and MetS after adjustments.ConclusionThese findings indicate that adiposity measurements and MetS are linked through circulating Nrg4 and adipsin levels in obese adults, suggesting that circulating Nrg4 and adipsin levels might be potential predictors for management of MetS.

Negative Correlation between Neuregulin-4 and IL-9 Serum Levels in Patients with Coronary Artery Disease

Background: Neuregulin 4 (Nrg4) is a novel adipocytokine which has been proposed to play a role in modulating energy metabolism and pathogeneses of atherosclerosis. However, no published research is available about the mechanisms underlying the anti-atherosclerotic effect of Nrg4. Regarding the close link between adipocytokines and immune system, we wonder whether there is a relation between Nrg4 and athero-protective cytokines. The aim of this study was to investigate the relationship between serum Nrg4 levels and type 2 cytokines in Iranian patients with coronary artery disease (CAD). Methods: In this case-control study, 125 CAD patients were compared to 55 healthy controls. Serum concentration of Nrg4, IL-5, IL-9 and IL-13 were measured using ELISA. The associations of circulating Nrg4 and IL-5, IL-9, IL-13 were assessed using linear regression analyses. Results: Serum concentration of IL-9 was significantly higher in patients compared to the healthy controls (317.9±139 versus 228.3±99.1, P= ˂0.0001). IL-13 and Nrg4 were significantly lower in patients compared to the healthy controls (4.3±3.7 versus 6.1±3.9, P=0.01 and 0.5 versus 1.3, P=0.001 respectively). Multiple linear regression analyses revealed that IL-9 was negatively correlated with Nrg4 (β= -0.3, P=0.009). Conclusion: Our study ،for the first-time, provides the clinical evidence revealing that circulating Nrg4 concentrations are inversely correlated with IL-9 in Iranian patients with CAD, suggesting that the protective role of Nrg4 on atherosclerosis may be, in part, mediated by the proatherogenic cytokine, IL-9.

Alteration of serum neuregulin 4 and neuregulin 1 in gestational diabetes mellitus

Context: Neuregulin 4 (Nrg4) and neuregulin 1 (Nrg1) have been shown to play vital roles in several disorders of glucose metabolism. The pathophysiological role of Nrg4 and Nrg1 in gestational diabetes mellitus (GDM), however, remains poorly understood. We assessed the clinical relevance of the two cytokines in patients with GDM. Methods: The study recruited 36 GDM patients and 38 age-matched, gestational age (24–28 weeks of gestation)–matched, and BMI (during pregnancy)–matched controls in this study. Serum Nrg4 and Nrg1 were measured using ELISA. Inflammatory factors such as IL-6, IL-1β, leptin, TNF-α, and monocyte chemotactic protein 1 (MCP-1) were determined via Luminex technique. Results: Serum Nrg4 in GDM patients was significantly lower than that in the controls, while Nrg1 was significantly higher in the GDM group ( p < 0.01). Inflammatory factors such as IL-6, leptin, and TNF-α were significantly increased in GDM patients, while MCP-1 and IL-1β were not significantly different between the two groups. In addition, serum Nrg4 was negatively correlated with fasting glucose ( r = −0.438, p = 0.008), HOMA-IR ( r = −0.364, p = 0.029), IL-6 ( r = −0.384, p = 0.021), leptin ( r = −0.393, p = 0.018), TNF-α ( r = −0.346, p = 0.039), and MCP-1 ( r = −0.342, p = 0.041), and positively correlated with high-density lipoprotein cholesterol (HDL-C) ( r = −0.357, p = 0.033) in GDM group. Serum Nrg1 was positively correlated with BMI ( r = 0.452, p = 0.006), fasting glucose ( r = 0.424, p = 0.010), HOMA-IR ( r = 0.369, p = 0.027), and triglyceride ( r = 0.439, p = 0.007). The decrease of Nrg4 and the increase of Nrg1 were significantly related to the increased prevalence of GDM. Finally, ROC curve results indicated that Nrg1 combined with IL-6 and TNF-α might be an effective means for GDM screening. Conclusions: Lower circulating Nrg4 and higher circulating Nrg1 serve risk factors of GDM. Nrg1 combined with IL-6 and TNF-α might be a potential tool for GDM screening.

Downregulation of Neuregulin 4 (NRG4) Inhibits Proliferation, Migration and Invasion of Glioma Cells

Objectives: Neuregulin 4 (NRG4) is a novel signaling protein involved in many physiological activities. This study aims to explore role of NRG4 in glioma. Study design: Serum samples were obtained from patients with glioma, RT-qPCR assay was performed to detect the levels of NRG4 and ErbB4 in the patient's serum and glioma cell lines U251, SHG44, LN229 and T98G. The results suggested that the expression of NRG4 and ErbB4 were markedly higher in patients and glioma cell lines, especially in U251. Further, Small interfering RNAs (si-RNAs) that targeting NRG4 (si-NRG4) and the overexpressed plasmid of ErbB4 (pcDNA-ErbB4) were transfected into U251 cells. Cell proliferation was measured by CCK8 assay, migration and invasion was assessed by wound healing and transwell assays respectively. And the expression of PI3 K-p110 , PI3 K-p110 , pAKT-ser473, pAKT-thr308 and AKT were measured by western blot assay. Results: We found that downregulation of NRG4 significantly inhibited proliferation, migration and invasion of U251 cells. Moreover, NRG4 inhibition markedly reduced the expression of PI3 K-p110 , pAKT-ser473 and pAKT-thr308. And overexpression of ErbB4 alleviated the impact caused by NRG4 inhibition. Conclusion: NRG4 inhibition suppresses proliferation, migration and invasion of U251 via blocking the PI3 K/AKT pathway. NRG4 could serve as a potential target of glioma treatment.