The Diurnal Variation in Mitochondrial Gene in Human Type 2 Diabetic Mesenchymal Stem Cell Grafts

Abstract

The application of regenerative therapy through stem cell transplantation has emerged as a promising avenue for the treatment of diabetes mellitus (DM). Transplanted tissue homeostasis is affected by disturbances in the clock genes of stem cells. The aim of this study is to investigate the diurnal variation in mitochondrial genes and function after transplantation of adipose-derived mesenchymal stem cells (T2DM-ADSCs) from type 2 diabetic patients into immunodeficient mice. Diurnal variation in mitochondrial genes was assessed by next-generation sequencing. As a result, the diurnal variation in mitochondrial genes showing troughs at ZT10 and ZT22 was observed in the group transplanted with adipose-derived mesenchymal stem cells derived from healthy individuals (N-ADSC). On the other hand, in the group transplanted with T2DM-ADSCs, diurnal variation indicative of troughs was observed at ZT18, with a large phase and amplitude deviation between the two groups. To evaluate the diurnal variation in mitochondrial function, we quantified mitochondrial DNA copy number using the Human mtDNA Monitoring Primer Set, measured mitochondrial membrane potential using JC-1, and evaluated mitophagy staining. The results showed a diurnal variation in mitochondrial DNA copy number, mitophagy, mitochondrial membrane potential, and NF-kB signaling in the N-ADSC transplant group. In contrast, no diurnal variation was observed in T2DM-ADSC transplants. The diurnal variation in mitochondrial function revealed in this study may be a new marker for the efficiency of T2DM-ADSC transplantation.

Keywords: NF-kB; diurnal variation; mesenchymal stem cells (MSC); mitochondria; mitophagy; type 2 diabetes mellitus.

Figure 1

Daily fluctuations in mitochondrial gene expressions. (a,b) Daily fluctuations in mitochondrial genes in adipose-derived mesenchymal stem cells derived from healthy individuals’ transplants. (c,d) Daily fluctuations in mitochondrial genes in adipose-derived mesenchymal stem cells derived from a patient with type 2 diabetes mellitus transplants. (a,c) Heat map of mitochondrial gene expression. (b,d) The 24-h sequential monitoring graph of mitochondrial gene expression. Abbreviations in figure: N-ADSC, adipose-derived mesenchymal stem cells derived from a healthy individual; T2DM-ADSC, adipose-derived mesenchymal stem cells derived from a patient with type 2 diabetes mellitus; ZT, Zeitgeber time; MT, mitochondrially encoded.

Figure 2

Daily fluctuations in the number of mitochondrial DNA copies. The number of mitochondrial DNA copies is represented by the mean of 2^ΔCt from the difference in the cycle threshold values of ND1/SLCO2B1 and ND5/SERPINA1. Light periods are indicated by the white column on the horizontal axis, and dark periods by the black column. **** p-values < 0.0001. *** p-values < 0.005. Data are shown as the mean ± SD; n = 6 for each sampling point, normally distributed; two-way ANOVA with Tukey–Kramer post hoc test.

Figure 3

Daily fluctuations in mitochondrial membrane potential. The difference in mitochondrial membrane potential is represented by the ratio of red fluorescence intensity to green fluorescence intensity. * p-values < 0.05. ** p-values < 0.01. **** p-values < 0.0001. Data are shown as the mean ± SD, n = 6 for each sampling point, normally distributed; two-way ANOVA with Tukey–Kramer post hoc test.

Figure 4

Daily fluctuations in mitophagy. Daily fluctuations in mitophagy, as displayed by the fluorescence intensity of the Mtphagy Dye that visualizes mitophagy. * p-values < 0.05. ** p-values < 0.01. Data are shown as the mean ± SD; n = 6 for each sampling point, normally distributed; two-way ANOVA with Tukey–Kramer post hoc test.

Figure 5

Daily fluctuations in nuclear factor-kB (NF-kB) signaling. (a) Schematic of the signaling pathway by next-generation sequencing. (b) Daily fluctuations in the NF-kB reporter activity. The NF-kB reporter activity is represented by absorbance levels measured using the NF-kB reporter assay. **** p-values < 0.0001. Data are shown as the mean ± SD; n = 6 for each sampling point, normally distributed; two-way ANOVA with Tukey–Kramer post hoc test.