How the metabolic dysregulation caused by insulin resistance leads to local nerve fiber damage in DN is unclear. In this study, we performed global gene expression profiling of sciatic nerve from 24-week-old db/db mice, which exhibit hyperglycemia, dyslipidemia, and DN typical of type 2 diabetes. We detected alterations in the expression of genes involved in carbohydrate and lipid metabolism, lipid transport, stress responses, and apoptosis. Furthermore, promoter sequences of coexpressed lipid metabolism–related DEGs exhibited significantly overrepresented TFBS motifs and are likely to share a common regulatory mechanism relevant to the development of DN.
Morphological and electrophysiological changes in the BKS db/db
mice closely mimic the changes observed in DN patients (8
). However, the db/db
mouse model does not fully duplicate human DN pathology; not all structural changes observed in human DN are seen in the db/db
). Duration of diabetes in mice is likely not long enough for the development of severe long-term degeneration associated with human DN. The development of hyperglycemia and DN in the BKS db/db
mice are considered to be consequences of hyperphagia and insulin resistance resulting from impaired leptin signaling in the hypothalamus (8
). However, the role of leptin signaling in peripheral nerve is not understood, and whether leptin receptor mutation and obesity have a compounding effect on the development of DN in these mice is not known.
Schwann cells (SC) are major contributors to the mRNA in the sciatic nerve biopsies, with a small contribution coming from axons, epineural fibroblasts, adipocytes, and vascular endothelial cells (30
). Our analyses revealed that genes encoding myelin structural proteins (P0, Pmp22, Connexin-32, Mag, E-Cadherin, and Periaxin), myelin structural lipid synthesis genes (Plp
, and Apolipoprotein D
), as well as Sox10, a TF required for myelination, are downregulated in db/db
nerve. Segmental demyelination has been observed in human DN (31
); mouse studies, however, do not report evidence of demyelination (27
). Downregulation of myelin protein-encoding genes and myelin lipid synthesis genes may represent SC abnormalities preceding the structural changes.
The TFs c-Jun and Krox-24, and other genes expressed by denervated SC to promote axonal regeneration (GAP-43
, and L1
), are downregulated, as are positive regulators of axonogenesis (Cdh4, Nefl, and Mapt) and axon guidance pathway genes (Netrin G1
), suggesting a lack of axonal regeneration. These changes are consistent with the axonal degeneration observed in morphological studies in the db/db
Loss of neurotrophic signal in DN may impair nerve generation and cause dying back of axons (3
). Neurotrophin Ngf; NT-3 receptor TrkC; Gdnf receptors Gfra1, Gfra3, and Gfra4; neuregulin receptor Erbb; and neurotrophin signaling pathway genes (Akt3
) are downregulated, indicating impaired neurotrophic support in the db/db
nerve. Downregulation of Ngf, TrkC, and GFRα1 is consistent with the results of studies in diabetic rats (35
). Other neurotrophins are not differentially regulated in our data. Some studies in rats with streptozotocin-induced diabetes report reduced expression of Bdnf, NT-3, NT-4/5, Igf-I, Igf-II, Cntf, Gdnf, and neurotrophin receptors p75LNGFR
and TrkB approximately 12 weeks after the induction of diabetes (35
), whereas others report no change in NT-3 expression in rat sciatic nerve (41
). The difference in the animal models used in the studies (rat model of type 1 diabetes vs. mouse model of type 2 diabetes) may explain this discrepancy.
The SC marker S100β as well as antiapoptotic genes (Mapk8ip1
) are downregulated, whereas proapoptotic genes (Fas
, and Brca1
) are upregulated in the db/db
samples (Supplementary Table 2
). The downregulation of SC marker and antiapoptotic genes along with upregulation of proapoptotic genes suggests SC apoptosis in db/db
nerve; however, no studies to date have demonstrated SC death in DN.
The causes of axonal and SC degradation in DN are not well understood, but several hypotheses have been developed in regard to the pathogenesis of the nerve injury (3
). Our analyses indicate increased glucose, energy, and lipid metabolism in the db/db
nerve and support the roles of hyperglycemia- and hyperlipidemia-induced oxidative stress, inflammatory response, and vascular ischemia in DN. In addition, our data indicate impaired axonal transport, neurotrophic signal, cell adhesion, and cell communication. In this study, we focused on hyperglycemia- and dyslipidemia-related gene expression changes in the nerve.
Hyperglycemia is a major factor in the development and progression of DN; current hypotheses suggest the effect of hyperglycemia is likely to be vascular, metabolic, or a combination of both (34
). The metabolic hypothesis of axonal and SC damage in diabetes suggests that activation of glucose metabolism pathways in hyperglycemia results in oxidative stress. In our data, glycolysis, TCA, and oxidative phosphorylation genes are upregulated (Supplementary Table 2
), suggesting activation of glucose and energy metabolism in the db/db
nerve. Upregulation of oxidative phosphorylation genes and downregulation of mitochondrial H+
transporting ATP synthases in the db/db
nerve suggest increased superoxide production (43
). The mitochondrial proton carrier Ucp2, known to be upregulated in response to elevated reactive oxygen species (ROSs) (44
), is highly upregulated; the oxidative stress-induced growth inhibitor Osgin1 is also upregulated in the db/db
nerve. Upregulation of antioxidant genes (Sod2
, and Catalase
) suggests cellular response to increased ROS production. Edwards et al. (3
) hypothesized that increased cellular oxidative stress results in activation of the Poly (ADP-ribose) polymerase (PARP) pathway, which in turn induces inflammatory responses in the nerve. Upregulation of PARP, inflammatory response, and MAPK signaling pathway genes in the db/db
nerve support the PARP pathway–mediated inflammatory response hypothesis. Nuclear factor-κB (NF-κB) induced–inflammatory response is also implicated in demyelination and axon degeneration (3
); however, NF-κB and genes regulated by NF-κB, such as iNOS
, are not differentially regulated in our data.
The vascular hypothesis of nerve damage in diabetes suggests that activation of glucose metabolism pathways causes functional and structural changes in the neuronal vasculature leading to endothelial hypoxia and axonal ischemia (45
). Bradley et al. (46
) noted thickening of perineurial cell basal lamina and increased endoneurial collagenization around SC in the sural nerve of DN patients. Activation of protein kinase C by hyperglycemia induces expression of vascular endothelial growth factor (VEGF)—an angiogenic gene and permeability factor—inhibits production of nitric oxide and alters Na+
-ATPase activity. Activation of glucose metabolism pathways induces TGF-β1 and Serpine1 expression, which results in endothelial fibrosis and thickening of vascular membranes (3
). VEGF-C, TGF-β1, and Serpine1 are upregulated; Na+
-ATPases (Atp1b2 and Atp1b3) are downregulated in the db/db
nerve. Upregulation of hypoxia-inducible factor Hif1a may indicate cellular response to ischemia (47
). These changes in gene expression support the hypothesis of neuronal ischemia and hypoxia. Imbalance in expression of nitric oxide synthase (Nos) isoforms has been implicated in diabetic endothelial dysfunction (48
); however, these are not differentially expressed in our data.
Dysregulation of lipid metabolism is implicated in peripheral neuropathy (7
), but the mechanism of its effect is not well understood. We observed upregulation of genes expressed by adipocytes (Adiponectin
, and Lpl
), possibly due to increased amounts of adipose tissue in the epineurium of the db/db
nerve. Verheijen et al. (30
) demonstrated that epineurium of adult mouse sciatic nerve contains adipose-like tissue, and both endoneurium and epineurium express lipid metabolism genes; they suggested that adipocytokine signaling from endoneurial adipocytes may regulate lipid metabolism in SC and axons. Our analysis indicates upregulation of fatty acid metabolism, glycerolipid metabolism, lipid transport, PPAR signaling, and adipocytokine signaling genes in the db/db
nerve (Supplementary Table 2
Vincent et al. (7
) hypothesized that increased production of ROS in hyperglycemia results in lipid peroxidation and that the formation of oxidized low density lipoproteins (oxLDL) contributes to the nerve injury in diabetic dyslipidemia. The apoptotic effect of oxLDL on vascular endothelial cells and neurons is mediated via oxLDL receptor (Olr1/LOX-1), which is induced by hyperglycemia, ROS, oxLDL, and TNF-α. In vitro studies demonstrate that binding of oxLDL to Olr1 leads to NADPH oxidase (Nox) activation, mitochondrial superoxide production, and neuronal oxidative stress in dorsal root ganglia of mice (50
). oxLDL receptor Olr1 is highly overexpressed and Nox2 is upregulated in our data, supporting the hypothesis of oxLDL-induced nerve injury in dyslipidemia, possibly via activation of Nox. oxLDL also acts on macrophages via scavenger receptor (Scarb1/CD36) and triggers immune responses via MAPK and NF-κB signaling pathways (7
). In our data, CD36 and MAPK signaling pathway genes (TNF-α
, and TGF-β1
) are upregulated, supporting the hypothesis of CD36-mediated inflammatory response in the db/db
Coexpression of genes suggests shared mechanism of regulation. DEGs associated with “lipid metabolism,” “mitochondrion,” and “axonogenesis” clustered together based on their correlated expression pattern (). Most of the lipid metabolism– and mitochondria-associated genes in the cluster are upregulated, whereas axonogenesis genes are downregulated. To investigate the potential coregulation mechanism, we analyzed promoter sequences of 22 DEGs from this cluster annotated with the term “lipid metabolism”; 5 of these genes are also annotated with “mitochondrion.”
It is interesting that TFs binding to the overrepresented TFBSs in promoter sequences of lipid metabolism genes are associated with neuron differentiation, axon guidance, and immune response, suggesting a possible common regulatory mechanism in lipid metabolism, stress response, and axonal degeneration. TFs annotated with “nervous system development”–related terms are downregulated in the db/db
nerve. The HOXF family, the HBOX family, and the LHXF family TFs are annotated with “motor axon guidance” and “regulation of neuron differentiation.” HOMF family TF Msx1 is annotated with “negative regulation of vascular endothelial growth factor receptor signaling pathway” and “neuron differentiation.” “Immune response”–associated ETSF family TFs are upregulated in the db/db
nerve (Supplementary Table 4
). OCT1 family TF Oct-6, an SC TF important in myelination (30
), is not among the DEGs.
In conclusion, gene expression changes suggest roles of multiple etiological factors in the development of DN. The changes are consistent with pathological characteristics of DN, such as axonal degeneration and potential loss of neurotrophic signal. Our findings support the role of hyperglycemia-induced oxidative stress and ischemia in nerve injury. Our results also support the hypothesis of oxidized lipid–mediated nerve injury and increased mitochondrial oxidative stress in dyslipidemia. In addition, our analyses revealed possible coregulation of lipid metabolism, stress response, and axonal degeneration genes and identified TFs that may modulate this coregulation. Further investigation into the signal mediated by these TFs is likely to provide more insight into dyslipidemia-induced nerve injury.