Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

ATF5 is a regulator of ER stress and ß-cell apoptosis in different mouse models of genetic- and diet-induced obesity and diabetes mellitus.

Endoplasmic reticulum (ER) stress is closely associated with type 2 diabetes (T2D). Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element binding protein (CREB) family whose levels are increased upon stress in pancreatic islets from mice. Intriguingly, ATF5 deficiency has been shown to contribute to increased ER stress and apoptosis in mouse islet micro-organs. We hypothesized that either deficiency or overexpression of ATF5 is equally deleterious for pancreatic islets in terms of ER stress and apoptosis. To test this, we used a number of in vitro and in vivo models whereby ATF5 levels were overexpressed. We also determined the regulation of ATF5 in the context of metabolic derangements by using various mouse models of obesity and T2D. Our in vitro results show that ATF5 overexpression promoted palmitic acid (PA)-induced lipotoxic apoptosis. In vivo, global ATF5 overexpression in mice was lethal and pancreas-specific ATF5 overexpressing mice exhibit increased ß-cell apoptosis. Interestingly, ATF5 is downregulated in all mouse models of severe obesity and T2D used in the current study. In conclusion, a tight control on ATF5 levels might be considered when developing novel agents targeting ATF5 for prevention and treatment of metabolic diseases.

Lowering apolipoprotein CIII protects against high-fat diet-induced metabolic derangements.

Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity.

Apolipoprotein CIII Reduction Protects White Adipose Tissues against Obesity-Induced Inflammation and Insulin Resistance in Mice.

Apolipoprotein CIII (apoCIII) is proinflammatory and increases in high-fat diet (HFD)-induced obesity and insulin resistance. We have previously shown that reducing apoCIII improves insulin sensitivity in vivo by complex mechanisms involving liver and brown adipose tissue. In this study the focus was on subcutaneous (SAT) and visceral (VAT) white adipose tissue (WAT). Mice were either given HFD for 14 weeks and directly from start also treated with antisense oligonucleotide (ASO) against apoCIII or given HFD for 10 weeks and HFD+ASO for an additional 14 weeks. Both groups had animals treated with inactive (Scr) ASO as controls and in parallel chow-fed mice were injected with saline. Preventing an increase or lowering apoCIII in the HFD-fed mice decreased adipocytes' size, reduced expression of inflammatory cytokines and increased expression of genes related to thermogenesis and beiging. Isolated adipocytes from both VAT and SAT from the ASO-treated mice had normal insulin-induced inhibition of lipolysis compared to cells from Scr-treated mice. In conclusion, the HFD-induced metabolic derangements in WATs can be prevented and reversed by lowering apoCIII.