Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: A new mechanism for the regulation of inflammation by vitamin D

Inflammation of adipose tissue is believed to be a contributing factor to many chronic diseases associated with obesity. Vitamin D (VD) is now known to limit this metabolic inflammation by decreasing inflammatory marker expression and leukocyte infiltration in adipose tissue. In this study, we investigated the impact of VD on microRNA (miR) expression in inflammatory conditions in human and mouse adipocytes, using high-throughput methodology (miRNA PCR arrays). Firstly, we identified three miRs (miR-146a, miR-150, and miR-155) positively regulated by TNFα in human adipocytes. Interestingly, the expression of these miRs was strongly prevented by 1,25(OH)₂D preincubation. These results were partly confirmed in 3T3-L1 adipocytes (for miR-146a and miR-150). The ability of VD to control the expression of these miRs was confirmed in diet-induced obese mice: the levels of the three miRs were increased following high fat (HF) diet in epididymal white adipose tissue and reduced in HF diet fed mice supplemented with VD. The involvement of NF-κB signaling in the induction of these miRs was confirmed in vitro and in vivo using aP2-p65 transgenic mice. Finally, the ability of VD to deactivate NF-κB signaling, via p65 and IκB phosphorylation inhibition in murine adipocyte, was observed and could constitute a driving molecular mechanism. This study demonstrated for the first time that VD modulates the expression of miRs in adipocytes in vitro and in adipose tissue in vivo through its impact on NF-κB signaling pathway, which could represent a new mechanism of regulation of inflammation by VD.     

Role of adiponectin and inflammation in insulin resistance of Mc3r and Mc4r knockout mice

Objective: To investigate the involvement of hypoadiponectinemia and inflammation in coupling obesity to insulin resistance in melanocortin‐3 receptor and melanocortin‐4 receptor knockout (KO) mice (Mc3/4rKO). Research Methods and Procedures: Sera and tissue were collected from 6‐month‐old Mc3rKO, Mc4rKO, and wild‐type C57BL6J litter mates maintained on low‐fat diet or exposed to high‐fat diet (HFD) for 1 or 3 months. Inflammation was assessed by both real‐time polymerase chain reaction analysis of macrophage‐specific gene expression and immunohistochemistry. Results: Mc4rKO exhibited hypoadiponectinemia, exacerbated by HFD and obesity, previously reported in murine models of obesity. Mc4r deficiency was also associated with high levels of macrophage infiltration of adipose tissue, again exacerbated by HFD. In contrast, Mc3rKO exhibited normal serum adiponectin levels, irrespective of diet or obesity, and a delayed inflammatory response to HFD relative to Mc4rKO. Discussion: Our findings suggest that severe insulin resistance of Mc4rKO fed a HFD, as reported in other models of obesity such as leptin‐deficient (Lep^ob/Lep^ob) and KK‐A^y mice, is linked to reduced serum adiponectin and high levels of inflammation in adipose tissue. Conversely, maintenance of normal serum adiponectin may be a factor in the relatively mild insulin‐resistant phenotype of severely obese Mc3rKO. Mc3rKO are, thus, a unique mouse model where obesity is not associated with reduced serum adiponectin levels. A delay in macrophage infiltration of adipose tissue of Mc3rKO during exposure to HFD may also be a factor contributing to the mild insulin resistance in this model.     

The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.     

Consumption of a high fat diet promotes protein O-GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.     

Downregulation of microRNA-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by targeting IRF1

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor.     

The a2b adenosine receptor modulates glucose homeostasis and obesity

Background

High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.

Methodology/Principal Findings

Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.

Conclusions/Significance

Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.     

Decreased Serum Level of miR-146a as Sign of Chronic Inflammation in Type 2 Diabetic Patients

Background

There is increasing evidence that chronic inflammation is an important determinant in insulin resistance and in the pathogenesis of type 2 diabetes (T2D). MicroRNAs constitute a newly discovered system of cell regulation and in particular two microRNAs (miR-146a and miR-155) have been described as regulators and biomarkers of inflammation.

Aim

To determine a putative association between the levels of miR-146a and miR-155 in serum of T2D patients, clinical parameters and serological indicators of inflammation.

Methods

We performed quantitative Real Time PCR (qPCR) of microRNAs from serum (56 Ecuadorian T2D ambulatory patients and 40 non-diabetic controls). In addition, we evaluated T2D-related serum cytokines.chemokines and growth factors using a commercially available multi-analyte cytometric bead array system. We correlated outcomes to clinical parameters, including BMI, HbA1c and lipid state.

Results

The Ecuadorian non-diabetic controls appeared as overweight (BMI>25: patients 85%, controls 82.5%) and as dyslipidemic (hypercholesterolemia: patients 60.7%, controls 67.5%) as the patients.

• The serum levels of miR-146a were significantly reduced in T2D patients as compared to these non-diabetic, but obese/dyslipidemic control group (mean patients 0.61, mean controls set at 1; p = 0.042), those of miR-155 were normal.
• The serum levels of both microRNAs correlated to each other (r = 0.478; p<0.001) and to leptin levels. The microRNAs did not correlate to BMI, glycemia and dyslipidemia.
• From the tested cytokines, chemokines and growth factors, we found IL-8 and HGF significantly raised in T2D patients versus non-diabetic controls (p = 0.011 and 0.023 respectively).

Conclusions

This study shows decreased serum anti-inflammatory miR-146a, increased pro-inflammatory IL-8 and increased HGF (a vascular/insular repair factor) as discriminating markers of failure of glucose control occurring on the background of obesity and dyslipidemia.     

Blockade of myeloid differentiation protein 2 prevents obesity‐induced inflammation and nephropathy

Obesity is a major and independent risk factor of kidney diseases. The pathogenic mechanisms of obesity‐associated renal injury are recognized to at least involve a lipid‐rich and pro‐inflammatory state of the renal tissues, but specific mechanisms establishing causal relation remain unknown. Saturated fatty acids are elevated in obesity, and known to induce chronic inflammation in kidneys. Myeloid differentiation protein 2 (MD2) is an important protein in lipopolysaccharide‐induced innate immunity response and inflammation. We suggested that obesity‐associated renal injury is regulated by MD2 thereby driving an inflammatory renal injury. The used three mouse models for in vivo study: MD2 knockout mice (KO) maintained on high fat diet (HFD), wild‐type mice on HFD plus L6H21, a specific MD2 inhibitor and KO mice given palmitic acid (PA) by IV injection. The in vitro studies were carried out in cultured renal tubular epithelial cells, mouse mesangial cells and primary macrophages, respectively. The HFD mice presented with increased hyperlipidemia, serum creatinine and proteinuria. Renal tissue from HFD mice had increased fibrosis, inflammatory cytokines, macrophage infiltration, and activation of NF‐κB and MAPKs. This HFD‐induced renal injury profile was not observed in KO mice or L6H21‐treated mice. Mice given PA mimmicked the HFD‐induced renal injury profiles, which were prevented by MD2 knockout. The in vitro data further confirmed MD2 mediates PA‐induced inflammation. MD2 is causally related with obesity‐associated renal inflammatory injury. We believe that MD2 is an attractive target for future therapeutic strategies in obesity‐associated kidney diseases.     

Inhibition of microRNA-103 attenuates inflammation and endoplasmic reticulum stress in atherosclerosis through disrupting the PTEN-mediated MAPK signaling

Atherosclerosis (AS), a chronic disorder of large arteries, is the underlying pathological process of heart disease and stroke. Former researchers have found that microRNAs (miRs) are involved in the several key processes of AS. Apolipoprotein E knockout (ApoE^−/−) mice fed a high-fat-diet (HFD) to establish AS model. The expression of miR-103 was characterized in the mice model. The effects of miR-103 on inflammation and endoplasmic reticulum stress (ERS) were analyzed when the expression of miR-103 was inhibited in ApoE ^−/− mice fed an HFD and human aortic endothelial cells (HAECs) exposed to oxidized low-density lipoprotein (ox-LDL). The relationship between miR-103 and phosphatase and tensin homolog (PTEN) was identified by luciferase activity detection and real-time quantitative polymerase chain reaction (RT-qPCR). Gain- and loss-function approaches were further applied for investigating the regulatory effects of miR-103 and PTEN on ERS. Role of MAPK signaling was then analyzed using PD98059 to block this pathway. miR-103 was highly expressed in the ApoEApoE ^−/− mice fed an HFD. Downregulation of miR-103 suppressed inflammation and ERS in endothelial cells isolated from ApoE ^−/− mice fed a HFD and ox-LDL-exposed HAECs. In addition, miR-103 can target PTEN and downregulate its expression. Overexpression of PTEN reversed the miR-103-induced activation of MAPK signaling. Moreover, PTEN upregulation or MAPK signaling inhibition ease miR-103-induced inflammation and ERS in vivo and in vitro. Thus, miR-103 depletion restrains the progression of AS through blocking PTEN-mediated MAPK signaling.     

Excessive dietary salt promotes neuroinflammation to worsen retinopathy in mice with streptozotocin-induced diabetes

ObjectiveDiabetic retinopathy (DR) includes vascular and neural tissue injury. Persistent low-grade inflammation may contribute to DR. Increased salt intake has been shown to promote autoimmunity in the brain. This study determined the role of salt intake in DR development.MethodsEight-week-old C57BL/6 J male mice received streptozotocin to induce diabetes. Diabetic or non-diabetic mice were fed a diet containing normal, low and high amounts of salt. The retinal function, structure and inflammatory response were determined 8 weeks after the establishment of diabetes. Interleukin (IL)-1β or a NLR family pyrin domain containing 3 (NLRP3) inhibitor was injected intravitreally and the retinal changes were evaluated.ResultsA high salt diet worsened the functional and structural damage of retinal cells and increased IL-1β in the retina of diabetic mice. IL-1β injection impaired the function of photoreceptors and retinal structure in the diabetic mice. Blocking NLRP3 inhibited IL-1β increase in the mouse bone marrow macrophages cultured in high sodium medium. NLRP3 inhibition attenuated retinal injury of diabetic mice on high salt diet. A low-salt diet also triggered inflammation and cell damage in the retina of diabetic mice but at a lower grade than those induced by high salt diet. A low or high salt diet for 8 weeks did not induce inflammation or cell injury in the retina of mice without diabetes.ConclusionThese results indicate that high salt intake has deleterious effects in DR development through NLRP3 inflammasome activation and the subsequent production of IL-1β. Limiting salt intake may not attenuate DR development.     

Maternal obesity induces gut inflammation and impairs gut epithelial barrier function in nonobese diabetic mice

Impairment of gut epithelial barrier function is a key predisposing factor for inflammatory bowel disease, type 1 diabetes (T1D) and related autoimmune diseases. We hypothesized that maternal obesity induces gut inflammation and impairs epithelial barrier function in the offspring of nonobese diabetic (NOD) mice. Four-week-old female NOD/ShiLtJ mice were fed with a control diet (CON; 10% energy from fat) or a high-fat diet (HFD; 60% energy from fat) for 8 weeks to induce obesity and then mated. During pregnancy and lactation, mice were maintained in their respective diets. After weaning, all offspring were fed the CON diet. At 16 weeks of age, female offspring were subjected to in vivo intestinal permeability test, and then ileum was sampled for biochemical analyses. Inflammasome mediators, activated caspase-1 and mature forms of interleukin (IL)-1β and IL-18 were enhanced in offspring of obese mothers, which was associated with elevated serum tumor necrosis factor α level and inflammatory mediators. Consistently, abundance of oxidative stress markers including catalase, peroxiredoxin-4 and superoxide dismutase 1 was heightened in offspring ileum (P<.05). Furthermore, offspring from obese mothers had a higher intestinal permeability. Morphologically, maternal obesity reduced villi/crypt ratio in the ileum of offspring gut. In conclusion, maternal obesity induced inflammation and impaired gut barrier function in offspring of NOD mice. The enhanced gut permeability in HFD offspring might predispose them to the development of T1D and other gut permeability-associated diseases.     

Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A<Subscript>2B</Subscript> adenosine receptors

Adenosine A_2B receptors (A_2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A_2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A_2BR localization was examined by immunofluorescence. The role of A_2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A_2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A_2BR antagonist MRS1754 enhanced electrically induced NK₁-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A_2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A_2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A_2BR expression. A_2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.     

Dysregulation of microRNA-125a contributes to obesity-associated insulin resistance and dysregulates lipid metabolism in mice

Obesity is associated with an increased risk of developing insulin resistance (IR) and type 2 diabetes (T2D). A diverse group of factors including miRNA has been implicated in the pathogenesis of these two metabolic conditions, although underlying molecular mechanisms involved are not well defined. Here, we provide evidence that hepatic miR-125a levels are diminished in both genetic as well as dietary mouse models of obesity. Overexpression of miR-125a enhanced insulin signaling and attenuated cellular lipid accumulation in HepG2 cells and Hepa1–6 cells. Likewise, treatment of mice with ago-miR-125a increased insulin sensitivity, similar to overexpression of miR-125a, whereas treatment of mice with antago-miR-125a blunted the insulin sensitivity. Furthermore, overexpression of miR-125a in mice previously fed a high-fat diet (HFD), significantly improved insulin sensitivity, and attenuated obesity-linked hepatic steatosis and hepatocyte lipid accumulation. In addition, we show that ELOVL fatty acid elongase 6 (Elovl6) is a direct target of miR-125a, and participates in miR-125a mediated regulation of insulin sensitivity and lipid metabolism. These data led us to conclude that dysregulated miR-125a expression augments the development of obesity-induced IR and that miR-125a might serve as a therapeutic target for the development of new drug(s) in the clinical management of metabolic diseases.     

microRNA-383 regulates cell viability and apoptosis by mediating Wnt/β-catenin signaling pathway in non–small cell lung cancer

The aim of this study was to investigate the roles of microRNA-383 (miRNA-383) in progression of non–small cell lung cancer (NSCLC) and the potential mechanism. The expressions of miR-383 and Wnt1 protein were detected in lung cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. After the transfection of miR-383 mimics, si-Wnt1 or miR-383+Wnt1, the viability and apoptosis of NSCLC cells were detected by cell counting kit-8 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling, respectively. The interaction between miR-383 and Wnt1 was investigated by luciferase activity and Western blot analysis. Cells stably transfected with miR-383 mimics were inoculated into the right axillary of nude mice by subcutaneous injection. The tumor volume and weight were measured, and the expressions of miR-383, Wnt1, β-catenin, and cyclin D1 were detected by qRT-PCR and Western blot analysis. The expression of miR-383 was significantly decreased, and the level of Wnt1 was significantly increased (P < 0.05) in lung cancer tissues and cells. Upregulation of miR-383 or inhibition of Wnt1 expression inhibited the cell viability and induce apoptosis in NSCLC cells. Moreover, Wnt1 was the target gene of miR-383, and its overexpression weakened the regulatory effect of miR-383 on cell viability and apoptosis in NSCLC cells. Besides, the addition of miR-383 decreased the tumor volume and size and inhibited the expressions of Wnt1, β-catenin, and cyclin D1 at the protein level in nude mice. Collectively, miR-383 induced apoptosis and inhibited cell viability as well as tumorigenic capacity in nude mice via regulating the Wnt/β-catenin signaling pathway.     

MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus

Background

Dysregulation of microRNA (miRNA) expression in various tissues and body fluids has been demonstrated to be associated with several diseases, including Type 2 Diabetes mellitus (T2D). Here, we compare miRNA expression profiles in different tissues (pancreas, liver, adipose and skeletal muscle) as well as in blood samples from T2D rat model and highlight the potential of circulating miRNAs as biomarkers of T2D. In parallel, we have examined the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients.

Methodology/Principal Findings

Employing miRNA microarray and stem-loop real-time RT-PCR, we identify four novel miRNAs, miR-144, miR-146a, miR-150 and miR-182 in addition to four previously reported diabetes-related miRNAs, miR-192, miR-29a, miR-30d and miR-320a, as potential signature miRNAs that distinguished IFG and T2D. Of these microRNAs, miR-144 that promotes erythropoiesis has been found to be highly up-regulated. Increased circulating level of miR-144 has been found to correlate with down-regulation of its predicted target, insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. We could also experimentally demonstrate that IRS1 is indeed the target of miR-144.

Conclusion

We demonstrate that peripheral blood microRNAs can be developed as unique biomarkers that are reflective and predictive of metabolic health and disorder. We have also identified signature miRNAs which could possibly explain the pathogenesis of T2D and the significance of miR-144 in insulin signaling.