TQ improves insulin sensitivity in HepG2 cells via a SIRT-1 dependent mechanism.

<p>HepG2 cells were cultured in high (20 mM) glucose or in growth media containing 5.5 mM glucose for 18 hours, starved with serum-free media for 2 hours, then pre-incubated with vehicle control (0.5% DMSO), nicotinamide (0.5 mM), compound C (20 μM), or with nicotinamide and compound C together for 30 mins, followed by incubation with TQ (10 μM) in the presence or absence of nicotinamide and compound C; or with TQ, resveratrol (50 μM), or AICAR (2 mM) alone for 24 hours in 20mM glucose media. Vehicle-treated cells in 5.5 mM glucose served as control. Insulin (100 nM) was added during the last 30 min. (A) Western blot images of p-Akt, Akt, and β-actin. (B) Protein band quantification using densitometry from three independent experiments. p≤ 0.05 where (*) is significantly different from 5.5G, (#) is significantly different from 20G, and (Δ) is significantly different from 20G + TQ using independent t-tests. 5.5 G: 5.5 mM glucose, 20G: 20 mM glucose, TQ: thymoquinone, R: resveratrol, AIC: AICAR, NIC: nicotinamide, C: compound C.</p

TQ normalizes glucose tolerance and insulin sensitivity.

<p>(A) Blood glucose levels in response to oral glucose tolerance test (OGTT). (B) Blood glucose levels in response to insulin tolerance test (ITT). p<0.05 when comparing HFD and LFD (+), and HFD and HFD+TQ (*), using a two-way ANOVA followed by Holm-Sidak post-test. Results are means ± SEM (n = 10–12 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

Effects of TQ on Akt and AMPKα protein expression in liver.

<p>(A) Western blot images of Akt, p-Akt, AMPKα and p-AMPKα protein in liver. β-actin was used as a loading control. Western blot images are representative of combined liver lysates from n = 10–12 mice per treatment group. (B) Protein band quantification using densitometry from three independent experiments. p≤0.05 when comparing (+) HFD and LFD, (*) HFD + TQ and HFD, and (#) LFD and LFD + TQ using independent t-tests. LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

Effects of TQ on triglyceride content in liver and muscle.

<p>(A) Triglyceride concentration in liver. (B) Triglyceride concentration in soleus muscle. (*) p<0.05 when comparing HFD + TQ and HFD using a one-way ANOVA followed by Sidak post-test. Results are means ± SEM (n = 8–12 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

Effects of TQ on NQO1 expression.

<p>NQO1 mRNA expression in liver (A) and soleus muscle (B). (C) Western blot images of NQO1 and β-actin protein in liver (D) Western blot images of NQO1 protein in soleus muscle. β-tubulin was used as a loading control. Statistical analysis (A and B): one-way ANOVA followed by Sidak post-test (p≤0.05). qPCR results are means ± SEM (n = 8–12 mice per treatment group). Western blot images are representative of combined liver and soleus muscle lysates from n = 10–12 mice per treatment group. LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

Effect of TQ on serum glycerol and fatty acids.

<p>Effect of TQ on serum glycerol and fatty acids.</p

Effects of TQ on NADH/NAD⁺ ratio in liver and soleus muscle.

<p>(A) NADH/NAD⁺ ratio in liver. (B) NADH/NAD⁺ ratio in soleus muscle. p ≤ 0.05 when comparing (+) HFD and LFD, (*) HFD + TQ and HFD, and (#) LFD and LFD + TQ using independent t-tests. Results are means ± SEM (n = 8–10 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

TQ ameliorates weight gain, lowers fasting blood glucose and insulin in DIO mice.

<p>(A) Effect of TQ on body weight (B) Effect of TQ treatment on fasting blood glucose after a 6 hour fast. (C) Effect of TQ on serum insulin. Total body weight was measured weekly for the duration of the study. p<0.05 when comparing HFD and LFD (+), and HFD and HFD+TQ (*), using a one-way ANOVA followed by Sidak post-test (A and B) or independent t-test (C). Results are means ± SEM (n = 10–12 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p

Effects of TQ on serum cholesterol.

<p>(A) Total cholesterol serum concentration. (B) HDL cholesterol serum concentration. (C) LDL/VLDL cholesterol serum concentration. p≤0.05 when comparing (+) HFD and LFD, (*) HFD + TQ and HFD using independent t-tests. Results are means ± SEM (n = 6–7 mice per treatment group). LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone, LDL: low-density lipoprotein, HDL: high-density lipoprotein, VLDL: very-low-density lipoprotein.</p

Effects of TQ on SIRT-1 protein expression.

<p>(A) Western blot images of SIRT-1 and p-SIRT-1 protein in liver. β-actin was used as a loading control. (B) Western blot images of SIRT-1 and p-SIRT-1 protein in soleus muscle. β-tubulin was used as a loading control. Western blot images are representative of combined liver and soleus muscle lysates from n = 10–12 mice per treatment group. (C and D) Protein band quantification using densitometry from three independent experiments. p ≤ 0.05 when comparing (+) HFD and LFD and (*) HFD + TQ and HFD using independent t-tests LFD: low fat diet, HFD: high fat diet, TQ: thymoquinone.</p