Knock-Down of IL-1Ra in Obese Mice Decreases Liver Inflammation and Improves Insulin Sensitivity

<div><p>Interleukin 1 Receptor antagonist (IL-1Ra) is highly elevated in obesity and is widely recognized as an anti-inflammatory cytokine. While the anti-inflammatory role of IL-1Ra in the pancreas is well established, the role of IL-1Ra in other insulin target tissues and the contribution of systemic IL-1Ra levels to the development of insulin resistance remains to be defined. Using antisense knock down of IL-1Ra in vivo, we show that normalization of IL-1Ra improved insulin sensitivity due to decreased inflammation in the liver and improved hepatic insulin sensitivity and these effects were independent of changes in body weight. A similar effect was observed in IL1-R1 KO mice, suggesting that at high concentrations of IL-1Ra typically observed in obesity, IL-1Ra can contribute to the development of insulin resistance in a mechanism independent of IL-1Ra binding to IL-1R1. These results demonstrate that normalization of plasma IL-1Ra concentration improves insulin sensitivity in diet- induced obese mice.</p></div

IL1Ra ASO treatment reduces liver triglyceride and improved liver inflammation.

<p>A) Liver weight (g), B) H&E stain of liver. C) Liver triglyceride content after 6 weeks of IL-1Ra ASO or control ASO treatment in HFD fed mice. D) QPCR analysis of inflammatory gene expression in the liver of obese mice treated with IL-1Ra ASO or control ASO, results are normalized to beta actin mRNA and expressed relative to control ASO treatment. * p<0.05 compared to HFD-fed control treated group, n = 5−8 mice per group.</p

IL-1Ra ASO treatment results in increased energy expenditure.

<p>Oxygen consumption (A) and energy expenditure (B) were measured in CLAMS cages over a 48 hr period in week 4 of the study. core body temperature (C) in HFD-fed IL-1Ra ASO treated mice compared with control ASO treated mice. D) Activity is unchanged between groups. E) Quantitative PCR analysis of uncoupling protein 1 (UCP-1) in BAT. * p<0.05 compared to HFD-fed control treated group, n = 5 mice per group, ns, not significant.</p

Antisense oligonucleotide (ASO) mediated knock-down of IL-1Ra transcript in vitro and in vivo using locked nucleic acids (LNAs).

<p>A) QPCR analysis of IL-1Ra mRNA expression after ASO treatment in Hepa 1–6 cells, each concentration was run in duplicate. B) QPCR analysis of IL-1Ra and IL-1β mRNA levels in liver and epididymal adipose (eWAT) from mice one week after IL-1Ra ASO administration. C) Blood glucose concentration in mice one week after IL-1Ra ASO treatment. D. QPCR analysis of IL-1Ra mRNA expression in liver and eWAT after 6 weeks of treatment of IL-1Ra ASO or control ASO. E. Plasma IL-1Ra concentration in obese mice treated with IL-1Ra ASO or control ASO for 6 weeks compared with aged matched lean control mice fed normal chow (NC). F. Analysis of liver enzyme levels after IL-1Ra ASO treatment in HFD-fed mice (ALT, alanine aminotransferase; AST, aspartate aminotransferase; GDH, glutamate dehydrogenase). *P<0.05 compared with control treated mice, QPCR results are normalized to beta actin mRNA and expressed relative to control treatment. Differences between groups were analyzed by one way ANOVA followed by Tukey post hoc tests, n = 5–8 mice per group, * p<0.05 compared to HFD-fed control treated group; ns, not significant.</p

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears

<div><p>Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans.</p></div

Fold change in expression between pooled RSA biopsies and controls.

<p>As a single pool, RSA biopsies are not significantly different from controls, though expression of pro-myogenic genes trended down while atrophic, adipogenic, and fibrotic genes trended up.</p

Heterogeneous muscle gene expression patterns in patients with massive rotator cuff tears - Fig 7

<p>Fold changes in expression between (A) HIGH and LOW expression muscle groups, (B) HIGH and NO-MUSCLE groups, (C) HIGH muscle and HI-FAT groups, (D) LOW muscle and HI-FAT groups, (E) LOW muscle and NO-MUSCLE groups, and (F) HI-FAT and NO-MUSCLE groups. Solid bars indicate significant up- or down-regulation (p<0.01 and p<0.05 indicated by ‘ = ‘, and ‘ * ‘, respectively).</p

Fold change in expression between the RSA biopsies that contain muscle compared to those without muscle.

<p>Solid bars indicate significant up- or down-regulation (p<0.01 and p<0.05 indicated by ‘ = ‘, and ‘ * ‘, respectively). With muscle present, nearly all genes of interest are significantly differentially regulated, with increased expression of muscle- and fat-related genes and decreased expression of fibrosis-related genes.</p

Principal component analysis employed to visualize variability between biopsies.

<p>Samples containing histological muscle are red, samples without muscle are blue, and controls are black. Of particular note are the cluster of blue samples in the lower center which correspond to the HI-FAT group in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190439#pone.0190439.g002" target="_blank">Fig 2</a>, and the high variability in expression among the muscle-containing samples.</p

Change in FMD during sitting conditions.

<p>Condition-associated change in FMD is represented by the ratio of FMD 2 (end of sitting period) to FMD 1 (baseline) and are shown by group (Panels A and B) and by individual (Panels C and D) using raw data (Panels A and C) and allometrically scaled data (Panels B and D) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188544#sec007" target="_blank">Materials and Methods</a> section). An FMD 2–to–FMD 1 ratio greater than 1 (dotted, horizontal line) indicates that the FMD response was greater at the completion of the sitting period relative to baseline. Box and whisker plots (Panels A and B): x = mean, line = median, dots above boxes are outliers. n = 10 for the control, 2-minute walking every hour, and 10-minute standing every hour conditions; n = 9 for the 2-minute standing every 20 minutes condition. <i>p</i>-value vs. the control condition. * Statistically significant after Bonferroni correction. Bonferroni-corrected cut-off for significance in 3-arm comparison with control was <i>p</i>< 0.0167.</p