Inhibition of Gluconeogenesis by Boldine in the Perfused Liver: Therapeutical Implication for Glycemic Control

Abstract

The alkaloid boldine occurs in the Chilean boldo tree (Peumus boldus). It acts as a free radical scavenger and controls glycemia in diabetic rats. Various mechanisms have been proposed for this effect, including inhibited glucose absorption, stimulated insulin secretion, and increased expression of genes involved in glycemic control. Direct effects on glucose synthesis and degradation were not yet measured. To fill this gap, the present study is aimed at ensuring several metabolic pathways linked to glucose metabolism (e.g., gluconeogenesis) in the isolated perfused rat liver. In order to address mechanistic issues, energy transduction in isolated mitochondria and activities of gluconeogenic key enzymes in tissue preparations were also measured. Boldine diminished mitochondrial ROS generation, with no effect on energy transduction in isolated mitochondria. It inhibited, however, at least three enzymes of the gluconeogenic pathway, namely, phosphoenolpyruvate carboxykinase, fructose-bisphosphatase-1, and glucose 6-phosphatase, starting at concentrations below 50 μM. Consistently, in the perfused liver, boldine decreased lactate-, alanine-, and fructose-driven gluconeogenesis with IC₅₀ values of 71.9, 85.2, and 83.6 μM, respectively. Conversely, the compound also increased glycolysis from glycogen-derived glucosyl units. The hepatic ATP content was not affected by boldine. It is proposed that the direct inhibition of hepatic gluconeogenesis by boldine, combined with the increase of glycolysis, could be an important event behind the diminished hyperglycemia observed in boldine-treated diabetic rats.

Figure 1

The effects of boldine on glycogen catabolism in the livers from fed rats. (a) Time courses of the changes caused by 200 μM boldine. Data are the means ± mean standard errors of 4 to 6 liver perfusion experiments. The time period of boldine infusion is indicated by the horizontal bar. (b) Concentration dependences of the effects of boldine on the rates of oxygen uptake, lactate and pyruvate productions, and glucose output. All parameters were evaluated before (10 minutes of perfusion time) and at 20 min after starting boldine infusion (30 minutes of perfusion time). The values corresponding to zero boldine concentration (controls) are the means ± mean standard errors of 12 perfusion experiments, computed just before initiating boldine infusion; rates in the presence of boldine represent the means of 4 liver perfusion experiments computed at 30 minutes of perfusion time. Asterisks (^∗) identify those rates that differ from the control condition, as indicated by the post hoc Student-Newman-Keuls testing (p ≤ 0.05).

Figure 2

Effect of boldine on the lactate gluconeogenesis in the perfused liver isolated from fasted rats. Time courses (a) and concentration-dependent effects of boldine (b) on lactate gluconeogenesis and related parameters in the livers isolated from fasted rats. Liver was perfused as described in the Materials and Methods. In (a), data are the means ± mean standard errors of 3 to 4 liver perfusion experiments. Boxes near the time scale indicate the lactate and boldine infusion periods and concentrations. In (b), the rates of pyruvate and glucose productions and oxygen uptake were evaluated at 36 minutes of perfusion time (control condition, zero boldine concentration; n = 10) and at 56 min perfusion time (20 minutes after starting boldine infusion; n = 4). Asterisks (^∗) in (b) indicate those rates that differ from the control condition (absence of boldine; p ≤ 0.05).

Figure 3

Effects of boldine on fructose gluconeogenesis in the perfused livers isolated from fasted rats. Time courses (a) and concentration-dependent effects of boldine (b) on fructose gluconeogenesis and related parameters. Livers were perfused as described in the Materials and Methods. In (a), data are the means ± mean standard errors of 4 liver perfusion experiments. Boxes near to the time scale indicate the fructose and boldine infusion periods and concentrations. In (b), the rates of pyruvate, lactate, and glucose productions and oxygen uptake were evaluated at 36 minutes of perfusion time (control condition, zero boldine concentration; n = 12) and at 56 min perfusion time (n = 4). Asterisks (^∗) in (b) indicate those rates that differ from the control condition (absence of boldine; p ≤ 0.05).

Figure 4

Effect of boldine on glycerol gluconeogenesis in the perfused livers isolated from fasted rats. (a) The time courses of the effects of glycerol and 200 μM boldine and (b) the concentration dependences of the effects of boldine. Livers were perfused as described in the Materials and Methods. In (a), data are the means ± mean standard errors of 3 to 4 liver perfusion experiments. Boxes near to the time scale indicate the glycerol and boldine infusion periods and concentrations. In (b), the rates of pyruvate, lactate, and glucose productions and oxygen uptake were evaluated at 36 minutes of perfusion time (control condition; n = 12) and at 56 min perfusion time (n = 4). Asterisks (^∗) in (b) indicate statistically significant differences (p ≤ 0.05) when compared to the control condition (absence of boldine), as given by the post hoc Student-Newman-Keuls testing.

Figure 5

Time course of the effects of boldine on the alanine gluconeogenesis in the perfused livers isolated from fasted rats. Livers were perfused as described in the Materials and Methods. Boxes near to the time scale indicate the alanine and boldine infusion periods and concentrations. The outflowing perfusate was sampled every 2 minutes and used to quantify glucose, pyruvate, lactate, ammonia, and urea. Oxygen uptake was monitored polarographically by a platinum electrode. Data are the means ± mean standard errors of 4 to 5 liver perfusion experiments.

Figure 6

Concentration dependence of the effects of boldine on alanine gluconeogenesis in the perfused livers from fasted rats. Livers were perfused as described in the Materials and Methods. The metabolic rates were calculated at 36 minutes of perfusion time (control condition, zero boldine concentration; n = 12) and at 5 minutes of perfusion time (n = 4). Asterisks (^∗) indicate statistically significant differences when compared to the control condition (absence of boldine), as indicated by post hoc testing according to Student-Newman-Keuls (p ≤ 0.05).

Figure 7

Concentration dependences of the effects of boldine on regulatory enzymes of the gluconeogenic pathway (a), ROS production (b), and mitochondrial respiration (c). Experimental procedures were described in the Materials and methods. (a) Values are means ± mean standard errors of 4 to 6 assays. Asterisks (^∗) indicate statistical significance in comparison with the control condition (Student-Newman-Keuls, p ≤ 0.05). (b, c) Mitochondria of the rat livers were isolated as described in the Materials and methods. Values are means ± mean standard errors of 4 to 6 assays. In (b), 0-rot: without boldine and rotenone; 0+rot: without boldine in the presence of rotenone; all boldine concentrations were evaluated in the presence of rotenone. The symbol # indicates statistical significance in comparison with 0-rot; ^∗ indicates statistical significance in comparison with the stimulated condition (0+rot) (Student-Newman-Keuls, p ≤ 0.05).

Figure 8

Schematic representation of the main sites of action of boldine on the pathways leading to glucose synthesis and degradation in the liver. The + sign denotes stimulation, and the x denotes inhibition.