HSG4112 regulates metabolic gene expression towards increased energy use in liver and muscle and affects pAMPK level while not affecting UCP1 level.
To better understand the molecular mechanism in which HSG4112 mediates the above beneficial effects, the expressions of genes involved in energy metabolism, leptin and insulin signaling, and inflammation were investigated. A total of 68 genes was selected through literary search (Hynes and Jones, 2001; Stern, Rutkowski and Scherer, 2016; Müller and Geisler, 2017), and we performed qRT-PCR on hypothalamus, liver, gastrocnemius muscle, and interscapular tissue (brown adipose tissue, or BAT) on the same animals as above. Genes with significant or relevant difference in expression levels are shown in Figure 4A-D ; their list and primer sequences are available in SupplementaryTable 1 .
Amongst muscle, liver, hypothalamus, and BAT, expressions of the selected genes were most affected by HSG4112 in muscle and liver. In muscle, genes related to fatty acid oxidation (ACOX1 ,PRKAA2 ) were significantly upregulated, while genes related to glucose transport and metabolism (SLC2A4 , PDK4 ) were normalized after HSG4112 treatment (Figure 4A ). In liver, genes related to glucose metabolism (FGF21 , PEPCK ), insulin signaling (FOXO1 , HNFA4 ), and lipid metabolism (CREB3L3 , APOA5 , SCD1 ) were induced by HSG4112 to either be normalized or increase towards the direction of increased energy expenditure (Figure 4B ). Relatively few changes were observed in hypothalamus and BAT. In hypothalamus, metabolism enhancingTTR expression was upregulated, and PTPN1 level denoting insulin resistance was downregulated (Figure 4C ), whileNPY , AGRP , POMC , CART level denoting leptin resistance showed normalizing trend (Supplementary Figure 1 ). In BAT, LEP (leptin) level was robustly normalized (Figure 4D ). mRNA level of UCP1 , which is one of the most well-known protein for enhancing energy expenditure through futile cycling of mitochondrial potential and consequent thermogenesis in BAT (Zaninovich, 2005), was not influenced by HSG4112 in all tissues.PGC-1ɑ , which is the master regulator of mitochondrial biogenesis (Scarpulla, 2011), was consistently upregulated in BAT, liver, and muscle, suggesting a possible role of this gene or its related pathway in the mechanism of action of HSG4112. Inflammation-related genes (CCL2 , NFE2L2 ) also had significant change in expression in liver and muscle.
Majority of the affected genes suggested enhancement of energy expenditure as HSG4112’s mode of action. Therefore, we investigated whether HSG4112 acts through two well-known enhancers of energy expenditure – AMPK and UCP1 – by measuring their protein levels at an early timepoint of 11 days, when body weight just begins to decrease. We found that phosphorylated hypothalamic AMPK markedly decreased to about half-fold in HSG4112 group (Figure 4E ); decrease in hypothalamic phospho-AMPK signifies activation of peripheral AMPK signaling and increased energy metabolism (Kim et al. , 2004). Consistent with transcriptome data, HSG4112 had no effect on UCP1 protein level in BAT (Figure 4F ), confirming that HSG4112’s mode of action does not converge with UCP1 pathway.