Myricetin Ameliorates Defective Post-Receptor Insulin Signaling via β-Endorphin Signaling in the Skeletal Muscles of Fructose-Fed Rats

Abstract

β-Endorphin plays a major role in the amelioration of insulin resistance. The present study documents that myricetin (3,5,7,3', 4', 5'-hexahydroxyflavone) ameliorates insulin resistance by enhancing β-endorphin production in insulin-resistant rats. The rats were induced for insulin resistance by feeding them a diet containing 60% fructose for 6 weeks. The degree of insulin resistance was measured by the homeostasis model assessment of basal insulin resistance (HOMA-IR). The plasma levels of insulin and β-endorphin were measured by an enzyme-linked immunosorbent assay. The insulin receptor-related signaling mediators in the soleus muscles of rats were evaluated by immunoprecipitation or immunoblotting. Myricetin was injected daily (1 mg kg(-1) per injection, thrice daily) for 14 days. Consequently, the high-glucose plasma levels in fructose-fed rats decreased significantly concomitant with an increase in plasma β-endorphin. The reduction of the elevated HOMA-IR index following treatment with myricetin was subsequently inhibited by the administration of β-funaltrexamine hydrochloride (β-FNA) at doses sufficient to block μ-opioid receptors (MOR). The myricetin treatment was also observed to affect the phosphorylation of the insulin receptor, insulin receptor substrate-1, Akt and Akt substrate of 160 kDa, with subsequent effects on glucose-transporter subtype 4 translocation, all of which were blocked by β-FNA pretreatment. These results indicated that enhancement of β-endorphin secretion, which in turn leads to peripheral MOR activation, is involved in the action of myricetin on the amelioration of impaired signaling intermediates downstream of insulin receptors.

Figure 1

Protein expression of insulin receptor-related signaling mediators in the soleus muscles of fructose-fed rats receiving 14-day treatment with myricetin (1 mg kg⁻¹ per i.v. injection, thrice daily) or myricetin plus β-FNA. β-FNA (10 μg kg⁻¹ per injection) was i.v. injected 30 min prior to administration of myricetin. Rats that did not receive any treatment were given the same volume of vehicle used to dissolve the test medications. Findings were reproduced on four separate occasions. The quantification of the data is shown in Table 2.

Figure 2

Tyrosine phosphorylation (pY) of insulin receptor (IR)(a) and insulin receptor substrate (IRS)-1(b) in the soleus muscles of fructose-fed rats receiving 14-day treatment with myricetin (1 mg kg⁻¹ per i.v. injection, thrice daily) or myricetin plus β-FNA. β-FNA (10 μg kg⁻¹ per injection) was i.v. injected 30 min prior to administration of myricetin. Rats that did not receive any treatment were given the same volume of vehicle used to dissolve the test medications. Findings were reproduced on four separate occasions. Quantification of protein levels are expressed as mean ± SEM (n = 5 per group) in each column. ^a P < .05 represents the level of significance compared to the basal values of the vehicle-treated group.

Figure 3

The amount of the p85 subunit of PI3-kinase associated with IRS-1(p85/IRS-1) in the soleus muscles of fructose-fed rats receiving 14-day treatment with myricetin (1 mg kg⁻¹ per i.v. injection, thrice daily) or myricetin plus β-FNA. β-FNA (10 μg kg⁻¹ per injection) was i.v. injected 30 min prior to administration of myricetin. Rats that did not receive any treatment were given the same volume of vehicle used to dissolve the test medications. Findings were reproduced on four separate occasions. Quantification of protein levels expressed as mean ± SEM (n = 5 per group) in each column. ^a P < .05 and ^b P < .01 represent the level of significance compared to the basal values of the vehicle-treated group.

Figure 4

Phosphorylation of Akt (a) and AS160 (b) in the soleus muscles of fructose-fed rats receiving 14-day treatment with myricetin (1 mg kg⁻¹ per i.v. injection, thrice daily) or myricetin plus β-FNA. β-FNA (10 μg kg⁻¹ per injection) was i.v. injected 30 min prior to administration of myricetin. Rats that did not receive any treatment were given the same volume of vehicle used to dissolve the test medications. Findings were reproduced on four separate occasions. Quantification of protein levels expressed as mean ± SEM (n = 5 per group) in each column. ^a P < .05 and ^b P < .01 represent the level of significance compared to the basal values of the vehicle-treated group.

Figure 5

Insulin-stimulated GLUT 4 protein expression in the membrane (a) and cytosolic (b) fractions in the soleus muscles of fructose-fed rats receiving 14-day treatment with myricetin (1 mg kg⁻¹ per i.v. injection, thrice daily) or myricetin plus β-FNA. β-FNA (10 μg kg⁻¹ per injection) was i.v. injected 30 min prior to administration of myricetin. Rats that did not receive any treatment were given the same volume of vehicle used to dissolve the test medications. Findings were reproduced on four separate occasions. Quantification of protein levels expressed as mean ± SEM (n = 5 per group) in each column. ^a P < .05 represents the level of significance compared to the values of the vehicle-treated group.

Figure 6

The possible mechanisms of myricetin action on the amelioration of defective post-receptor insulin signaling in the skeletal muscle of fructose-fed rats.