Long-lasting effect of obesity on skeletal muscle transcriptome

Abstract

Background: Reduced physical activity and increased intake of calorically-dense diets are the main risk factors for obesity, glucose intolerance, and type 2 diabetes. Chronic overnutrition and hyperglycemia can alter gene expression, contributing to long-term obesity complications. While caloric restriction can reduce obesity and glucose intolerance, it is currently unknown whether it can effectively reprogram transcriptome to a pre-obesity level. The present study addressed this question by the preliminary examination of the transcriptional dynamics in skeletal muscle after exposure to overnutrition and following caloric restriction.

Results: Six male rhesus macaques of 12-13 years of age consumed a high-fat western-style diet for 6 months and then were calorically restricted for 4 months without exercise. Skeletal muscle biopsies were subjected to longitudinal gene expression analysis using next-generation whole-genome RNA sequencing. In spite of significant weight loss and normalized insulin sensitivity, the majority of WSD-induced (n = 457) and WSD-suppressed (n = 47) genes remained significantly dysregulated after caloric restriction (FDR ≤0.05). The Metacore^TM pathway analysis reveals that western-style diet induced the sustained activation of the transforming growth factor-β gene network, associated with extracellular matrix remodeling, and the downregulation of genes involved in muscle structure development and nutritional processes.

Conclusions: Western-style diet, in the absence of exercise, induced skeletal muscle transcriptional programing, which persisted even after insulin resistance and glucose intolerance were completely reversed with caloric restriction.

Keywords: Caloric restriction; High-fat diet; Insulin resistance; Obesity; Skeletal muscle.

Fig. 1

WSD-induced weight gain and insulin resistance are reversed by CR. a Study design. Animals were maintained in individual housing while consuming chow diet for the first 2 months, followed by Western-style diet (WSD) for 6 months, and caloric restriction (CR) on chow for 4 months. Experimental procedures (DEXA, GTT and muscle biopsies) were performed at the end of each dietary period. Body weight (b), total body fat (c), and lean mass (d) were determined by DEXA, and AUC glucose (e), AUC insulin (f), fasting glucose (g) and fasting insulin (h) were determined by GTT, as described in “Materials and Methods”. HOMA-IR (i) was calculated as described [92]. HbA1c (j) was determined during GTT. Error bars are Means ± SEM, n = 6. *p < 0.05, **p < 0.01 by repeated-measure one-way ANOVA

Fig. 2

WSD induces sustained alterations in skeletal muscle transcription. Soleus muscle biopsies were collected longitudinally, before and after exposure to the WSD and after CR, and then subjected to RNAseq gene expression analysis, as described in “Materials and Methods”. DEGs were identified using three independent comparisons: a–c WSD vs chow (WSD/CHOW); e CR vs WSD (CR/WSD); and a, b and d CR vs chow (CR/CHOW). a The number of upregulated (brown) and downregulated (blue) genes from WSD/CHOW and CR/CHOW categories in rhesus macaque genome. b Venn diagram shows an overlap between WSD/CHOW and CR/CHOW genes. Heat maps of top DEGs during the transition from chow to WSD (c), CR vs chow (d), and CR vs. WSD (e) Each column represents an individual animal

Fig. 3

TGFβ pathway genes remain upregulated after CR. a Functional enrichment of DEGs with the highest FDR values common for WSD/CHOW and CR/CHOW categories. Heat maps of top DEGs involved in ECM organization (b) and cell adhesion (c). Each column represents an individual animal. d Anatomical structure development common gene network indicates upregulated (red) and downregulated (blue) DEGs. Positive and negative interactions between genes are represented by green and red arrows, respectively. Cellular compartmentalization of gene products is indicated

Fig. 4

CR-specific gene regulation. a Functional enrichment of CR/CHOW-specific DEGs with the highest FDR values that excludes DEGs present in WSD/CHOW and CR/WSD categories. Heat maps of top CR-specific DEGs involved in muscle structural development (b) and nutritional processes (c). Each column represents an individual animal. d CR-specific regulation and development gene network indicates upregulated (red) and downregulated (blue) DEGs. Positive and negative interactions between genes are represented by green and red arrows, respectively. Cellular compartmentalization of gene products is indicated

Fig. 5

qRT-PCR validation of RNAseq analysis of gene expression. Changes in SM mRNA levels were determined as described in “Materials and Methods”. Graphs represent logarithm mean fold changes for WSD Ct values normalized to CHOW (filled bars) and CR Ct values normalized to CHOW (open bars). Error bars are Means ± SEM, n = 5. *p < 0.05, **p < 0.01 by repeated-measure one-way ANOVA