Synergistic effects of metformin, resveratrol, and hydroxymethylbutyrate on insulin sensitivity

Figures & data

Figure 1 Synergistic effects of metformin and resveratrol-HMB on fatty acid oxidation in C2C12 muscle cells and 3T3L1 adipocytes. Differentiated C2C12 muscle cells (A) and 3T3L1 adipocytes (B) were treated with the treatments indicated for 24 hours.

Notes: OCR was measured after palmitate injection (point A and C) and represented as a percentage of baseline. The AUC is calculated by subtracting the starting rate (third baseline point) from each average rate point. Data are expressed as the mean ± standard deviation (n = 4). Bars with the same letter superscripts are not significantly different from each other; bars with nonmatching letter superscripts indicate significant differences between groups (P < 0.04).
Abbreviations: AUC, area under curve; CTRL, control; HMB, hydroxymethylbutyrate; MET, metformin; OCR, oxygen consumption rate; RESV, resveratrol.

Figure 2 Synergistic effects of metformin and resveratrol-HMB on AMPK and Sirt1 activity in C2C12 muscle cells and 3T3L1 adipocytes. Differentiated muscle cells and adipocytes were treated with the treatments indicated. (A and B) AMPK activity in C2C12 cell lysates. (C) Sirt1 activity in 3T3L1 cell lysates. (D) Quantitative band analysis of AMPK phosphorylation in 3T3L1 adipocytes. (E) Western blots of AMPK in 3T3L1 cell lysates using anti-phospho-AMPK-α (Thr172) and anti-AMPKα antibodies. AICAR was used as a positive control. (F) P-AMPK/AMPK ratio in 3T3L1 adipocytes.

Notes: Data are expressed as the mean ± standard error of the mean (n = 4). Bars with the same letter superscripts are not significantly different from each other; bars with nonmatching letter superscripts indicate significant differences between groups (P < 0.05).
Abbreviations: AFU, arbitrary fluorescent units; AMPK, AMP-activated protein kinase; HMB, hydroxymethylbutyrate; MET, metformin; RESV, resveratrol.

Table 1 Effects of indicated treatments on body weight, visceral fat mass, liver weight, plasma glucose, and food intake in mice

	Control	Standard metformin	Low-dose metformin	Very low-dose metformin	Low-dose metformin + resveratrol-HMB	Very low-dose metformin + resveratrol-HMB
Body weight (g) (start)	45.0 ± 0.6^a	45.2 ± 0.8^a	45.1 ± 0.6^a	44.4 ± 0.9^a	45.6 ± 1.0^a	46.7 ± 0.6^a
Body weight (g) (end)	46.8 ± 0.7^a	48.6 ± 0.6^a	48.2 ± 0.4^a	46.3 ± 1.1^a	48.3 ± 1.0^a	47.9 ± 1.0^a
Plasma glucose (mg/dL)	481.3 ± 25.0^a	460.1 ± 24.7^a	482.3 ± 15.4^a	503.0 ± 17.0^a	433.7 ± 51.6^a	529.1 ± 18.6^a
Visceral fat (g)	4.5 ± 0.18^a	4.3 ± 0.29^a	4.6 ± 0.14^a	4.4 ± 0.13^a	3.8 ± 0.19^b	3.6 ± 0.23^b
Liver (g)	2.78 ± 0.16^a	2.76 ± 0.09^a	2.65 ± 0.09^a	2.68 ± 0.12^a	2.35 ± 0.13^b	2.41 ± 0.08^b

Notes:db/db mice were fed the indicated diets for 2 weeks. Data are expressed as the mean ± standard error of the mean (n = 10). Values with the same letter superscripts in each row are not significantly different from each other; values with nonmatching letter superscripts in each row indicate significant differences between groups (P < 0.03).

Abbreviation: HMB, hydroxymethylbutyrate.

Figure 3 Synergistic effects of metformin and resveratrol-HMB on insulin sensitivity in mice. Effects of standard dose, low-dose, and very low-dose of metformin compared with low-dose metformin-resveratrol-CaHMB and with very low-dose metformin-resveratrol-CaHMB on (A) plasma insulin and (B) HOMA_IR in db/db mice.

Notes: Data are expressed as the mean ± standard error of the mean (n = 10). Bars with the same letter superscripts are not significantly different from each other; bars with nonmatching letter superscripts indicate significant differences between groups (P < 0.025).
Abbreviations: HOMA, homeostatic assessment of insulin resistance; HMB, hydroxymethylbutyrate; MET, metformin; RESV, resveratrol.

Figure 4 Synergistic effects of metformin and resveratrol-HMB on insulin tolerance test in mice.

Notes: Effects of standard dose, low-dose, and very low-dose metformin compared with low-dose metformin-resveratrol-CaHMB and with very low-dose metformin-resveratrol-CaHMB on 30-minute plasma glucose response to insulin (0.75 U/kg body weight) in db/db mice. Data are expressed as the mean ± standard error of the mean (n = 10). Bars with the same letter superscripts are not significantly different from each other; bars with nonmatching letter superscripts indicate significant differences between groups (P < 0.02).
Abbreviations: HMB, hydroxymethylbutyrate; Met, metformin; RESV, resveratrol.