Synchronized human skeletal myotubes of lean, obese and type 2 diabetic patients maintain circadian oscillation of clock genes

Abstract

Cell and animal studies have demonstrated that circadian rhythm is governed by autonomous rhythmicity of clock genes. Although disturbances in circadian rhythm have been implicated in metabolic disease development, it remains unknown whether muscle circadian rhythm is altered in human models of type 2 diabetes. Here we used human primary myotubes (HPM) to investigate if rhythmicity of clock- and metabolic gene expression is altered in donors with obesity or type 2 diabetes compared to metabolically healthy donors. HPM were obtained from skeletal muscle biopsies of four groups: type 2 diabetic patients and their BMI- and age-matched obese controls and from lean, healthy and young endurance trained athletes and their age-matched sedentary controls. HPM were differentiated for 7 days before synchronization by serum shock followed by gene expression profiling over the next 72 hours. HPM display robust circadian rhythms in clock genes, but REVERBA displayed dampened rhythmicity in type 2 diabetes. Furthermore, rhythmicity in NAMPT and SIRT1 expression was only observed in HPM from trained athletes. Rhythmicity in expression of key-regulators of carbohydrate and lipid metabolism was modest. We demonstrate that in human skeletal muscle REVERBA/B, NAMPT and SIRT1 circadian rhythms are affected in donors of sedentary life style and poor health status.

Figure 1. Rhythmic gene expression of core components of the molecular clock.

The relative transcript abundance of the positive (A) and negative limb (B) components of the molecular clock was quantified by RT-PCR and normalized to the corresponding geometric mean of RPL26, CYPB and GUSB. Each value consists of the average of the independent cultures from three different donors. The value of the sample taken immediately after serum shock (0 h) was normalized to 1. Expression profiles of synchronized differentiated primary myotube cultures from trained lean (black triangle), untrained lean (open diamond), obese (black circle) and type 2 diabetes patients (open circle) are shown. Data are mean ± SEM.

Figure 2. Rhythmic gene expression of molecular clock components.

The relative abundance of transcripts that are under the control of the Ebox domain was quantified by RT-PCR and normalized to the corresponding geometric mean of RPL26, CYPB and GUSB. Each value consists of the average of the independent cultures from three different donors. The value of the sample taken immediately after serum shock (0 h) was normalized to 1. Expression profiles of synchronized differentiated primary myotube cultures from trained lean (black triangle), untrained lean (open diamond), obese (black circle) and type 2 diabetes patients (open circle) are shown. Data are mean ± SEM.

Figure 3. Expression profiles of cellular glucose metabolism genes.

The relative abundance of transcripts that encode proteins of cellular glucose and glycogen metabolism was quantified by RT-PCR and normalized to the corresponding geometric mean of RPL26, CYPB and GUSB. Each value consists of the average of the independent cultures from three different donors. The value of the sample taken immediately after serum shock (0 h) was normalized to 1. Expression profiles of synchronized differentiated primary myotube cultures from trained lean (black triangle), untrained lean (open diamond), obese (black circle) and type 2 diabetes patients (open circle) are shown. Data are mean ± SEM.

Figure 4. Expression profiles of genes involved in mitochondrial metabolism.

The relative transcript abundance of transcripts that encode proteins involved in mitochondrial function and biogenesis was quantified by RT-PCR and normalized to the corresponding geometric mean of RPL26, CYPB and GUSB. Each value consists of the average of the independent cultures from three different donors. The value of the sample taken immediately after serum shock (0 h) was normalized to 1. Expression profiles of synchronized differentiated primary myotube cultures are plotted as follows: trained lean (black triangle) against untrained lean (open diamond) and obese (black circle) against type 2 diabetics (open circle). Data are mean ± SEM.

Figure 5. Correlative analysis of circadian rhythmicity with the metabolic phenotype of the donor.

The plot displays the association (Spearman’s Rho test for nominal data) between the metabolic phenotype of the donor with the amplitude of the JTK_CYCLE based peak and nadir for REVERBA. Data are relative expression amplitude with 95% CI as error bars.