In this report, we confirm that high-fat feeding in mice produces neuropathy and increased tissue and systemic oxidative stress prior to the development of frank diabetes. We postulated that these metabolic changes would generate oxLDLs and that these could contribute to neuronal injury mechanisms. We demonstrate that DRG neurons express the oxLDL receptor LOX-1 and that activation of this receptor in vitro leads to NAD(P)H oxidase activity that injures DRG neurons via generation of oxidative stress.
After 12 weeks on a high-fat diet, weight was modestly increased while fasting blood glucose remained normal. The hindpaw latency was increased and sciatic NCV decreased, consistent with peripheral neuropathy. Previous studies (14
) established that a high-fat diet leads to type 2 diabetes in mice. These studies focused on the mechanisms leading to insulin resistance, although one study (43
) examined the consequences of a high-fat diet on the onset of nephropathy and one (17
) on neuropathy. No previous study has shown that a high-fat (specifically high cholesterol) diet leads to NCV deficits and reduced hindpaw withdrawal response prior to impaired glucose tolerance. Our current data suggest that high-fat–induced neuronal deficits may precede the development of diabetes.
By 34 weeks on the high-fat diet, the mice displayed impaired glucose tolerance, a significant increase in plasma insulin levels, and an increase in GHb. These increases are small compared with mice with frank type 2 diabetes, where the plasma insulin may be elevated fourfold and GHb by several percent, but the extent of neuropathy is comparable (26
). This provides supporting evidence for a role for dyslipidemia in the development of neuropathy. Clinical studies support this finding. In the EURODIAB trial, of 1,200 subjects who did not have diabetic neuropathy at baseline, hypertension, serum lipids, and BMI were each independently associated with the risk of developing diabetic neuropathy during a 7-year follow-up period. Of these risk factors, dyslipidemia was closely linked with the onset and progression of diabetic neuropathy (44
). In parallel, we recently reported that dyslipidemia, not hyperglycemia, was more closely correlated with neuropathy progression in 427 trial participants (10
We demonstrate not only that the high-fat diet produced dyslipidemia, with high levels of HDL and LDL cholesterol and triglycerides, but also that LDLs and other circulating lipids and proteins are oxidized. Oxidatively modified proteins and lipids are toxic to complication-prone tissues, such as renal tubules (20
) and the retina (45
). In particular, oxLDLs mediate vascular injury via interaction with LOX-1 on endothelial cells (46
). This led us to postulate that oxLDLs may injure DRG neurons.
We report strong expression of LOX-1 on the adult DRG neurons by Western blotting and immunocytochemistry. Next, we characterized the effects of high glucose and oxLDLs on DRG neuron oxidative stress. Both insults lead to mitochondrial superoxide formation, the key mediator of DRG neuron injury in high glucose (1
). The effects of high glucose and oxLDLs on mitochondrial superoxide were additive. Furthermore, antioxidant potential is depleted over time by oxLDLs. Using a neutralizing antibody to LOX-1, we demonstrate that the effects of oxLDLs are largely mediated via the LOX-1 receptor. This protection has been shown for vascular injury in dyslipidemia (48
) but not previously for neurons. Our data suggest that multiple metabolic deficits in dyslipidemia and early diabetes combine to produce oxidative stress and accelerate the onset and progression of neuropathy. These data strongly argue for an antioxidant component to therapies for diabetic neuropathy (49
). Our work also raises the interesting question of how obesity, immobility, and the metabolic syndrome contribute to neuropathy.
High glucose or oxLDLs insults increased DRG neuron injury, evidenced by the activation of caspase 3 and by nuclear DNA degradation. The injury produced by either stressor was blocked by the antioxidant α-lipoic acid. These data support the consensus that pathways leading to cellular injury in diabetes (mainly shown for hyperglycemia to date) converge upon oxidative stress. Antioxidant therapies remain the most promising strategy for diabetic neuropathy, but clearly greater understanding of the long-term application of antioxidant therapy needs to be explored.
We also found that inhibition of NAD(P)H oxidase could block oxLDL-induced, but not high-glucose–induced, injury. Supporting evidence that oxLDLs led to NAD(P)H oxidase activity was obtained by observing subunit expression and localization and by measuring apocyanin-inhibitable NADPH oxidation. Thus, NAD(P)H oxidase activation and subsequent generation of superoxide is the primary injury mechanism in oxLDL-treated DRG neurons. This activation of NAD(P)H oxidase is mediated via LOX-1 signaling, since blocking LOX-1 abrogates oxLDL-induced DRG neuron injury. Our data agree with studies showing LOX-1–mediated activation of NAD(P)H oxidase in vascular endothelial cells through recruitment of NAD(P)H oxidase subunits (50
). Collectively, these data demonstrate a pivotal role for NAD(P)H oxidase in the injury of DRG neurons and the progression of neuropathy (51
This study demonstrates a mechanism by which dyslipidemia produces DRG neuron injury that may underlie emerging clinical evidence that dyslipidemia is an independent risk factor for diabetic neuropathy. The data suggest why glycemic control alone is insufficient to prevent complications in type 2 diabetes and argue for combination therapies targeting multiple metabolic imbalances and receptor-mediated signaling that leads to oxidative injury. Prevention of oxLDL formation may be one strategy, but targeting oxLDL receptors may be an important alternative approach.