The beneficial effects of inhibition of hepatic APPL2 on metabolic homeostasis

Abstract

APPL2 (Adaptor Protein, Phosphotyrosine Interacting with PH Domain and Leucine Zipper 2) can interact with adiponectin receptors and negatively regulate adiponectin signaling. Human genome studies displayed correlation between single nucleotide polymorphisms (SNPs) of APPL2 gene and obesity or type 2 diabetes mellitus (T2DM). In animal studies, APPL2 was proved to interact with TBC1D1 and then regulate insulin-stimulated glucose transporter type 4 (Glut4) translocation in muscle cells, thereby fine-tuning glucose homeostasis. In other tissues, APPL2 was shown to regulate insulin exocytosis from β-cell and enhance subcutaneous white adipose tissue beiging. In the liver, hepatocyte-specific APPL2 deficiency enhanced adiponectin signaling and protected mice against diet-induced insulin resistance. As a novel therapy, antisense oligonucleotides (ASOs) for the treatment of genetic disorders have been approved for years. The primary purpose of this study is to examine whether and how selectively hepatic APPL2 knockdown by GalNAc-APPL2-ASO contributes to glucose, lipid, and energy metabolism. Key findings: 1. Subcutaneous injection of N-Acetylgalactosamine GalNAc-APPL2-ASO was able to selectively silence hepatic APPL2 on mRNA and protein levels of mice, with acceptable safety and not obvious off-target effect. 2. An animal model with high-fat-diet (HFD) induced obesity and insulin resistance was used to investigate the effect of GalNAc-APPL2-ASO treatment on glucose metabolism. The results showed that the treatment can improve glucose tolerance and increase insulin sensitivity, as well as suppressing gluconeogenesis. In vitro study suggested that the inactivation of hepatic APPL2 by both two sequences of GalNAc-APPL2-ASO substantially reduced glucagon-induced glucose production in primary hepatocytes. 3. RNA-Sequencing (RNA-Seq) analysis results with various approaches, including Gene Ontology, Kyoto Encyclopedia of Genes and Genome, and Gene Set Enrichment Analysis, indicated that liver-specifically knockdown APPL2 regulated several pathways associated with glucose and lipid metabolism. Some of the most remarkable ones included fatty acids metabolism and amino acid metabolism. 4. GalNAc-APPL2-ASO treatment alleviated diet-induced hypercholesterolemia, via decreasing total cholesterol and low-density-lipoprotein cholesterol. 5. GalNAc-APPL2-ASO treatment improved energy expenditure. With the trigger of cold exposure, the treatment also protected against hepatic steatosis and adipocyte hypertrophy induced by high-fat-diet. Taken together, this study presented here revealed that, for the first time, hepatic APPL2 knockdown by GalNAc-APPL2-ASOs improved glucose metabolism (insulin sensitivity and gluconeogenesis). The suppression of gluconeogenesis was due to the inhibitory action of APPL2 on glucose production from pyruvate as a precursor. In addition, GalNAc-APPL2-ASO treatment also alleviated lipid metabolism (hypercholesterolemia), and enhanced energy expenditure in HFD-induced mice.