Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy

Abstract

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit(+)Sca-1(+)lineage(-) (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.

Keywords: Cell–cell fusion; Hematopoietic stem cell; Hyperglycemia; Neuropathy; Stem cell abnormalities.

Fig. 1

Electrophysiological tests and immunofluorescent overlap analysis of bone marrow-derived cells (BMDCs) and DRG neurons in control (Ctrl) and in STZ and high fat diet (HFD) diabetic mice. (A) Relative ratio of sensory nerve conduction velocity in Ctrl (n = 6) compared to STZ (n = 10) and Ctrl (n = 5) compared to HFD (n = 6) mice. Data are means ± S.E. **P < 0.01. (B) Immunofluorescent co-staining of MAP2 (Red), Insulin (Red) or TNF-α (Red) with GFP (Green) and Nuclei (Blue) were carried out in DRG tissues. Scale bars = 20 μm.

Fig. 2

Immunohistochemical analysis of insulin- and TNF-α-positive cells in the bone marrow. (A) Immunohistochemical staining of insulin- and TNF-α-positive cells in the bone marrow. Arrows indicate positive staining for insulin or TNF-α. Scale bars = 10 μm. (B) Immunofluorescent overlap staining of TNF-α (Red)/Insulin (Green)/Nuclei (Blue) of bone marrow cells. Double-positive cells expressing both TNF-α and Insulin are shown surrounded with an interrupted line. Scale bars = 5 μm.

Fig. 3

The characteristics of BMDCs and their gene expression of insulin and TNF-α in Ctrl and in STZ and HFD mice. (A) Bands show the RT-PCR products of the insulin 1 gene from the hematopoietic cell lineage fraction. Beta-actin was used as the internal control. TBM, total bone marrow cells; Mono, mononuclear cells; Gra, granulocytes; Lin (−), lineage negative cells; KSL, KSL cells. (B) Quantitative real-time PCR of TNF-α mRNA expression in KSL cells. The value for the reference RNAs is arbitrarily assigned a value of 1.0 in Ctrl mice. Expression is shown as a standardized quotient divided by the value simultaneously obtained for beta-actin. Data are means ± S.E. *P < 0.05, **P < 0.01; Ctrl: n = 13, STZ: n = 13, HFD: n = 5. C: FACS analysis of KSL cells. The insulin-positive fraction of KSL cells (top panels). The arrow in the plot area indicates the population of insulin-positive cells. The TNF-α positive fraction of KSL cells (bottom panels). The arrow in the plot area indicates TNF-α positive cells. (D) The percentages of insulin- or TNF-α positive cells of KSL cells in Ctrl and STZ and HFD mice. For insulin analysis Ctrl (n = 8), STZ (n = 5) and HFD (n = 7). For TNF-α analysis Ctrl (n = 9), STZ (n = 9) and HFD (n = 3).

Fig. 4

Immunofluorescent co-staining of DRG neurons in the KSL-BMT study. Immunofluorescent co-staining of MAP2 (Red), Insulin (Red) or TNF-α (Red) with GFP (KSL, Green) and Nuclei (Blue) was performed in DRG neurons in non-diabetic mice after BMT with KSL cells from control (Ctrl → KSL-BMT) or STZ (STZ → KSL-BMT) mice. Scale bars = 20 μm.