Pierre Watcho

Endoplasmic Reticulum Stress Plays a Key Role in the Pathogenesis of Diabetic Peripheral Neuropathy

Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins impairs metabolism, transcriptional regulation, and gene expression, and it is a key mechanism of cell injury. Endoplasmic reticulum stress plays an important role in cardiovascular and neurodegenerative diseases, cancer, and diabetes. We evaluated the role for this phenomenon in diabetic peripheral neuropathy. Endoplasmic reticulum stress manifest in upregulation of multiple components of unfolded protein response was identified in neural tissues (sciatic nerve, spinal cord) of streptozotocin diabetic rats and mice. A chemical chaperone, trimethylamine oxide, administered for 12 weeks after induction of diabetes (110 mg⋅kg−1⋅d−1, a prevention paradigm) attenuated endoplasmic reticulum stress, peripheral nerve dysfunction, intraepidermal nerve fiber loss, and sciatic nerve and spinal cord oxidative-nitrative stress in streptozotocin diabetic rats. Similar effects on diabetes-induced endoplasmic reticulum stress and peripheral nerve dysfunction were observed with a structurally unrelated chemical chaperone, 4-phenylbutyric acid (100 mg⋅kg−1⋅d−1, intraperitoneal). CCAAT/enhancer-binding protein homologous protein (CHOP)−/− mice made diabetic with streptozotocin displayed less severe sciatic nerve oxidative-nitrative stress and peripheral neuropathy than the wild-type (C57Bl6/J) mice. Neither chemical chaperones nor CHOP gene deficiency reduced diabetic hyperglycemia. Our findings reveal an important role of endoplasmic reticulum stress in the development of diabetic peripheral neuropathy and identify a potential new therapeutic target.

Analgesic and Anti-Inflammatory Properties of Extracts from the Bulbils of Dioscorea bulbifera L. var sativa (Dioscoreaceae) in Mice and Rats

The aqueous and methanol extracts from the dry bulbils of Dioscorea bulbifera L. var sativa (Dioscoreaceae)—evaluated orally at the doses of 300 and 600 mg/kg against pain induced by acetic acid, formalin, pressure and against inflammation induced by carrageenan, histamine, serotonin and formalin in mice and rats, showed a dose dependant inhibition of pain and inflammation with a maximum effect of 56.38%, 73.06% and 42.79% produced by the aqueous extract, respectively on pain induced by acetic acid, formalin and pressure while the methanol extract at the same dose respectively inhibited these models of pain by 62.70%, 84.54% and 47.70%. The oral administration of aqueous and methanol extracts caused significant anti-inflammatory activity on paw oedema induced by histamine, serotonin and formalin. The present results show that the bulbils of Dioscorea bulbifera var sativa possess potent analgesic and anti-inflammatory activities. These activities may results from the inhibition of inflammatory mediators such as histamine, serotonin and prostaglandins. Thus, the analgesic activity of the bulbils of Dioscorea bulbifera may be at least partially linked to its anti-inflammatory activity.

Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy.

Growing evidence suggests that prediabetes and metabolic syndrome are associated with increased risk for the development of microvascular complications including retinopathy, nephropathy, and, most commonly, peripheral painful neuropathy and/or autonomic neuropathy. The etiology of these disabling neuropathies is unclear, and several clinical and experimental studies implicated obesity, impaired fasting glycemia/impaired glucose tolerance, elevated triglyceride and non-esterified fatty acids, as well as oxidative-nitrative stress. Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins and leading to the impairment of metabolism, transcriptional regulation, and gene expression, is emerging as a key mechanism of metabolic diseases including obesity and diabetes. We evaluated the role for this phenomenon in prediabetic neuropathy using two animal models i.e., Zucker (fa/fa) rats and high-fat diet fed mice which displayed obesity and impaired glucose tolerance in the absence of overt hyperglycemia. Endoplasmic reticulum stress manifest in upregulation of the glucose-regulated proteins BiP/GRP78 and GRP94 of unfolded protein response was identified in the sciatic nerve of Zucker rats. A chemical chaperone, trimethylamine oxide, blunted endoplasmic reticulum stress and alleviated sensory nerve conduction velocity deficit, thermal and mechanical hypoalgesia, and tactile allodynia. A selective inhibitor of eukaryotic initiation factor-2α dephosphorylation, salubrinal, improved glucose intolerance and alleviated peripheral nerve dysfunction in high-fat diet fed mice. Our findings suggest an important role of endoplasmic reticulum stress in the neurobiology of prediabetic peripheral neuropathy, and identify a new therapeutic target.

Triglyceride, nonesterified fatty acids, and prediabetic neuropathy: role for oxidative–nitrosative stress

Peripheral neuropathy develops in human subjects with prediabetes and metabolic syndrome before overt hyperglycemia. The contributions of impaired glucose tolerance and insulin signaling, hypertriglyceridemia and/or increased nonesterified fatty acids (NEFA), and hypercholesterolemia to this condition remain unknown. Niacin and its derivatives alleviate dyslipidemia with a minor effect on glucose homeostasis. This study evaluated the roles of impaired glucose tolerance versus dyslipidemia in prediabetic neuropathy using Zucker fatty (fa/fa) rats and the niacin derivative acipimox, as well as the interplay of hypertriglyceridemia, increased NEFA, and oxidative-nitrosative stress. Sixteen-week-old Zucker fatty rats with impaired glucose tolerance, obesity, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and increased NEFA displayed sensory nerve conduction velocity deficit, thermal and mechanical hypoalgesia, and tactile allodynia. Acipimox (100 mg kg(-1) day(-1), 4 weeks) reduced serum insulin, NEFA, and triglyceride concentrations without affecting glucose tolerance and hypercholesterolemia. It alleviated sensory nerve conduction velocity deficit and changes in behavioral measures of sensory function and corrected oxidative-nitrosative stress, but not impaired insulin signaling, in peripheral nerve. Elevated NEFA increased total and mitochondrial superoxide production and NAD(P)H oxidase activity in cultured human Schwann cells. In conclusion, hypertriglyceridemia and/or increased NEFA concentrations cause prediabetic neuropathy through oxidative-nitrosative stress. Lipid-lowering agents and antioxidants may find a use in the management of this condition.

High-Fat Diet-Induced Neuropathy of Prediabetes and Obesity: Effect of PMI-5011, an Ethanolic Extract of Artemisia dracunculus L.

Artemisia species are a rich source of herbal remedies with antioxidant and anti-inflammatory properties. We evaluated PMI-5011, an ethanolic extract of Artemisia dracunculus L., on neuropathy in high-sfat diet-fed mice, a model of prediabetes and obesity developing oxidative stress and proinflammatory changes in peripheral nervous system. C57Bl6/J mice fed high-fat diet for 16 weeks developed obesity, moderate nonfasting hyperglycemia, nerve conduction deficit, thermal and mechanical hypoalgesia, and tactile allodynia. They displayed 12/15-lipoxygenase overexpression, 12(S)-hydroxyeicosatetraenoic acid accumulation, and nitrosative stress in peripheral nerve and spinal cord. PMI-5011 (500 mgkg−1d−1, 7 weeks) normalized glycemia, alleviated nerve conduction slowing and sensory neuropathy, and reduced 12/15-lipoxygenase upregulation and nitrated protein expression in peripheral nervous system. PMI-5011, a safe and nontoxic botanical extract, may find use in treatment of neuropathic changes at the earliest stage of disease.

Prediabetic Nephropathy as an Early Consequence of the High-Calorie/High-Fat Diet: Relation to Oxidative Stress

This study evaluated early renal functional, structural, and biochemical changes in high-calorie/high-fat diet fed mice, a model of prediabetes and alimentary obesity. Male C57BL6/J mice were fed normal (11 kcal% fat) or high-fat (58 kcal% fat) diets for 16 wk. Renal changes were evaluated by histochemistry and immunohistochemistry, Western blot analysis, ELISA, enzymatic assays, and chemiluminometry. High-fat diet consumption led to increased body and kidney weights, impaired glucose tolerance, hyperinsulinemia, polyuria, a 2.7-fold increase in 24-h urinary albumin excretion, 20% increase in renal glomerular volume, 18% increase in renal collagen deposition, and 8% drop of glomerular podocytes. It also resulted in a 5.3-fold increase in urinary 8-isoprostane excretion and a 38% increase in renal cortex 4-hydroxynonenal adduct accumulation. 4-hydroxynonenal adduct level and immunoreactivity or Sirtuin 1 expression in renal medulla were not affected. Studies of potential mechanisms of the high-fat diet induced renal cortex oxidative injury revealed that whereas nicotinamide adenine dinucleotide phosphate reduced form oxidase activity only tended to increase, 12/15-lipoxygenase was significantly up-regulated, with approximately 12% increase in the enzyme protein expression and approximately 2-fold accumulation of 12(S)-hydroxyeicosatetraenoic acid, a marker of 12/15-lipoxygenase activity. Accumulation of periodic acid-Schiff -positive material, concentrations of TGF-β, sorbitol pathway intermediates, and expression of nephrin, CAAT/enhancer-binding protein homologous protein, phosphoeukaryotic initiation factor-α, and total eukaryotic initiation factor-α in the renal cortex were indistinguishable between experimental groups. Vascular endothelial growth factor concentrations were reduced in high-fat diet fed mice. In conclusion, systemic and renal cortex oxidative stress associated with 12/15-lipoxygenase overexpression and activation is an early phenomenon caused by high-calorie/high-fat diet consumption and a likely contributor to kidney disease associated with prediabetes and alimentary obesity.

Metanx Alleviates Multiple Manifestations of Peripheral Neuropathy and Increases Intraepidermal Nerve Fiber Density in Zucker Diabetic Fatty Rats

Metanx is a product containing l-methylfolate, pyridoxal 5′-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in vascular endothelium and peripheral nerve. This study evaluates Metanx on diabetic peripheral neuropathy in ZDF rats, a model of type 2 diabetes. Metanx was administered to 15-week-old ZDF and ZDF lean rats at either 4.87 mg ⋅ kg−1 ⋅ day−1 (a body weight–based equivalent of human dose) or 24.35 mg ⋅ kg−1 ⋅ day−1 by oral gavage two times a day for 4 weeks. Both doses alleviated hind limb digital sensory, but not sciatic motor, nerve conduction slowing and thermal and mechanical hypoalgesia in the absence of any reduction of hyperglycemia. Low-dose Metanx increased intraepidermal nerve fiber density but did not prevent morphometric changes in distal tibial nerve myelinated fibers. Metanx treatment counteracted endothelial nitric oxide synthase uncoupling, inducible nitric oxide synthase upregulation, and methylglyoxal-derived advanced glycation end product, nitrotyrosine, and nitrite/nitrate accumulation in the peripheral nerve. In conclusion, Metanx, at a body weight–based equivalent of human dose, increased intraepidermal nerve fiber density and improved multiple parameters of peripheral nerve function in ZDF rats. Clinical studies are needed to determine if Metanx finds use in management of diabetic peripheral neuropathy.

Poly(ADP-ribose) polymerase-1 (PARP-1) gene deficiency alleviates diabetic kidney disease

Poly(ADP-ribose)polymerase (PARP) inhibitors prevent or alleviate diabetic nephropathy. This study evaluated the role for PARP-1 in diabetic kidney disease using the PARP-1-deficient mouse. PARP-1-/- and the wild-type (129S1/SvImJ) mice were made diabetic with streptozotocin, and were maintained for 12 weeks. Final blood glucose concentrations were increased ∼ 3.7-fold in both diabetic groups. PARP-1 protein expression (Western blot analysis) in the renal cortex was similar in non-diabetic and diabetic wild-type mice (100% and 107%) whereas all knockouts were PARP-1-negative. PARP-1 gene deficiency reduced urinary albumin (ELISA) and protein excretion prevented diabetes-induced kidney hypertrophy, and decreased mesangial expansion and collagen deposition (both assessed by histochemistry) as well as fibronectin expression. Renal podocyte loss (immunohistochemistry) and nitrotyrosine and transforming growth factor-β₁ accumulations (both by ELISA) were slightly lower in diabetic PARP-1-/- mice, but the differences with diabetic wild-type group did not achieve statistical significance. In conclusion, PARP-1-/- gene deficiency alleviates although does not completely prevent diabetic kidney disease.

Anti-Nociceptive and Anti-Inflammatory Activities of Extracts from the Stem Bark of Croton macrostachyus (Euphorbiaceae) in Mice and Rats

The present study evaluates the anti-nociceptive and anti-inflammatory properties of the aqueous and methylene chloride/methanol (CH2Cl2 / CH3OH) extracts of the stem bark of Croton macrostachyus. The extracts administered orally at the doses of 150, 300 and 600 mg/kg were examined against pain induced by acetic acid, formalin and pressure and against inflammation induced by carragenan, histamine and formalin. Both extracts induced a significant dose-dependent (P < 0.001) reduction in the number of abdominal constrictions induced by acetic acid. The three doses of the two extracts also significantly reduced (P < 0.001) the two phases of pain induced by formalin. At the dose of 600 mg/kg, the aqueous and the CH2Cl2 / CH3OH extracts exhibited a significant analgesic activity against pressure-induced pain. The two extracts also exhibited anti-inflammatory activity, the CH2Cl2 / CH3OH extract being the most active, inhibited acute inflammation induced by carrageenan, histamine and formalin. Both extracts also significantly reduced the chronic inflammation induced by formalin. These results show that the aqueous and CH2Cl2 / CH3OH extracts of the stem bark of Croton macrostachyus possess analgesic and anti-inflammatory properties. These findings are in accordance with the traditional use of the plant and indicate that Croton macrostachyus is a potent source of analgesic and anti-inflammatory principles.

Evaluation of In Vitro Uterotonic Activities of Fruit Extracts of Ficus asperifolia in Rats

The aim of the present study was to determine the uterotonic activities of Ficus asperifolia and investigate its mechanism. The effects of aqueous and methanol extracts of the dried fruits of F. asperifolia (0.05–1.60 mg mL−1) were evaluated on estrogenized isolated rat uterus in the presence and absence of atropine (1.73–55.27 nM), pyrilamine maleate (1.25 × 10−3 to 40 × 10−3 M), indomethacin (0.06 × 10−5 to 2.00 × 10−5 M) or hexamethonium (0.66 × 10−4 to 21.43 × 10−4 M). Aqueous (EC50, 0.36 mg mL−1) and methanol (EC50, 0.22 mg mL−1) extracts as well as oxytocin (EC50, 0.02 nM), acetylcholine (EC50, 7.87 nM) and histamine (EC50, 0.76 nM) evoked concentration-dependent contractions of the uterus. Atropine, pyrilamine maleate and indomethacin concentration dependently blocked the response of the uterus to acetylcholine (IC50, 4.82 nM), histamine (IC50, 2.49 nM) and oxytocin (IC50, 0.07 nM), respectively, and to aqueous extract. Hexamethonium produced graded decreases in oxytocin-induced uterine contractions (IC50, 0.37 μM), but did not prevent the contractile effects of the aqueous extract (IC50, 9.88 μM). These results suggest that F. asperifolia-induced uterotonic effect is related to the release of prostaglandins and contraction of the myometrial cells through muscarinic, oxytocic and H1 histamine receptors. These data further give added value to the ethnic use of F. asperifolia for its abortificient and contraceptive properties.