Fig. 3

Hesperetin treatment reduces fat and improves glucose homeostasis in old WT mice. A–D Hesperetin treatment for 6 months (from 22- to 28-month old). A, B Representative Micro-CT analysis and quantification of total body fat (volume) and visceral fat (volume) in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3–10 mice per group). Yellow circles indicate the area of visceral fat for quantification. C, D Representative Micro-CT analysis and quantification of total body lean (volume) and percent of body lean (%) in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3–10 mice per groups). E Body weight in 3-month WT, 28-month Veh-treated and 28-month hesperetin-treated WT mice (n = 3–10 mice per groups). F, G Basal levels of blood glucose (fasting 6 h), and blood glucose levels at 120 min measured during glucose tolerance test (GTT) after hesperetin treatment for 6 months (from 20.5- to 26.5-month old) in the Veh-treated and hesperetin-treated mice. H The mRNA levels of key enzymes involved in the pathway of glycogenolysis in the livers of 3-month WT, 26-month Veh-treated and 26-month hesperetin-treated WT mice (n = 3 mice per group) after hesperetin treatment for 5 months (from 21- to 26-month old). The mRNA levels were quantified by RNA-seq analysis. I Schematic pathway of the enzymes and metabolites involved in hepatic insulin signaling and glucose metabolism, including glycolysis, glycogen synthesis, gluconeogenesis, and glycogenolysis, in naturally aged Veh-treated and hesperetin-treated WT mice. The nuclear factor kappa-B kinase subunit β (IKKβ) is able to inhibit insulin signaling via phosphorylation of insulin receptor substrate 1. AKT is central to regulating hepatic insulin action and glucose metabolism. Glycolysis: The key enzymes involved in glycolysis are glucokinase (Gck), phosphofructokinase (Pfkl) and pyruvate kinase (Pklr). Glycogen synthesis: The key enzymes involved in glycogen synthesis are glycogen synthase (Gys2) and glycogen branching enzyme (Gbe1). In addition, glycogen synthase kinase 3β (Gsk3β) is able to phosphorylate and inhibit glycogen synthase activity, whereas the protein phosphatase 1 (PP1) is able to dephosphorylate and promote glycogen synthase activity. Gluconeogenesis: The key enzymes involved in gluconeogenesis are pyruvate carboxylase (Pcx), phosphoenolpyruvate carboxykinase (Pck1/Pepck), fructose 1,6-bisphosphatase (Fbp1) and glucose-6-phosphatase (G6pc). Glycogenolysis: The key enzymes involved in glycogenolysis are glycogen debranching enzyme (Agl) and glycogen phosphorylase (Pygl). In addition, protein kinase A alpha (PKAα) is able to phosphorylate and activate the α subunit of phosphorylase kinase (PhKα); subsequently, the activated PhK phosphorylates Pygl to increase its enzymatic activity. Phosphorylase kinase is one of the three main families of Ca²⁺/Calmodulin-dependent protein kinases. Moreover, the δ subunit of phosphorylase kinase (PhKδ) is the endogenous calmodulin, the activity of which is able to be regulated by intracellular Ca²⁺ levels. G6P, glucose-6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; PEP, phosphoenolpyruvate; OAA, oxaloacetate; G1P, glucose 1-phosphate; Pgm2, phosphoglucomutase 2. Results are presented as mean ± SD. *p < 0.05; **p < 0.005; not significant (n.s.). In (B), (D), (E) and (H), the statistical analyses were performed by one-way ANOVA with Bonferroni multiple comparison test. In (F) and (G), the statistical analyses were performed by Student’s t test. All the mice used in this study are males. UT untreated