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.