Dorsal Root Ganglia Mitochondrial Biochemical Changes in Non-diabetic and Streptozotocin-Induced Diabetic Mice Fed with a Standard or High-Fat Diet.

BACKGROUND: Mitochondrial dysfunction is purported as a contributory mechanism underlying diabetic neuropathy, but a defined role for damaged mitochondria in diabetic nerves remains unclear, particularly in standard diabetes models. Experiments here used a high-fat diet in attempt to exacerbate the severity of diabetes and expedite the time-course in which mitochondrial dysfunction may occur. We hypothesized a high-fat diet in addition to diabetes would increase stress on sensory neurons and worsen mitochondrial dysfunction. METHODS: Oxidative phosphorylation proteins and proteins associated with mitochondrial function were quantified in lumbar dorsal root ganglia. Comparisons were made between non-diabetic and streptozotocin-induced (STZ) C57Bl/6 mice fed a standard or high-fat diet for 8 weeks. RESULTS: Complex III subunit Core-2 and voltage dependent anion channel were increased (by 36% and 28% respectively, p<0.05) in diabetic mice compared to nondiabetic mice fed the standard diet. There were no differences among groups in UCP2, PGC-1α, PGC-1ß levels or Akt, mTor, or AMPK activation. These data suggest compensatory mitochondrial biogenesis occurs to offset potential mitochondrial dysfunction after 8 weeks of STZ-induced diabetes, but a high-fat diet does not alter these parameters. CONCLUSION: Our results indicate mitochondrial protein changes early in STZ-induced diabetes. Interestingly, a high-fat diet does not appear to affect mitochondrial proteins in either nondiabetic or STZ- diabetic mice.

Central or peripheral delivery of an adenosine A1 receptor agonist improves mechanical allodynia in a mouse model of painful diabetic neuropathy.

Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8 weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N(6)-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of painful diabetic neuropathy. Moreover, central and peripheral activation of A1R significantly improved mechanical sensitivity, warranting further investigation into this important antinociceptive pathway as a novel therapeutic option for the treatment of painful diabetic neuropathy.

Vaginal hypersensitivity and hypothalamic-pituitary-adrenal axis dysfunction as a result of neonatal maternal separation in female mice.

Early life stress can permanently alter functioning of the hypothalamic-pituitary-adrenal (HPA) axis, which regulates the stress response and influences the perception of pain. Chronic pelvic pain patients commonly report having experienced childhood neglect or abuse, which increases the likelihood of presenting with comorbid chronic pain and/or mood disorders. Animal models of neonatal stress commonly display enhanced anxiety-like behaviors, colorectal hypersensitivity, and disruption of proper neuro-immune interactions in adulthood. Here, we tested the hypothesis that early life stress impacts vaginal sensitivity by exposing mice to neonatal maternal separation (NMS) for 3h/day during the first two (NMS14) or three (NMS21) postnatal weeks. As adults, female mice underwent vaginal balloon distension (VBD), which was also considered an acute stress. Before or after VBD, mice were assessed for anxiety-like behavior, hindpaw sensitivity, and changes in gene and protein expression related to HPA axis function and regulation. NMS21 mice displayed significantly increased vaginal sensitivity compared to naïve mice, as well as significantly reduced anxiety-like behavior at baseline, which was heightened following VBD. NMS21 mice exhibited significant thermal and mechanical hindpaw hypersensitivity at baseline and following VBD. NMS14 mice displayed no change in anxiety-like behavior and only exhibited significantly increased hindpaw mechanical and thermal sensitivity following VBD. Centrally, a significant decrease in negative regulation of the HPA axis was observed in the hypothalamus and hippocampus of NMS21 mice. Peripherally, NMS and VBD affected the expression of inflammatory mediators in the vagina and bladder. Corticotropin-releasing factor (CRF) receptor and transient receptor potential (TRP) channel protein expression was also significantly, and differentially, affected in vagina, bladder, and colon by both NMS and VBD. Together these data indicate that NMS affects both central and peripheral aspects of the HPA axis, which may drive changes in vaginal sensitivity and the development of comorbid chronic pain and mood disorders.

Protection from diabetes-induced peripheral sensory neuropathy--a role for elevated glyoxalase I?

Diabetic neuropathy is a common complication of diabetes mellitus with over half of all patients developing neuropathy symptoms due to sensory nerve damage. Diabetes-induced hyperglycemia leads to the accelerated production of advanced glycation end products (AGEs) that alter proteins, thereby leading to neuronal dysfunction. The glyoxalase enzyme system, specifically glyoxalase I (GLO1), is responsible for detoxifying precursors of AGEs, such as methylglyoxal and other reactive dicarbonyls. The purpose of our studies was to determine if expression differences of GLO1 may play a role in the development of diabetic sensory neuropathy. BALB/cJ mice naturally express low levels of GLO1, while BALB/cByJ express approximately 10-fold higher levels on a similar genetic background due to increased copy numbers of GLO1. Five weeks following STZ injection, diabetic BALB/cJ mice developed a 68% increase in mechanical thresholds, characteristic of insensate neuropathy or loss of mechanical sensitivity. This behavior change correlated with a 38% reduction in intraepidermal nerve fiber density (IENFD). Diabetic BALB/cJ mice also had reduced expression of mitochondrial oxidative phosphorylation proteins in Complexes I and V by 83% and 47%, respectively. Conversely, diabetic BALB/cByJ mice did not develop signs of neuropathy, changes in IENFD, or alterations in mitochondrial protein expression. Reduced expression of GLO1 paired with diabetes-induced hyperglycemia may lead to neuronal mitochondrial damage and symptoms of diabetic neuropathy. Therefore, AGEs, the glyoxalase system, and mitochondrial dysfunction may play a role in the development and modulation of diabetic peripheral neuropathy.

Phenotypic changes in diabetic neuropathy induced by a high-fat diet in diabetic C57BL/6 mice.

Emerging evidence suggests that dyslipidemia is an independent risk factor for diabetic neuropathy (DN) (reviewed by Vincent et al. 2009). To experimentally determine how dyslipidemia alters DN, we quantified neuropathic symptoms in diabetic mice fed a high-fat diet. Streptozotocin-induced diabetic C57BL/6 mice fed a high-fat diet developed dyslipidemia and a painful neuropathy (mechanical allodynia) instead of the insensate neuropathy (mechanical insensitivity) that normally develops in this strain. Nondiabetic mice fed a high-fat diet also developed dyslipidemia and mechanical allodynia. Thermal sensitivity was significantly reduced in diabetic compared to nondiabetic mice, but was not worsened by the high-fat diet. Moreover, diabetic mice fed a high-fat diet had significantly slower sensory and motor nerve conduction velocities compared to nondiabetic mice. Overall, dyslipidemia resulting from a high-fat diet may modify DN phenotypes and/or increase risk for developing DN. These results provide new insight as to how dyslipidemia may alter the development and phenotype of diabetic neuropathy.

Insulin receptor substrate 2 expression and involvement in neuronal insulin resistance in diabetic neuropathy.

Insulin signaling depends on tyrosine phosphorylation of insulin receptor substrates (IRSs) to mediate downstream effects; however, elevated serine phosphorylation of IRS impairs insulin signaling. Here, we investigated IRS protein expression patterns in dorsal root ganglia (DRG) of mice and whether their signaling was affected by diabetes. Both IRS1 and IRS2 are expressed in DRG; however, IRS2 appears to be the prevalent isoform and is expressed by many DRG neuronal subtypes. Phosphorylation of Ser(731)IRS2 was significantly elevated in DRG neurons from type 1 and type 2 diabetic mice. Additionally, Akt activation and neurite outgrowth in response to insulin were significantly decreased in DRG cultures from diabetic ob/ob mice. These results suggest that DRG neurons express IRS proteins that are altered by diabetes similar to other peripheral tissues, and insulin signaling downstream of the insulin receptor may be impaired in sensory neurons and contribute to the pathogenesis of diabetic neuropathy.

Characterisation of glyoxalase I in a streptozocin-induced mouse model of diabetes with painful and insensate neuropathy.

AIMS/HYPOTHESIS: Diabetic peripheral neuropathy (DN) is a common complication of diabetes; however, the mechanisms producing positive or negative symptoms are not well understood. The enzyme glyoxalase I (GLO1) detoxifies reactive dicarbonyls that form AGEs and may affect the way sensory neurons respond to heightened AGE levels in DN. We hypothesised that differential GLO1 levels in sensory neurons may lead to differences in AGE formation and modulate the phenotype of DN. METHODS: Inbred strains of mice were used to assess the variability of Glo1 expression by quantitative RT-PCR. Non-diabetic C57BL/6 mice were used to characterise the distribution of GLO1 in neural tissues by immunofluorescence. Behavioural assessments were conducted in diabetic A/J and C57BL/6 mice to determine mechanical sensitivity, and GLO1 abundance was determined by western blot. RESULTS: GLO1 immunoreactivity was found throughout the nervous system, but selectively in small, unmyelinated peptidergic dorsal root ganglia (DRG) neurons that are involved in pain transmission. GLO1 protein was present at various levels in DRG from different inbred mice strains. Diabetic A/J and C57BL/6 mice, two mouse strains with different levels of GLO1, displayed dramatically different behavioural responses to mechanical stimuli. Diabetic C57BL/6 mice also had a reduced abundance of GLO1 following diabetes induction. CONCLUSIONS/INTERPRETATION: These findings reveal that the abundance of GLO1 varies between different murine strains and within different sensory neuron populations. These differences could lead to different responses of sensory neurons to the toxic effects of hyperglycaemia and reactive dicarbonyls associated with diabetes.

Neurotrophic modulation of myelinated cutaneous innervation and mechanical sensory loss in diabetic mice.

Human diabetic patients often lose touch and vibratory sensations, but to date, most studies on diabetes-induced sensory nerve degeneration have focused on epidermal C-fibers. Here, we explored the effects of diabetes on cutaneous myelinated fibers in relation to the behavioral responses to tactile stimuli from diabetic mice. Weekly behavioral testing began prior to streptozotocin (STZ) administration and continued until 8 weeks, at which time myelinated fiber innervation was examined in the footpad by immunohistochemistry using antiserum to neurofilament heavy chain (NF-H) and myelin basic protein (MBP). Diabetic mice developed reduced behavioral responses to non-noxious (monofilaments) and noxious (pinprick) stimuli. In addition, diabetic mice displayed a 50% reduction in NF-H-positive myelinated innervation of the dermal footpad compared with non-diabetic mice. To test whether two neurotrophins nerve growth factor (NGF) and/or neurotrophin-3 (NT-3) known to support myelinated cutaneous fibers could influence myelinated innervation, diabetic mice were treated intrathecally for 2 weeks with NGF, NT-3, NGF and NT-3. Neurotrophin-treated mice were then compared with diabetic mice treated with insulin for 2 weeks. NGF and insulin treatment both increased paw withdrawal to mechanical stimulation in diabetic mice, whereas NT-3 or a combination of NGF and NT-3 failed to alter paw withdrawal responses. Surprisingly, all treatments significantly increased myelinated innervation compared with control-treated diabetic mice, demonstrating that myelinated cutaneous fibers damaged by hyperglycemia respond to intrathecal administration of neurotrophins. Moreover, NT-3 treatment increased epidermal Merkel cell numbers associated with nerve fibers, consistent with increased numbers of NT-3-responsive slowly adapting A-fibers. These studies suggest that myelinated fiber loss may contribute as significantly as unmyelinated epidermal loss in diabetic neuropathy, and the contradiction between neurotrophin-induced increases in dermal innervation and behavior emphasizes the need for multiple approaches to accurately assess sensory improvements in diabetic neuropathy.

NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides.

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.

A systematic approach to biochemical profiling.

Sequencing of the Arabidopsis thaliana genome is complete. The analytical tools for determining gene function by altering and monitoring gene expression are relatively well developed, and are generating large volumes of valuable data. Recent advances in techniques for the analysis of small molecules allow researchers to apply biochemical profiling as another powerful approach to functional genomics and metabolic research.

Inhibition of protoporphyrinogen oxidase expression in Arabidopsis causes a lesion-mimic phenotype that induces systemic acquired resistance.

We have used an antisense expression technology in Arabidopsis based on the yeast GAL4/UAS transactivation system (Guyer et al., Genetics, 1998; 149:633-639) to reduce levels of protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll. Plants expressing the antisense PPO gene presented growth alterations and their leaves showed necrotic lesions that appeared similar to lesions characteristic of the pathogen-induced hypersensitive reaction, and seen in the so-called lesion-mimic mutants. Plants expressing the antisense gene also had high endogenous salicylic acid levels, constitutive expression of the PR-1 gene, and were resistant to Peronospora parasitica, consistent with the activation of systemic acquired resistance (SAR). Treatment of wild-type plants with sublethal concentrations of herbicides that inhibit PPO also induced defence responses that conferred enhanced tolerance to P. parasitica. This effect was not observed in NahG and nim1 plants, which are compromised in their ability to activate SAR. These results demonstrate that genetic or chemical disruption of a metabolic pathway can lead to the induction of a set of defence responses including activation of SAR.

Wheat genes encoding two types of PR-1 proteins are pathogen inducible, but do not respond to activators of systemic acquired resistance.

Wheat cDNAs that encode proteins PR-1.1 and PR-1.2 were cloned. Deduced amino acid sequences were homologous to those of pathogen-induced, basic PR-1 proteins from plants. Although expression of PR1.1 and PR1.2 genes was induced upon infection with either compatible or incompatible isolates of the fungal pathogen Erysiphe graminis, these genes did not respond to activators of systemic acquired resistance (SAR), such as salicylic acid (SA), benzothiadiazole (BTH), or isonicotinic acid (INA).

Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis.

The Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-kappa B. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.

Impaired fungicide activity in plants blocked in disease resistance signal transduction.

Fungicide action is generally assumed to be dependent on an antibiotic effect on a target pathogen, although a role for plant defense mechanisms as mediators of fungicide action has not been excluded. Here, we demonstrate that in Arabidopsis, the innate plant defense mechanism contributes to the effectiveness of fungicides. In NahG and nim1 (for noninducible immunity) Arabidopsis plants, which normally exhibit increased susceptibility to pathogens, the fungicides metalaxyl, fosetyl, and Cu(OH)2 are much less active and fail to control Peronospora parasitica. In contrast, the effectiveness of these fungicides is not altered in Arabidopsis mutants defective in the ethylene or jasmonic acid signal transduction pathways. Application of the systemic acquired resistance activator benzothiadiazole (BTH) in combination with these fungicides results in a synergistic effect on pathogen resistance in wild-type plants and an additive effect in NahG and BTH-unresponsive nim1 plants. Interestingly, BTH treatment normally induces long-lasting pathogen protection; however, in NahG plants, the protection is transient. These observations suggest that BTH treatment can compensate only partially for an impaired signal transduction pathway and support the idea that pathogen defense mechanisms are under positive feedback control. These observations are strikingly reminiscent of the reduced efficacy of antifungal agents in immunocompromised animals.

Mechanosensitive expression of a lipoxygenase gene in wheat.

Touch stimulation of wheat (Triticum aestivum L.) seedlings led to a strong and dose-dependent increase in the level of lipoxygenase mRNA transcripts. The touch-induced response occurred within 1 h and was transient. A similar response was observed after wind treatment and wounding. The mechanical strain-regulated lipoxygenase might translate mechanical strain into lipoxygenase pathway-dependent growth responses.

Salicylate-independent lesion formation in Arabidopsis lsd mutants.

In many interactions of plants with pathogens, the primary host defense reaction is accompanied by plant cell death at the site of infection. The resulting lesions are correlated with the establishment of an inducible resistance in plants called systemic acquired resistance (SAR), for which salicylic acid (SA) accumulation is a critical signaling event in Arabidopsis and tobacco. In Arabidopsis, the lesions simulating disease (lsd) mutants spontaneously develop lesions in the absence of pathogen infection. Furthermore, lsd mutants express SAR marker genes when lesions are present and are resistant to the same spectrum of pathogens as plants activated for SAR by necrogenic pathogen infection. To assess the epistatic relationship between SA accumulation and cell death, transgenic Arabidopsis unable to accumulate SA due to the expression of the salicylate hydroxylase (nahG) gene were used in crosses with the dominant mutants lsd2 or lsd4. Progeny from the crosses were inhibited for SAR gene expression and disease resistance. However, these progeny retained the spontaneous cell death phenotype similar to siblings not expressing nahG. Because lesions form in the absence of SA accumulation for isd2 and lsd4, a model is suggested in which lesion formation in these two mutants is determined prior to SA accumulation in SAR signal transduction. By contrast, the loss of SAR gene expression and disease resistance in nahG-expressing lsd mutants indicates that these traits are dependent upon SA accumulation in the SAR signal transduction pathway.

The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa B.

The NIM1 (for noninducible immunity) gene product is involved in the signal transduction cascade leading to both systemic acquired resistance (SAR) and gene-for-gene disease resistance in Arabidopsis. We have isolated and characterized five new alleles of nim1 that show a range of phenotypes from weakly impaired in chemically induced pathogenesis-related protein-1 gene expression and fungal resistance to very strongly blocked. We have isolated the NIM1 gene by using a map-based cloning procedure. Interestingly, the NIM1 protein shows sequence homology to the mammalian signal transduction factor I kappa B subclass alpha. NF-kappa B/I kappa B signaling pathways are implicated in disease resistance responses in a range of organisms from Drosophila to mammals, suggesting that the SAR signaling pathway in plants is representative of an ancient and ubiquitous defense mechanism in higher organisms.

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.