COMPARATIVE EFFECT OF A HIGH FAT WITH OR WITHOUT HIGH LEVELS OF SUCROSE DIETS ON PERIPHERAL NEUROPATHY IN C57BL/6J MICE.

Objective: Feeding mice a diet containing high fat and high sucrose has been promoted as a good model for type 2 diabetes. This study sought to determine the effect of feeding mice a high fat and high sucrose diet on neuropathy compared to mice fed only a high fat diet and mice fed a high diet and treated with streptozotocin. Methods: C57Bl/6J mice were divided into five groups and fed the following diets for 20 weeks: Normal (Control); Sucrose enriched (Control + Sucrose), High Fat (Diet-induced obesity (DIO)), High Fat and High Sucrose (DIO + sucrose) and High Fat diet/streptozotocin treated (Diabetic). The endpoints evaluated included motor and sensory nerve conduction velocity, thermal and mechanical sensitivity and innervation of sensory nerves in the cornea and skin. Results: Diabetic mice were hyperglycemic at the end of the study and along with DIO mice with or without Sucrose had impaired glucose utilization. DIO mice had slowed sensory nerve conduction velocity, mechanical allodynia and decreased innervation of the cornea and skin. DIO + Sucrose and to a greater extent diabetic mice were thermal hypoalgesic, had mechanical allodynia, reduced motor and sensory nerve conduction velocities and decrease innervation of the cornea and skin. Conclusions: Development of peripheral neuropathy was more severe in High Fat and High Sucrose fed mice compared to high fat fed mice but fasting hyperglycemia and impaired glucose utilization was similar for these two models. Peripheral neuropathy was most severe in diabetic mice.

Characterization of Mice Ubiquitously Overexpressing Human 15-Lipoxygenase-1: Effect of Diabetes on Peripheral Neuropathy and Treatment with Menhaden Oil.

To rigorously explore the role of omega-3 polyunsaturated fatty acids (PUFA) in the treatment of diabetic peripheral neuropathy (DPN), we have created a transgenic mouse utilizing a Cre-lox promoter to control overexpression of human 15-lipoxygenase-1 (15-LOX-1). In this study, we sought to determine the effect of treating type 2 diabetic wild-type mice and transgenic mice ubiquitously overexpressing 15-LOX-1 with menhaden oil on endpoints related to DPN. Wild-type and transgenic mice on a C57Bl/6J background were divided into three groups. Two of each of these groups were used to create a high-fat diet/streptozotocin model for type 2 diabetes. The remaining mice were control groups. Four weeks later, one set of diabetic mice from each group was treated with menhaden oil for twelve weeks and then evaluated using DPN-related endpoints. Studies were also performed using dorsal root ganglion neurons isolated from wild-type and transgenic mice. Wild-type and transgenic diabetic mice developed DPN as determined by slowing of nerve conduction velocity, decreased sensory nerve fibers in the skin and cornea, and impairment of thermal and mechanical sensitivity of the hindpaw compared to their respective control mice. Although not significant, there was a trend for the severity of these DPN-related deficits to be less in the diabetic transgenic mice compared to the diabetic wild-type mice. Treating diabetic wild-type and transgenic mice with menhaden oil improved the DPN-related endpoints with a trend for greater improvement or protection by menhaden oil observed in the diabetic transgenic mice. Treating dorsal root ganglion neurons with docosahexanoic acid but not eicosapentaenoic acid significantly increased neurite outgrowth with greater efficacy observed with neurons isolated from transgenic mice. Targeting pathways that will increase the production of the anti-inflammatory metabolites of omega-3 PUFA may be an efficacious approach to developing an effective treatment for DPN.

Effect of mitoquinone on liver metabolism and steatosis in obese and diabetic rats.

Previous work by ourselves and others showed that mitoquinone (mitoQ) reduced oxidative damage and prevented hepatic fat accumulation in mice made obese with high-fat (HF) feeding. Here we extended these studies to examine the effect of mitoQ on parameters affecting liver function in rats treated with HF to induce obesity and in rats treated with HF plus streptozotocin (STZ) to model a severe form of type 2 diabetes. In prior reported work, we found that mitoQ significantly improved glycemia based on glucose tolerance data in HF rats but not in the diabetic rats. Here we found only non-significant reductions in insulin and glucose measured in the fed state at sacrifice in the HF mice treated with mitoQ. Metabolomic data showed that mitoQ altered several hepatic metabolic pathways in HF-fed obese rats toward those observed in control normal chow-fed non-obese rats. However, mitoQ had little effect on pathways observed in the diabetic rats, wherein diabetes itself induced marked pathway aberrations. MitoQ did not alter respiration or membrane potential in isolated liver mitochondria. MitoQ reduced liver fat and liver hydroperoxide levels but did not improve liver function as marked by circulating levels of aspartate and alanine aminotransferase (ALT). In summary, our results for HF-fed rats are consistent with past findings in HF-fed mice indicating decreased liver lipid hydroperoxides (LPO) and improved glycemia. However, in contrast to the HF obese mice, mitoQ did not improve glycemia or reset perturbed metabolic pathways in the diabetic rats.

Progressive Loss of Corneal Nerve Fibers and Sensitivity in Rats Modeling Obesity and Type 2 Diabetes Is Reversible with Omega-3 Fatty Acid Intervention: Supporting Cornea Analyses as a Marker for Peripheral Neuropathy and Treatment.

PURPOSE: To determine whether cornea nerve fiber density and/or corneal function are valid markers for early detection and treatment of peripheral neuropathy in rats modeling prediabetes and type 2 diabetes. METHODS: High-fat feeding combined without or with low-dose streptozotocin was used to create rat models for prediabetes and type 2 diabetes that were longitudinally studied for loss of structure and function of sensory nerves in the cornea and skin as well as nerve conduction velocity and vascular reactivity of epineurial arterioles. There were three time points examined in each of the three conditions with 12 rats per group. The latest time point (24 weeks of high-fat diet with or without 16 weeks of hyperglycemia) was used to examine reversibility of neuro and vascular pathology following 16 weeks of treatment with menhaden oil, a natural source of long-chain omega-3 polyunsaturated fatty acids. The number of rats in the intervention study ranged from 6 to 17. RESULTS: Our longitudinal study demonstrated that vascular and neural dysfunction associated with obesity or type 2 diabetes occur early and are progressive. Decrease in cornea nerve fiber length and function were valid markers of disease in both the pre-diabetic and diabetic rat models and were more sensitive than decrease in intraepidermal nerve fiber density of the skin and thermal nociception of the hindpaw. Late intervention with menhaden oil significantly reversed both vascular and peripheral nerve damage induced by chronic obesity or type 2 diabetes. CONCLUSION: These studies provide support for examination of corneal structure and function as an early marker of peripheral neuropathy in prediabetes and type 2 diabetes. Furthermore, we demonstrate that omega-3 polyunsaturated fatty acids derived from fish oil are an effective treatment for peripheral neuropathy that occurs with chronic obesity or type 2 diabetes.

Effect of mitoquinone (Mito-Q) on neuropathic endpoints in an obese and type 2 diabetic rat model.

This study sought to determine whether the addition of mitoquinone (Mito-Q) in the diet is an effective treatment for peripheral neuropathy in animal models of diet-induced obesity (pre-diabetes) and type 2 diabetes. Unlike other anti-oxidative stress compounds investigated as a treatment for peripheral neuropathy, Mito-Q specifically targets mitochondria. Although mito-Q has been shown to reduce oxidative stress generated by mitochondria there have been no studies performed of the effect of Mito-Q on peripheral neuropathy induced by diet-induced obesity or type 2 diabetes. Diet-induced obese (12 weeks after high fat diet) or type 2 diabetic rats (12 weeks of high fat diet and 4 weeks after the onset of hyperglycemia) were treated via the diet with Mito-Q (0.93 g/kg diet) for 12 weeks. Afterwards, glucose utilization, vascular reactivity of epineurial arterioles to acetylcholine and peripheral neuropathy related endpoints were examined. The addition of Mito-Q to the diets of obese and diabetic rats improved motor and/or sensory nerve conduction velocity, cornea and intraepidermal nerve fibre density, cornea sensitivity and thermal nociception. Surprisingly, treating obese and diabetic rats with Mito-Q did not improve glucose utilization or vascular reactivity by epineurial arterioles to acetylcholine. These studies imply that mitochondrial dysfunction contributes to peripheral neuropathy in animal models of pre-diabetes and late-stage type 2 diabetes. However, improvement in peripheral neuropathy following treatment with Mito-Q was not associated with improvement in glucose utilization or vascular reactivity of epineurial arterioles to acetylcholine.

Effect of Early and Late Interventions with Dietary Oils on Vascular and Neural Complications in a Type 2 Diabetic Rat Model.

AIMS: Determine the effect of dietary oils enriched in different mono- or polyunsaturated fatty acids, i.e., olive oil (18 : 1, oleic acid), safflower oil (18 : 2 n-6, linoleic acid), flaxseed oil (18 : 3 n-3, alpha linolenic acid), evening primrose oil (18 : 3 n-6, gamma linolenic acid), or menhaden oil (20:5/22 : 6 n-3 eicosapentaenoic/docosahexaenoic acids), on vascular and neural complications in high-fat-fed low-dose streptozotocin-treated Sprague-Dawley rats, an animal model for late-stage type 2 diabetes. MATERIALS AND METHODS: Rats were fed a high-fat diet (45% kcal as fat primarily derived from lard) for 8 weeks and then treated with a low dose of streptozotocin (30 mg/kg) in order to induce hyperglycemia. After an additional 8 (early intervention) or 20 (late intervention) weeks, the different groups of rats were fed diets with 1/2 of the kcal of fat derived from lard replaced by the different dietary oils. In addition, a control group fed a standard diet (4.25% kcal as fat) and a diabetic group maintained on the high-fat diet were maintained. The treatment period was approximately 16 weeks. The endpoints evaluated included vascular reactivity of epineurial arterioles, motor and sensory nerve conduction velocity, thermal and corneal sensitivity, and innervation of sensory nerves in the cornea and skin. RESULTS: Our findings show that menhaden and flaxseed oil provided the greatest benefit for correcting peripheral nerve damage caused by diabetes, whereas enriching the high-fat diet with menhaden oil provided the most benefit to acetylcholine-mediated vascular relaxation of epineurial arterioles of the sciatic nerve. Enriching the diets with fatty acids derived from the other oils provided none to partial improvements. CONCLUSIONS: These studies imply that long-chain n-6 and n-3 polyunsaturated fatty acids could be an effective treatment for diabetic peripheral neuropathy with n-3 polyunsaturated fatty acids derived from fish oil being the most effective.

Dietary fats modify vascular fat composition, eNOS localization within lipid rafts and vascular function in obesity.

We tested whether dietary fatty acids alter membrane composition shifting localization of signaling pathways within caveolae to determine their role in vascular function. Wild type (WT) and caveolin-1-deficient mice (cav-1 KO), required for vascular caveolae formation, were fed low fat (LF), high saturated fat (HF, 60% kcal from lard), or high-fat diet with 50:50 lard and n-3 polyunsaturated fatty acid-enriched menhaden oil (MO). HF and MO increased body weight and fat in WT but had less effect in cav-1 KO. MO increased unsaturated fatty acids and the unsaturation index of aorta from WT and cav-1 KO. In LF WT aorta, endothelial nitric oxide synthase (eNOS) was localized to cav-1-enriched low-density fractions which shifted to actin-enriched high-density fractions with acetylcholine (ACh). HF and MO shifted eNOS to high-density fractions in WT aorta which was not affected by ACh. In cav-1 KO aorta, eNOS was localized in low-density non-caveolar fractions but not shifted by ACh or diet. Inducible NOS and cyclooxygenase 1/2 were not localized in low-density fractions or affected by diet, ACh or genotype. ACh-induced dilation of gracilis arteries from HF WT was similar to dilation in LF but the NOS component was reduced. In WT and cav-1 KO, dilation to ACh was enhanced by MO through increased role for NOS and cyclooxygenase. We conclude that dietary fats affect vascular fatty acid composition and membrane localization of eNOS but the contribution of eNOS and cyclooxygenase in ACh-mediated vascular responses is independent of lipid rafts.

Effect of Dietary Content of Menhaden Oil with or without Salsalate on Neuropathic Endpoints in High-Fat-Fed/Low-Dose Streptozotocin-Treated Sprague Dawley Rats.

In this study, we wanted to extend our investigation of the efficacy of fish oil with or without salsalate on vascular and neural complications using a type 2 diabetic rat model. Four weeks after the onset of hyperglycemia, diabetic rats were treated via the diet with 3 different amounts of menhaden oil with or without salsalate for 12 weeks. Afterwards, vascular reactivity of epineurial arterioles and neuropathy-related endpoints were examined. The addition of salsalate to high-fat diets enriched with 10% or 25% kcal of menhaden oil protected vascular reactivity to acetylcholine and calcium gene-related peptide, motor and sensory nerve conduction velocity, thermal nociception, intraepidermal nerve fiber density, and cornea sensitivity to a greater extent than 10% or 25% menhaden oil alone. Vascular and neural function was maximally protected with diet containing 45% kcal as menhaden oil, and adding salsalate did not provide any additional benefit. Salsalate alone in the high-fat diet of diabetic rats provided minimal protection/improvement of vascular and neural dysfunction. These studies imply that dietary salsalate in combination with lower amounts of menhaden oil can provide greater benefit toward diabetes-induced vascular and neural impairment than menhaden oil alone.

Vascular and Neural Complications in Type 2 Diabetic Rats: Improvement by Sacubitril/Valsartan Greater Than Valsartan Alone.

Previously, we had shown that a vasopeptidase inhibitor drug containing ACE and neprilysin inhibitors was an effective treatment for diabetic vascular and neural complications. However, side effects prevented further development. This led to the development of sacubitril/valsartan, a drug containing angiotensin II receptor blocker and neprilysin inhibitor that we hypothesized would be an effective treatment for diabetic peripheral neuropathy. Using early and late intervention protocols (4 and 12 weeks posthyperglycemia, respectively), type 2 diabetic rats were treated with valsartan or sacubitril/valsartan for 12 weeks followed by an extensive evaluation of vascular and neural end points. The results demonstrated efficacy of sacubitril/valsartan in improving vascular and neural function was superior to valsartan alone. In the early intervention protocol, sacubitril/valsartan treatment was found to slow progression of these deficits and, with late intervention treatment, was found to stimulate restoration of vascular reactivity, motor and sensory nerve conduction velocities, and sensitivity/regeneration of sensory nerves of the skin and cornea in a rat model of type 2 diabetes. These preclinical studies suggest that sacubitril/valsartan may be an effective treatment for diabetic peripheral neuropathy, but additional studies will be needed to investigate these effects further.

Effect of dietary oils on peripheral neuropathy-related endpoints in dietary obese rats.

PURPOSE: This study aimed to determine the effect of dietary oils (olive, safflower, evening primrose, flaxseed, or menhaden) enriched in different mono unsaturated fatty acids or polyunsaturated fatty acids on peripheral neuropathies in diet-induced obese Sprague-Dawley rats. MATERIALS AND METHODS: Rats at 12 weeks of age were fed a high-fat diet (45% kcal) for 16 weeks. Afterward, the rats were fed diets with 50% of the kilocalories of fat derived from lard replaced by the different dietary oils. In addition, a control group fed a standard diet (4% kcal fat) and a high fat fed group (45% kcal) were maintained. The treatment period was 32 weeks. The endpoints evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and vascular relaxation by epineurial arterioles. RESULTS: Menhaden oil provided the greatest benefit for improving peripheral nerve damage caused by dietary obesity. Similar results were obtained when we examined acetylcholine-mediated vascular relaxation of epineurial arterioles of the sciatic nerve. Enriching the diets with fatty acids derived from the other oils provided minimal to partial improvements. CONCLUSION: These studies suggest that omega-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for neural and vascular complications associated with obesity.

Determination of peripheral neuropathy in high-fat diet fed low-dose streptozotocin-treated female C57Bl/6J mice and Sprague-Dawley rats.

AIMS/INTRODUCTION: Peripheral neuropathy is a common complication of diabetes and also occurs in 30% of human obese individuals with impaired glucose tolerance. Even though peripheral neuropathy affects both sexes, most pre-clinical studies have been carried out using male rodents. The aim of the present study was to create diet-induced obesity and type 2 diabetes in female rats and mice in order to examine the development of peripheral neuropathy. MATERIALS AND METHODS: At 12 weeks-of-age, rats and mice were separated into three groups. Two groups or rats and mice were fed a 60-kcal% high-fat diet for 12 weeks (rats) or 8 weeks (mice). To induce type 2 diabetes, one group of high-fat diet-fed rats and mice were treated with a low dose of streptozotocin. Analyses of multiple neural end-points were carried out 12 weeks later. RESULTS: Glucose utilization was impaired in diet-induced obese female rats and mice, as was a number of neurological end-points including nerve conduction velocity, intraepidermal and subepithelial corneal nerve fiber densities, and thermal and mechanical sensitivity. When female diet-induced obese rats or mice were made hyperglycemic, glucose utilization and sensory nerve density of the skin and cornea, as well as thermal and mechanical sensitivity, were more significantly impaired compared with diet-induced obese female rodents. CONCLUSIONS: These studies show that diet-induced obese and type 2 diabetic female rodents develop peripheral neuropathy that is similar to that occurring in male rodents. However, for female rats, more aggressive treatment is required to induce dietary obesity.

Impaired Corneal Sensation and Nerve Loss in a Type 2 Rat Model of Chronic Diabetes Is Reversible With Combination Therapy of Menhaden Oil, α-Lipoic Acid, and Enalapril.

PURPOSE: This study investigated the efficacy of monotherapy versus combination of menhaden oil, α-lipoic acid, and enalapril on corneal sensation and morphometry and other neuropathy-related endpoints in a rat model of type 2 diabetes. METHODS: Male Sprague-Dawley rats (aged 12 weeks) were fed a high-fat diet for 8 weeks followed by 30 mg/kg streptozotocin. After 16 weeks of hyperglycemia, 12-week treatments consisting of menhaden oil, α-lipoic acid, enalapril, or their combination were initiated. Before and after treatments, we performed analyses of multiple neural and vascular endpoints including corneal sensitivity, corneal nerve density, vascular reactivity of epineurial arterioles, motor and sensory nerve conduction velocity, intraepidermal nerve fiber density, and thermal nociception. RESULTS: Before treatment, all the neural and vascular endpoints in diabetic rats were impaired. Treating diabetic rats with monotherapy was effective in improving neural and vascular deficits with menhaden oil being most efficacious. However, the combination therapy provided the greatest benefit and improved/reversed all nerve and vascular deficits. The effect of combination therapy on corneal relative sensitivity and structure (in mm/mm), primary endpoints for this study, for control, diabetic, and diabetic treated rats was 4.2 ± 1.4 and 7.5 ± 0.5, 12.1 ± 1.3* and 3.8 ± 0.2*, and 6.6 ± 2.3 and 7.3 ± 0.5, respectively (*P < 0.05 compared with control rats; P < 0.05 compared with diabetic rats). CONCLUSIONS: These studies suggest that a combination therapeutic approach may be most effective for treating vascular and neural complications of type 2 diabetes.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (l50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Effect of tempol on peripheral neuropathy in diet-induced obese and high-fat fed/low-dose streptozotocin-treated C57Bl6/J mice.

In this study, we sought to determine the efficacy of tempol on multiple neuropathic endpoints in a diet-induced obese mouse, a model of pre-diabetes, and a high-fat fed low-dose streptozotocin treated mouse, a model of type 2 diabetes. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperdine -1-oxyl) is a low molecular weight, water soluble, membrane permeable, and metal-independent superoxide dismutase mimetic that has been widely used in cellular studies for the removal of intracellular and extracellular superoxide. This in vivo study was designed to be an early intervention. Fourteen weeks post-high-fat diet (6 weeks post-hyperglycemia) control, obese, and diabetic mice were divided into no treatment and treatment groups. The treated mice received tempol by gavage (150 mg/kg in water), while the untreated mice received vehicle. The diet-induced obese and the diabetic mice were maintained on the high-fat diet for the duration of the study, while the control group was maintained on the standard diet. Obesity and diabetes caused slowing of motor and sensory nerve conduction, reduction in intraepidermal nerve fiber density, thermal hypoalgesia, and mechanical allodynia. Treatment with tempol partially or completely protected obese and diabetic mice from these deficits. These studies suggest that tempol or other effective scavengers of reactive oxygen species may be a viable option for treating neural complications associated with obesity or type 2 diabetes.

Effect of Fish Oil vs. Resolvin D1, E1, Methyl Esters of Resolvins D1 or D2 on Diabetic Peripheral Neuropathy.

OBJECTIVE: Fish oil is enriched in omega-3 polyunsaturated fatty acids primarily eicosapentaenoic and docosahexaenoic fatty acids. Metabolites of these two polyunsaturated fatty acids include the E and D series resolvins. Omega-3 polyunsaturated fatty acids and resolvins have been reported to have anti-inflammatory and neuroprotective properties. The objective of this study was to evaluate the efficacy of menhaden oil, a fish oil derived from the menhaden, resolvins D1 and E1 and the methyl esters of resolvins D1 and D2 on diabetic peripheral neuropathy. Hypothesis being examined was that the methyl esters of resolvins D1 and D2 would be move efficacious than resolvins D1 or E1 due to an extended half-life. METHODS: A model of type 2 diabetes in C57BL/6J mice was created through a combination of a high fat diet followed 8 weeks later with treatment of low dosage of streptozotocin. After 8 weeks of untreated hyperglycemia type 2 diabetic mice were treated for 8 weeks with menhaden oil in the diet or daily injections of 1 ng/g body weight resolvins D1, E1 or methyl esters of resolvins D1 or D2. Afterwards, multiple neurological endpoints were examined. RESULTS: Menhaden oil or resolvins did not improve hyperglycemia. Untreated diabetic mice were thermal hypoalgesic, had mechanical allodynia, reduced motor and sensory nerve conduction velocities and decreased innervation of the cornea and skin. These endpoints were significantly improved with menhaden oil or resolvin treatment. However, the methyl esters of resolvins D1 or D2, contrary to our hypothesis, were generally less potent than menhaden oil or resolvins D1 or E1. CONCLUSION: These studies further support omega-3 polyunsaturated fatty acids derived from fish oil via in part due to their metabolites could be an effective treatment for diabetic neuropathy.

Early vs. late intervention of high fat/low dose streptozotocin treated C57Bl/6J mice with enalapril, α-lipoic acid, menhaden oil or their combination: Effect on diabetic neuropathy related endpoints.

We have previously demonstrated that enalapril, α-lipoic acid and menhaden (fish) oil has potential as a treatment for diabetic peripheral neuropathy. In this study we sought to determine the efficacy of these treatments individually or in combination on multiple neuropathic endpoints in a high fat fed low dose streptozotocin treated mouse, a model of type 2 diabetes, following early or late intervention. Four or twelve weeks after the onset of hyperglycemia, diabetic mice were treated with enalapril, α-lipoic acid, menhaden oil or their combination for 12 weeks. Afterwards, endpoints including glucose tolerance, motor and sensory nerve conduction velocity, thermal nociception, and intraepidermal and cornea nerve fiber density was determined. Glucose clearance was impaired in diabetic mice and significantly improved only with combination treatment and early intervention. Diabetes caused steatosis, slowing of motor and sensory nerve conduction velocity, thermal hypoalgesia and reduction in intraepidermal and cornea nerve fiber density. Treating diabetic mice with enalapril, α-lipoic acid or menhaden oil partially protected diabetic mice from these deficits, whereas the combination of these three treatments was more efficacious following early or late intervention. These studies suggest that a combination therapy may be more effective for treating neural complications of type 2 diabetes.

Effect of Treatment with Salsalate, Menhaden Oil, Combination of Salsalate and Menhaden Oil, or Resolvin D1 of C57Bl/6J Type 1 Diabetic Mouse on Neuropathic Endpoints.

Aims. In this study a streptozotocin induced type 1 diabetes mouse model was used to assess the effectiveness of salsalate, menhaden oil, the combination of salsalate and menhaden oil, or resolvin D1 on neuropathic endpoints. Materials and Methods. Changes in body weight, blood glucose, serum markers for triglycerides, free fatty acids, cholesterol, and resolvin D1, motor and sensory nerve conduction velocities and thermal sensitivity were assessed, as well as performing in vivo confocal microscopy of subepithelial corneal nerves and immunohistochemistry of nerves in the cornea and foot pad. Results. Diabetic animals failed to gain weight and had elevated blood glucose levels. Diabetic mice had slowed nerve conduction velocity, reduced innervation of the foot pad and cornea subepithelial and epithelial layers, and reduced thermal sensitivity. Monotherapy treatment with salsalate, menhaden oil, and resolvin D1 reduced the pathological signs of diabetic neuropathy. The combination of salsalate and menhaden oil also reduced signs of pathology and generated elevated plasma levels of resolvin D1 compared to other groups. Conclusions. Additional studies are needed to determine whether the combination of salsalate and menhaden oil may be more efficacious than monotherapy alone for the treatment of diabetic peripheral neuropathy.

Corneal Sensitivity to Hyperosmolar Eye Drops: A Novel Behavioral Assay to Assess Diabetic Peripheral Neuropathy.

PURPOSE: Diagnosis of peripheral neuropathy (PN), which affects approximately 50% of the diabetic population, is subjective, with many patients seeking a diagnosis only after presenting with symptoms. Recently, in vivo confocal microscopy of subepithelial corneal nerve density has been promoted as a surrogate marker for early detection of PN, but imaging of corneal nerves requires sophisticated instrumentation, expertise in confocal imaging, cooperative patients, and automated analysis tools to derive corneal nerve density. As an alternative, we developed a simple screening method that is based on the sensitivity of corneal nerves to cause reflex eyelid squinting in response to hyperosmolar eye drops. METHODS: Eyes of control and type 2 diabetic rats were given an eye drop of a 290- to 900-mOsm solution, and the ocular response was video recorded. Other neuropathic end points including nerve conduction velocity and subepithelial cornea nerve density were determined. RESULTS: Motor and sensory nerve conduction velocity and total nerve fiber length of corneal nerves in the subepithelial layer were significantly decreased in diabetic rats. Applying the hyperosmotic solutions to the ocular surface caused an osmolarity-dependent increase in squinting of the treated eye in control rats. Squinting was almost totally blocked by preapplication of proparacaine or N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide, a transient receptor potential melastatin-8 channel blocker. Squinting in response to the 900-mOsm solution was significantly reduced in diabetic rats. CONCLUSIONS: Preclinical studies show that evaluation of corneal sensitivity may be an alternative method for the early detection of PN and has potential for translation to clinical studies.

Nicotinamide Riboside Opposes Type 2 Diabetes and Neuropathy in Mice.

Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.

Effect of combination therapy consisting of enalapril, α-lipoic acid, and menhaden oil on diabetic neuropathy in a high fat/low dose streptozotocin treated rat.

We have previously demonstrated that treating diabetic rats with enalapril, an angiotensin converting enzyme (ACE) inhibitor, α-lipoic acid, an antioxidant, or menhaden oil, a natural source of omega-3 fatty acids can partially improve diabetic peripheral neuropathy. In this study we sought to determine the efficacy of combining these three treatments on vascular and neural complications in a high fat fed low dose streptozotocin treated rat, a model of type 2 diabetes. Rats were fed a high fat diet for 8 weeks followed by a 30 mg/kg dose of streptozotocin. Eight weeks after the onset of hyperglycemia diabetic rats were treated with a combination of enalapril, α-lipoic acid and menhaden oil. Diabetic rats not receiving treatment were continued on the high fat diet. Glucose clearance was impaired in diabetic rats and significantly improved with treatment. Diabetes caused steatosis, elevated serum lipid levels, slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber profiles, decrease in cornea sub-basal nerve fiber length and corneal sensitivity and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineurial arterioles of the sciatic nerve. Treating diabetic rats with the combination of enalapril, α-lipoic acid and menhaden oil reversed all these deficits to near control levels except for motor nerve conduction velocity which was also significantly improved compared to diabetic rats but remained significantly decreased compared to control rats. These studies suggest that a combination therapeutic approach may be most effective for treating vascular and neural complications of type 2 diabetes.