Figure 1.
Dietary carbohydrates (carbs) are not necessary for the high fat diet (HFD)-mediated body weight gain but can promote the HFD-mediated weight gain in a dose-dependent manner.
C57BL/6 (B6) mice were fed the regular chow diet (CD) ad libitum or HFD plus 0.1%, 5%, 10% or 25.5% carbs for 5 weeks. (A–B) Food and calorie intakes were recorded weekly. The ratio of food calories over body weight daily was calculated. *: P<0.05 CD vs. either HFD +5% carbs or HFD +10% carbs. **: P<0.01 vs. CD. ##: P<0.01 vs. HFD +0.1% carbs. $$: P<0.01 HFD +5% carbs vs. HFD +0.1% or HFD +25.5% carbs. &: P<0.05 HFD +10% carbs vs. HFD +25.5% carbs. (C) Body weight was measured weekly. *: P<0.05 vs. CD. **: P<0.01 vs. CD. #: P<0.05 vs. HFD +0.1% carbs. ##: P<0.01 vs. HFD +0.1% carbs. $: P<0.05 HFD +5% carbs vs. HFD +25.5% carbs. $$: P<0.01 HFD +5% carbs vs. HFD +25.5% carbs. &: P<0.05 HFD +10% carbs vs. HFD +25.5% carbs. (D) Epididymis fat pads were collected and weighed. The ratio of epididymis fat over body weight was calculated. Results represent mean ± SD of 10 mice/group. **: P<0.01 vs. CD. ##: P<0.01 vs. HFD +0.1% carbs. $$: P<0.01 vs. HFD +5% carbs. ++: P<0.01 vs. HFD +10% carbs.
Figure 2.
Dietary carb is not necessary for the HFD-induced insulin resistance but a small amount of it can induce a maximal level of insulin resistance in mice on HFD.
(A) Fasting blood glucose was measured once a week after an 8-hour fast. (B) Plasma level of insulin was evaluated at the end of the 5-week experiment after an 8 hour fast. (C–D) Insulin tolerance test (ITT) was performed at the end of the 5-week experiment after an 8 hour fast and the area under curve was calculated. Results represent mean ± SD of 8 mice/group. *: P<0.05 vs. CD. **: P<0.01 vs. CD. #: P<0.05 vs. HFD +0.1% carbs. ##: P<0.01 vs. HFD +0.1% carbs. $$: P<0.01 vs. HFD +5% carbs. ++: P<0.01 vs. HFD +10% carbs.
Figure 3.
Dietary carb is not necessary for the HFD-induced insulin resistance.
At the end of the 5-week experiment, animals were fasted for 8 h. Liver (A) and skeletal muscle samples (B) were collected promptly after animals were sacrificed. Levels of total and phosphorylated IRS1, total and phosphorylated Akt, total and phosphorylated mTOR, and β-actin were detected with immunoblotting. The level of each target protein was quantified and normalized to control. The average of 3 mice was set at 1. Results were presented as mean ± SD of 6 animals/group for Akt and 3 animals for IRS1. *: P<0.05 vs. no acute insulin treatment in control. **: P<0.01 vs. no acute insulin treatment in control.
Figure 4.
Fatty acid is essential for the development of insulin resistance induced by chronic exposure to insulin.
Hep1c1c7 (A) and differentiated C1C12 (B) cells were pretreated with TOFA (30.8 µM) for 50 min in media containing 25 mM glucose before the incubation with insulin (1 nM, chronic insulin treatment) as noted in the continuous presence or absence of TOFA for another 12 h. After an extensive washing with warm PBS, cells were exposed to insulin (5 nM) for 15 min (acute insulin treatment) as noted, followed by evaluations of phosphorylated and total target proteins by using immunoblotting and quantification. No sera were added to the media throughout the whole experiment. Results represent mean ± SE of 3 independent experiments. *: P<0.05 vs. no acute insulin treatment. **: P<0.01 vs. no acute insulin treatment.
Figure 5.
Dietary carb is not necessary for the HFD-induced ectopic fat accumulation in liver and skeletal muscle.
At the end of the 5-week experiment, animals were fasted for 8 h and liver and skeletal muscle samples were collected. Content of triglyceride (TG) in liver (A) and (B) gastrocnemius were quantified. The mRNA levels of key lipogenic genes (Srebp-1c, Srebp-2, and fatty acid synthase (Fas)) in liver (C–E) and gastrocnemius (F–H) were quantified by using Real Time RT/PCR. Results represent mean ± SD of 4 mice per group. *: P<0.05 vs. CD. #: P<0.05 vs. HFD +0.1% carbs. $: P<0.05 vs. HFD +5% carbs. +: P<0.05 vs. HFD +10% carbs.
Figure 6.
Dietary carb is not necessary for the HFD-induced oxidative stress.
Levels of GSH/GSSG ratio, MnSOD activity, malondialdehyde (MDA) in liver (A, C, E) and gastrocnemius (B, D, F) of the mice described in Fig. 1 were measured and normalized to protein levels of the same samples as detailed in “Methods and Materials”. Results represent results of mean ± SD of 10 animals per group. *: P<0.05 vs. CD. **: P<0.01 vs. CD. #: P<0.05 vs. HFD +0.1% carbs. ##: P<0.01 vs. HFD +0.1% carbs. $$: P<0.01 vs. HFD +5% carbs.
Figure 7.
Inhibition of hepatic gluconeogenesis prevented the HFD-induced insulin resistance.
(A) Levels of key hepatic gluconeogenic genes (PEPCK and glucose-6-phosphatase (G6P) were measured by using RT-PCR as detailed in “Methods and Materials”. Results represent mean ± SD of 4 mice per group. *: P<0.05 vs. CD. #: P<0.05 vs. HFD +0.1% carbs. (B) B6 mice were fed either the regular chow diet (CD) ad libitum or HFD plus 0.1% carbs for 5 weeks. Some mice were treated with adenoviral shRNA against p300 (109 pfu/mouse) or the control shRNA at the beginning of week 5. After an 8 h fast, IIT was performed. Protein levels of p300 in liver were verified by using immunoblotting. Results represent mean ± SD of 5 mice per group. *: P<0.05 vs. CD. #: P<0.05 vs. HFD + shRNAp300 or CD.