The importance of glycemic control in patients with diabetes is well known. But neurological studies suggest that dyslipidemia is actually a more significant contributor to the development of peripheral neuropathy in the same patient population.
by Andrea M. Vincent, PhD, Lucy M. Hinder, PhD, and Eva L. Feldman, MD, PhD
Diabetes is defined by the inability to regulate the levels of blood glucose and results from either a lack of insulin (type 1) or failure to respond to insulin (type 2). Over time, episodes of, or prolonged, hyperglycemia causes the severe complications of eye, kidney, cardiovascular disease and foot problems.1 Most foot problems are related to diabetic peripheral neuropathy2 and result from a loss of sensation and reduced blood flow. These factors lead to recurrent infections, ulcers and amputations, and Charcot neuroarthropathy.3 It is estimated that more than 15% of patients with diabetes will develop at least one foot ulcer,4 and one recent study observed an annual incidence of nearly 2%.5 A significant number of neuropathic diabetic foot ulcers are accompanied by cellulitis or osteomyelitis (around 15%), and these conditions contribute to the annual incidence of lower extremity amputation in patients with diabetes, which has been estimated to be 0.6%.6 In fact, diabetes is the leading cause of non-traumatic lower extremity amputations.
Hyperglycemia and complications
There is a clear correlation between glucose control and the development of complications in type 1 diabetes.7,8 However, diabetes complications continue to occur, even in well-controlled patients. Furthermore, this close correlation between glycemia and complications is less clear-cut for type 2 patients, who account for around 95% of cases. Clinical effort is focused on regulating blood glucose levels, but the rates of foot disease remain far too high.
Researchers in our laboratory and other diabetes centers have demonstrated that acute hyperglycemia produces several effects on peripheral sensory neurons that lead to oxidative stress, decreased function, and neuronal cell death.9-13 Several mechanisms subsequent to hyperglycemia all culminate in this oxidative injury, including acute mitochondrial injury.13-15 In brief, mitochondria are the cellular center for energy metabolism, and excess energy substrates in diabetes lead to inefficient electron transfer that damages the mitochondrial electron transfer components. A second mechanism is that excess glucose produces non-enzymic glycosylation and glycation of proteins to form advanced glycation end-products (AGE).16 AGE accumulation, a normal part of aging that is accelerated in diabetes, can produce long-lived protein modifications that both alter their functions and activate the cellular receptor known as RAGE.17 RAGE signaling leads to activation of NAD(P)H oxidase that generates superoxide and produces cellular oxidative stress.18 Finally, excess glucose is shunted through alternate metabolic pathways such as the aldose reductase, hexose, and lactate pathways, all of which alter redox balance and deplete cellular antioxidant capacity.19,20 These findings provide a clear rationale for continuing to focus on hyperglycemia as a primary target for the prevention of diabetes complications, but there are additional metabolic parameters that require attention.
Dyslipidemia and diabetes
Recent clinical data have added a new perspective to the care of patients with diabetes. A number of long-term studies of large cohorts of patients with type 1 diabetes have been completed. In these studies differing degrees of glycemia control were achieved. However, over the course of seven or more years of follow-up, it is not glycemia but hypertension, serum lipids, and body mass index that are independently associated with the risk of developing diabetic neuropathy.21-26 In our own study of type 1 diabetic patients, we examined the progression of neuropathy in sural nerve biopsies over a period of two years.27 The only clinical parameter in these patients that correlated with rapid progression of diabetic neuropathy was elevated plasma triglycerides. In case studies in the absence of diabetes, hypertriglyceridemia has been associated with subclinical peripheral neuropathy28 that could underlie accelerated problems with added hyperglycemia. Dyslipidemia develops later in the course of type 1 than in type 2 diabetes, and these abnormal lipid profiles coincide with the delayed onset and progression of diabetic neuropathy.29,30 Because type 2 diabetes is already associated with disorders of lipid metabolism as well as glycemia, these findings help to explain why glycemic control is less well correlated with neuropathy in the patients with type 2 diabetes, as a high number of cases of peripheral neuropathy (as many as 10% to 20% of patients31) present at the time of diagnosis of diabetes. All of these clinical findings, in addition to further data in type 2 studies,23,32 have led us to develop new hypotheses for the role of dyslipidemia in the development of diabetic neuropathy.
Our primary hypothesis is that increased levels of plasma lipids, either causative of or subsequent to the development of diabetes, act in concert with both oxidative stress and glucose to produce peripheral sensory neuron and microvessel injury leading to diabetic neuropathy. Initially, we tested this hypothesis by feeding mice a high fat diet and assessing the onset of diabetes and behavioral and nerve conduction measures of neuropathy.33 After 12 weeks on a high fat diet, when there was not yet an increase in fasting blood glucose, the mice had decreased sural nerve conduction velocities and slower foot withdrawal in response to a heat stimulus compared with littermates on a control diet.
Dyslipidemia and neuropathy in mice
Metabolic profiling in the high fat fed mice revealed an increase in plasma low density lipoproteins (LDL) carrying increased cholesterol and triglycerides.33 Furthermore, these LDLs were highly oxidized. This led us to a more mechanistic hypothesis that these oxLDLs that carry lipids to the periphery could be one factor that acts in concert with glucose to increase sensory nerve injury. We demonstrated that oxLDL enters the dorsal root ganglia, the tissues containing the cell bodies for peripheral sensory neurons, and binds to a cell surface receptor on the neurons known as LOX-1. This receptor is linked to an intracellular signaling pathway that leads to the activation of the previously mentioned enzyme NAD(P)H oxidase, which is associated with the generation of superoxide and oxidative stress.34 Activation of NAD(P)H oxidase also occurs downstream of intracellular hyperglycemia and the activation of the AGE receptor RAGE, suggesting that NAD(P)H oxidase is a common pathway for cellular injury in diabetes and dyslipidemia.16,18 These same mechanisms also occur in the blood microvessels, leading to multiple neurovascular targets for oxidative stress mediated injury.35 The interplay between these pathways progresses even further because LOX-1 is a multiligand receptor that also binds AGE.36
Mechanisms of dyslipidemia-induced injury
To further characterize how oxLDL lead to dorsal root ganglia neuron injury, we are examining the effects of oxLDL exposure on dorsal root ganglia neuron mitochondrial activity and injury in basal and high glucose. Both oxLDL and glucose are taken up by the neurons, providing an abundance of metabolic substrates. The resulting additive effects of these substrates are decreased mitochondrial efficiency and increased oxidative stress. In contrast to oxLDL, preliminary data from our laboratory suggest that non-oxidized LDL do not produce oxidative stress and injury in dorsal root ganglia neurons, despite the expression of the LDL receptor LDLR and LDL uptake.
Because myelin is a lipid-rich and dynamic component of nerves, we and others predict that dyslipidemia will have profound effects on myelin biology. Despite these hypotheses, few data are available. One study in mice homozygous for the autosomal recessive fatty liver dystrophy (fld) mutation demonstrated a peripheral neuropathy that appears related to dysmyelination.37 This defect persists in the mice, despite the fact that neonatal hypertriglyceridemia (but not hypercholesterolemia) resolves in the adult mice. We are exploring alterations in lipid metabolic proteins in the leptin receptor deficient db/db mice and have found alterations in Schwann cell lipid metabolism (Hinder et al, manuscript in preparation). We previously demonstrated that Schwann cells are relatively resistant to hyperglycemia-induced injury,38 however diabetic neuropathy is associated with demyelination and loss of large myelinated fibers.39 Further exploration of dyslipidemia-induced changes in Schwann cells and myelin biology is clearly warranted.
Therapeutic targets for diabetic neuropathy
Taken together, our data point to a number of important preventive and therapeutic strategies that should be considered in addition to the regulation of glycemia in patients with both type 1 and type 2 diabetes.
Lipid lowering. Plasma lipid profiling will inform clinicians of risk for neuropathy. This profiling is already routinely performed for screening for risk of cardiovascular disease,40,41 but closer attention to triglycerides will provide opportunities for early intervention in a subset of diabetic patients.27 In this regard, we have explored the effects of lipid lowering therapy on diabetic neuropathy in mice. In a model of streptozotocin-induced type 1 diabetes, we demonstrated some improvement in sural nerve conduction velocity and hind paw responses to heat stimulus in diabetic mice receiving fenofibrate when compared with untreated mice with diabetes (unpublished data). In addition to diet and exercise counseling, discussed below, statins are a popular choice for lipid lowering in diabetic patients.42 These prevent cardiovascular events in diabetes and because these agents are thought to improve endothelial function and reduce oxidative stress, they are predicted to improve microvascular function.23 One study in diabetic rats suggested that statins improved neurovascular function.32 Interestingly, statins may decrease LOX-1 expression in vascular endothelium, so this may produce superior protection against dyslipidemia-induced microvascular disease.36,43
Antioxidants. As we have emphasized, oxidative stress is a key component of diabetes complications.12,44 Oxidative mechanisms are involved in the generation of oxLDL and advanced glycation end-products and also result from oxLDL- and glucose-induced disturbances in cellular metabolism. For this reason, maintenance and enhancement of antioxidant capacity is important. We previously reviewed the literature on antioxidant therapy in diabetes.12 In rodent models of diabetes, antioxidants clearly afford protection against the development of macro- and microvascular complications.45-50 However, single antioxidants are not generally effective in patient trials, probably because of the long term requirements for drug efficacy and the counterproductive effects of long term use of high dose single antioxidants.51,52 To further this therapeutic approach to diabetic neuropathy, we recently completed a trial of a triple antioxidant cocktail in patients with type 1 diabetes, for which we anticipate completion of the data analysis in the near future. The data to date indicate that patients with diabetes should be encouraged to eat a diet rich in antioxidant-boosting components such as grains, fruits and vegetables, and supplements including vitamin E, vitamin C, and N-acetylcysteine are appropriate. Alpha lipoic acid has been extensively tested as a potent antioxidant with additional metabolic benefits, and was effective against neuropathic symptoms in trials in Europe.53
LOX-1 inhibitors. The expression and/or activity of LOX-1 can be reduced by botanical compounds such as curcumin, found in turmeric54, and epigallocatechin-3-gallate, which is abundant in green tea55. These compounds are considered safe and well tolerated. They are available as dietary supplements and have the added benefit of anti-inflammatory and antioxidant effects. These compounds could be an excellent addition to a treatment regime against diabetic neuropathy. A soluble form of LOX-1 comprising the extracellular domain is known to be present in the plasma and highly elevated in macrovascular disease. It has not been determined whether exogenous application of soluble LOX-1 can scavenge LOX-1 ligands and so provide cytoprotection.
NAD(P)H oxidase inhibition. Apocynin is widely used as an NAD(P)H oxidase inhibitor in many experimental models. Despite effective inhibition of the target enzyme, outcomes are variable and this appears to be related to the prooxidant properties of this compound.56 To date, a safe and specific NAD(P)H oxidase inhibitor has not been identified for clinical use, although this appears to be an important target that could prevent cellular injury downstream of multiple pathways that are activated in the diabetic milieu.57 Interestingly, a new study demonstrates that AMP-activated protein kinase (AMPK), the cellular energy sensor, functions as a physiological suppressor of NAD(P)H oxidase expression.58 Thus, activation of AMPK, already indicated as a potential therapeutic target to improve metabolic status in diabetes sensitive tissues, may have added benefit to block the downstream NAD(P)H oxidase injury mechanism.
Diet and exercise counseling. To date, only preliminary and uncontrolled trials have been performed to address the benefits of exercise and diet for the treatment of diabetic neuropathy. In one study, intraepidermal nerve fiber density was used as a marker for changes in neuropathy in patients with impaired glucose tolerance.59 In this study, subjects received individualized counseling with goals of reducing weight by 7% and increasing weekly exercise to 150 minutes. After one year of treatment, there was a statistically significant 0.3 + 1.1 fibers/mm improvement in distal intraepidermal nerve fiber density and 1.4 + 2.3 fibers/mm improvement in proximal intraepidermal nerve fiber density, which correlated with a decrease in neuropathic pain. These data, along with other preliminary studies, suggest that diet and exercise counseling may be a useful treatment strategy. Diet and exercise regimens in patients with impaired glucose tolerance reduces risk of progression to diabetes, and those with neuropathy experience a short-term improvement in small fiber function with sustained benefit for pain.60
Increasing evidence supports the concept that dyslipidemia contributes to, or may be central to, the development of neuropathy. This finding provides critical understanding of why current therapies against diabetic neuropathy do not provide long term prevention or improvement. Closer attention to plasma lipids is warranted. Furthermore, as we continue to define the mechanisms of dyslipidemia-induced peripheral sensory neuron and microvascular injury, we anticipate that we can combine novel treatment strategies to target both the metabolic imbalances and cellular oxidative stress mechanisms that lead to diabetic foot complications.
Andrea M. Vincent, PhD, is a research assistant professor of neurology at the University of Michigan. Lucy M. Hinder, PhD, is a postdoctoral fellow and Eva L. Feldman, MD, PhD, is the Russell N. DeJong professor of neurology and director of the JDRF Center for the Study of Complications in Diabetes at the same institution.
Supported by Juvenile Diabetes Research Foundation (AMV, ELF), the American Diabetes Association, (AMV), the Animal Models of Diabetes Complications Consortium (AMDCC; NIH UO1 DK076160, ELF), NIH RC1 NS068182-01 (ELF), the Program for Neurology Research and Discovery, and the A. Alfred Taubman Medical Institute.
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