A High-Protein Diet Reduces Weight Gain, Decreases Food Intake, Decreases Liver Fat Deposition, and Improves Markers of Muscle Metabolism in Obese Zucker Rats

Abstract

A primary factor in controlling and preventing obesity is through dietary manipulation. Diets higher in protein have been shown to improve body composition and metabolic health during weight loss. The objective of this study was to examine the effects of a high-protein diet versus a moderate-protein diet on muscle, liver and fat metabolism and glucose regulation using the obese Zucker rat. Twelve-week old, male, Zucker (fa/fa) and lean control (Fa/fa) rats were randomly assigned to either a high-protein (40% energy) or moderate-protein (20% energy) diet for 12 weeks, with a total of four groups: lean 20% protein (L20; n = 8), lean 40% protein (L40; n = 10), obese 20% protein (O20; n = 8), and obese 40% protein (O40; n = 10). At the end of 12 weeks, animals were fasted and euthanized. There was no difference in food intake between L20 and L40. O40 rats gained less weight and had lower food intake (p < 0.05) compared to O20. O40 rats had lower liver weight (p < 0.05) compared to O20. However, O40 rats had higher orexin (p < 0.05) levels compared to L20, L40 and O20. Rats in the L40 and O40 groups had less liver and muscle lipid deposition compared to L20 and L40 diet rats, respectively. O40 had decreased skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1) phosphorylation and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to O20 (p < 0.05), with no difference in 5' AMP-activated protein kinase (AMPK), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), protein kinase B (Akt) or p70 ribosomal S6 kinase (p70S6K) phosphorylation. The data suggest that high-protein diets have the potential to reduce weight gain and alter metabolism, possibly through regulation of an mTORC1-dependent pathway in skeletal muscle.

Keywords: body composition; diabetes; diet; liver; muscle; obesity; protein.

Figure 1

Body weight, food intake and body composition following 12 weeks of consuming either a 20% protein or 40% protein diet in lean control (L) or obese Zucker (O) rats. (A) Body weight gain over time; (B) Total weight gain over the 12-week diet intervention; (C) Average daily food consumption; (D) Muscle weight is a combination of gastrocnemius, soleus and plantaris muscles from the right hindlimb; (E) Liver weight; (F) Epididymal fat pad weight. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein.

Figure 1

Body weight, food intake and body composition following 12 weeks of consuming either a 20% protein or 40% protein diet in lean control (L) or obese Zucker (O) rats. (A) Body weight gain over time; (B) Total weight gain over the 12-week diet intervention; (C) Average daily food consumption; (D) Muscle weight is a combination of gastrocnemius, soleus and plantaris muscles from the right hindlimb; (E) Liver weight; (F) Epididymal fat pad weight. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein.

Figure 2

Biomarkers of type 2 diabetes following 12 weeks of consuming either a 20% protein or 40% protein diet in lean control (L) or obese Zucker (O) rats. (A) Fasting glucose; (B) Fasting insulin; (C) Fasting glucagon; (D) Fasting cholesterol; (E) Fasting triglycerides; (F) Fasting free fatty acids; (G) Fasting leptin; (H) Fasting FGF-21; (I) Fasting orexin. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. FGF-21, fibroblast growth factor-21; L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein.

Figure 3

Effects of dietary protein concentration on gene expression in skeletal muscle, liver and epididymal adipose tissue following 12 weeks of consuming either a 20% protein or 40% protein diet in lean control (L) or obese Zucker (O) rats. Relative expressions of target genes were determined using the 2-ΔΔCt method. All genes are expressed relative to 18S, the control gene. (A) Fatty acid synthase (FAS)expression in skeletal muscle; (B) FAS expression in liver; (C) FAS expression in adipose tissue; (D) Sirtuin 1 (SIRT1) expression in skeletal muscle; (E) SIRT1 expression in liver; (F) SIRT1 expression in adipose tissue; (G) Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression in skeletal muscle; (H) PGC1α expression in liver; (I) PGC1α expression in adipose tissue; (J) Peroxisome proliferator-activated receptor-γ (PPARγ) expression in skeletal muscle; (K) PPARγ expression in liver; (L) PPARγ expression in adipose tissue. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. Values with an * were analyzed via t-test and are significantly different within a group (20% versus 40% protein), p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein.

Figure 4

Representative skeletal muscle (gastrocnemius; 8 µm thickness and 200× magnification; 4.17 pixels/um) and liver sections (12 µm thickness and 200× magnification; 4.17 pixels/um) were obtained from lean control (L) or obese Zucker (O) rats following 12 weeks of consuming either a 20% protein or 40% protein diet and stained for fat deposition using Oil Red O staining. (A) Skeletal muscle lipid deposition, scale indicates 0.08 mm; (B) Quantification of lipid deposition in skeletal muscle, scale indicates 0.08 mm; (C) Liver lipid deposition; (D) Quantification of lipid deposition in liver. Obese rats had higher fat deposition in muscle and liver compared to lean rats. Rats receiving the 40% protein diet tended to have less fat deposition than those fed the 20% protein diet. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein.

Figure 5

Changes in mTORC1 signaling in gastrocnemius muscle following 12-weeks of consuming either a 20% protein (n = 8 per group) or 40% protein diet (n = 10 per group) in lean control (L) or obese Zucker (O) rats. Representative blots are shown, all bands are from the same blot. (A) Mammalian target of rapamycin complex 1 (mTORC1) phosphorylation (S2448) over total protein; (B) 5′ AMP-activated protein kinase (AMPK) phosphorylation (T172) over total protein; (C) Eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) phosphorylation (S65) over total protein; (D) Protein kinase B (Akt) phosphorylation (S473) over total protein; (E) Ribosomal protein S6 kinase 1 (P70) phosphorylation (T389) over total protein. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein; P/T, phosphorylated/total protein.

Figure 6

Changes in mammalian target of rapamycin complex 1 (mTORC1) signaling in liver following 12 weeks of consuming either a 20% protein (n = 8 per group) or 40% protein diet (n = 10 per group) in lean control (L) or obese Zucker (O) rats. Representative blots are shown, all bands are from the same blot. (A) Mammalian target of rapamycin complex 1 (mTORC1) phosphorylation (S2448) over total protein; (B) 5′ AMP-activated protein kinase (AMPK) phosphorylation (T172) over total protein; (C) Eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) phosphorylation (S65) over total protein; (D) Protein kinase B (Akt) phosphorylation (S473) over total protein; (E) Ribosomal protein S6 kinase 1 (P70) phosphorylation (T389) over total protein. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. Values with an * were analyzed via t-test and are significantly different within a group (20% versus 40% protein), p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein; P/T, phosphorylated/total protein.

Figure 7

Changes in mammalian target of rapamycin complex 1 (mTORC1) signaling in epididymal adipose tissue following 12-weeks of consuming either a 20% protein (n = 8 per group) or 40% protein diet (n = 10 per group) in lean control (L) or obese Zucker (O) rats. Representative blots are shown, all bands are from the same blot. (A) Mammalian target of rapamycin complex 1 (mTORC1) phosphorylation (S2448) over total protein; (B) 5′ AMP-activated protein kinase (AMPK) phosphorylation (T172) over total protein; (C) Eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) phosphorylation (S65) over total protein; (D) Protein kinase B (Akt) phosphorylation (S473) over total protein; (E) Ribosomal protein S6 kinase 1 (P70) phosphorylation (T389) over total protein. Values are means + SEM. Data was analyzed using one-way ANOVA. Values without a common letter differ, p < 0.05. L20, lean 20% protein; L40, lean 40% protein; O20, obese 20% protein; O40, obese 40% protein; P/T, phosphorylated/total protein. Figure without boxes separating treatment groups can be found in Supplementary Data Figure S2.