John W. Wiley

The emerging role of autophagy in the pathophysiology of diabetes mellitus

An emerging body of evidence supports a role for autophagy in the pathophysiology of type 1 and type 2 diabetes mellitus. Persistent high concentrations of glucose lead to imbalances in the antioxidant capacity within the cell resulting in oxidative stress-mediated injury in both disorders. An anticipated consequence of impaired autophagy is the accumulation of dysfunctional organelles such as mitochondria within the cell. Mitochondria are the primary site of the production of reactive oxygen species (ROS), and an imbalance in ROS production relative to the cytoprotective action of autophagy may lead to the accumulation of ROS. Impaired mitochondrial function associated with increased ROS levels have been proposed as mechanisms contributing to insulin resistance. In this article we review and interpret the literature that implicates a role for autophagy in the pathophysiology of type 1 and type 2 diabetes mellitus as it applies to β-cell dysfunction, and more broadly to organ systems involved in complications of diabetes including the cardiovascular, renal and nervous systems.

Voltage-dependent calcium currents are enhanced in dorsal root ganglion neurones from the Bio Bred/Worchester diabetic rat.

1. Whole‐cell, high‐threshold, voltage‐dependent calcium currents (ICa) were enhanced in acutely dissociated, capsaicin‐sensitive dorsal root ganglion neurones from diabetic Bio Bred/Worchester (BB/W) rats, compared with those from age‐matched, non‐diabetic controls. The magnitude of the enhancement increased with the duration of diabetes, and reached significance at diabetic durations of 6 months (diabetic: 6.3 +/‐ 0.4 nA; current density (CD), 157 +/‐ 12 pA pF‐1; means +/‐ S.E.M., n = 9, P < 0.01; control: 3.9 +/‐ 0.6 nA; CD, 116 +/‐ 11 pA pF‐1; n = 18) and 8 months (diabetic: 7.6 +/‐ 0.4 nA; CD, 177 +/‐ 25 pA pF‐1; n = 11, P < 0.005; control: 5.1 +/‐ 0.5 nA; CD, 111 +/‐ 26 pA pF‐1; n = 15). Low‐threshold, voltage‐dependent ICa were also enhanced in neurones from animals diabetic for 8 months (diabetic: 2.5 +/‐ 0.7 nA, n = 4, P < 0.05; control: 0.7 +/‐ 0.5 nA, n = 6). 2. The ICa enhancement was prevented by long‐term treatment of diabetic animals with an aldose reductase inhibitor (ARI; peak ICa at 6 months: 4.41 +/‐ 0.48 nA, n = 2; at 8 months: 4.32 +/‐ 0.60 nA, n = 9). 3. The ICa enhancement was not due to a shift in the voltage dependence of either the current‐voltage relationship or steady‐state inactivation. 4. The L channel antagonist nifedipine and preferential N channel antagonist omega‐conotoxin GVIA (omega‐CgTX) caused a greater inhibition of high‐threshold ICa in diabetic neurones compared with controls (nifedipine: control: 25 +/‐ 3%, n = 26; diabetic: 36 +/‐ 7%, n = 11; omega‐CgTX: control: 40 +/‐ 4%, n = 21; diabetic: 50 +/‐ 7%, n = 7). Diabetic neurones also demonstrated a significantly greater residual current (2.44 +/‐ 0.34 nA, n = 7) in the presence of both antagonists vs. controls (1.28 +/‐ 0.30 nA, n = 8, P < 0.05), suggesting that N‐, L‐ and additional non‐N‐, non‐L‐type high‐threshold ICa were enhanced.

Sodium Channel β2 Subunits Regulate Tetrodotoxin-Sensitive Sodium Channels in Small Dorsal Root Ganglion Neurons and Modulate the Response to Pain

Voltage-gated sodium channel (Nav1) β2 subunits modulate channel gating, assembly, and cell-surface expression in CNS neurons in vitro and in vivo. β2 expression increases in sensory neurons after nerve injury, and development of mechanical allodynia in the spared nerve injury model is attenuated in β2-null mice. Thus, we hypothesized that β2 modulates electrical excitability in dorsal root ganglion (DRG) neurons in vivo. We compared sodium currents (INa) in small DRG neurons from β2+/+ and β2−/− mice to determine the effects of β2 on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Nav1 in vivo. Small-fast DRG neurons acutely isolated from β2−/− mice showed significant decreases in TTX-S INa compared with β2+/+ neurons. This decrease included a 51% reduction in maximal sodium conductance with no detectable changes in the voltage dependence of activation or inactivation. TTX-S, but not TTX-R, INa activation and inactivation kinetics in these cells were slower in β2−/− mice compared with controls. The selective regulation of TTX-S INa was supported by reductions in transcript and protein levels of TTX-S Nav1s, particularly Nav1.7. Low-threshold mechanical sensitivity was preserved in β2−/− mice, but they were more sensitive to noxious thermal stimuli than wild type whereas their response during the late phase of the formalin test was attenuated. Our results suggest that β2 modulates TTX-S Nav1 mRNA and protein expression resulting in increased TTX-S INa and increases the rates of TTX-S Nav1 activation and inactivation in small-fast DRG neurons in vivo. TTX-R INa were not significantly modulated by β2.

Participation of gastric mechanoreceptors and intestinal chemoreceptors in the gastrocolonic response

In this study we investigated the site and nature of the signal responsible for the generation of the gastrocolonic response at the rectosigmoid region. Sixteen healthy subjects participated in this study. Motor activities were recorded with pressure transducers placed in the rectosigmoid colon. Balloon distention of the stomach with 100, 200, or 300 ml of water caused a volume-dependent increase in rectosigmoid motility that was abolished by atropine. To investigate the intestinal phase of the gastrocolonic response, we infused into the duodenum isocaloric (178 kcal) solutions of normal saline, lipid, glucose, or essential amino acids at 1.5 ml/min for 30 min in random order on separate days. Only lipid infusion caused an increase in rectosigmoid pressure and this was accompanied by an elevation of plasma cholecystokinin from 1.2 +/- 0.1 to 4.3 +/- 1.0 fmol/ml. The increase in motility associated with lipid infusion was antagonized (58% +/- 7%) by atropine. To further investigate the possible role of cholecystokinin as a mediator of the gastrocolonic response, we infused cholecystokinin-octapeptide intravenously at doses of 5, 10, and 20 ng/kg.h. A significant increase in rectosigmoid motility was observed only at 20 ng/kg.h and this was accompanied by an increase in plasma cholecystokinin levels to 12 +/- 2 fmol/ml, a value threefold greater than that produced by lipid infusion. These studies demonstrate that gastric distention and intestinal lipid are potent stimuli for the generation of the gastrocolonic response involving the rectosigmoid region. The gastric phase can be generated by mechanoreceptors utilizing the cholinergic pathways, whereas the intestinal phase is nutrient-specific, partially atropine-sensitive, and independent of cholecystokinin.

Serum from Patients with Type 2 Diabetes with Neuropathy Induces Complement-independent, Calcium-dependent Apoptosis in Cultured Neuronal Cells

We hypothesized that sera from type 2 diabetic patients with neuropathy contains an autoimmune immunoglobulin that promotes complement-independent, calcium-dependent apoptosis in neuronal cell lines. Neuronal cells were cultured in the presence of complement-inactivated sera obtained from patients with type 2 diabetes with and without neuropathy and healthy adult control patients. Serum from diabetic patients with neuropathy was associated with a significantly greater induction of apoptosis, compared to serum from diabetic patients without neuropathy and controls. In the presence of calcium channel antagonists, induction of apoptosis was reduced by approximately 50%. Pretreatment of neuronal cells with serum from diabetic patients with neuropathy was associated with a significant increase in elevated K+-evoked cytosolic calcium concentration. Serum-induced enhancement in cytosolic calcium and calcium current density was blocked by treatment with trypsin and filtration of the serum using a 100,000-kd molecular weight filter. Treatment with an anti-human IgG antibody was associated with intense fluorescence on the surface of neuronal cells exposed to sera from patients with type 2 diabetes mellitus with neuropathy. We conclude that sera from type 2 diabetic patients with neuropathy contains an autoimmune immunoglobulin that induces complement-independent, calcium-dependent apoptosis in neuronal cells.

Corticosterone Mediates Reciprocal Changes in CB 1 and TRPV1 Receptors in Primary Sensory Neurons in the Chronically Stressed Rat

BACKGROUND & AIMS Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. METHODS Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486 and cannabinoid-receptor agonist WIN55,212-2. The visceromotor response to colorectal distension was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings were used to assess transient receptor potential vanilloid type 1 (TRPV1) currents in L6-S2 dorsal root ganglion (DRG) neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. RESULTS Chronic WA stress was associated with visceral hyperalgesia in response to colorectal distension, increased stool output and reciprocal changes in cannabinoid receptor 1 (CB1) (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ showed a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1, and decreased CB1 receptor protein levels, which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ, which was prevented by co-treatment with RU-486 or WIN55,212-2. CONCLUSIONS These results support a novel role for corticosterone to modulate CB1 and TRPV1-receptor pathways in L6-S2 DRGs in the chronic WA stressed rat, which contributes to visceral hyperalgesia observed in this model.

Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat

Abstract  We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut function: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus. A validated streptozotocin‐induced diabetic rat model and age‐matched healthy controls were studied. After 4–8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase‐3 and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL)‐positive cells in adjacent sections in neurones (PGP 9.5‐positive cells). The level of apoptosis was confirmed using double‐label assessment of caspase‐3 and TUNEL in DRG preparations. Caspase‐3 immunoreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6–8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2–0.5% in controls. Neurones staining positive for both caspase‐3 and TUNEL accounted for 1–2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1–0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL‐positive neurones in diabetic rats to control levels. Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder.

Decreased expression of nitric oxide synthase in the colonic myenteric plexus of aged rats

Nitric oxide (NO) is a major non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the gastrointestinal tract. NO released from the myenteric plexus enhances colonic transit and facilitates propulsion of the colonic contents by mediating descending relaxation. Although it has been suggested that colonic transit delays with aging, the mechanism of delayed colonic transit in aging remains unclear. We hypothesized that advanced age is associated with decreased expression of neuronal NO synthase (nNOS) and concomitant reduction in synthesis of NO in the rat colon. We studied nNOS mRNA expression, nNOS-immunohistochemistry, nNOS-immunoblotting and NOS catalytic activity in the mid-colon obtained from young (age 4-8 months) and aged (age 22-28 months) Fisher (F344xBN)F1 rats. Western blot analysis of PGP 9.5, a generic neuronal marker, of the colonic tissues were employed to study whether the total number of neurons of the myenteric plexus is reduced with aging. The number of nNOS-immunoreactive cells and nNOS synthesis in the colonic myenteric plexus were significantly reduced in aged rats. In contrast, expression of PGP 9.5 in colonic tissues was not affected in aged rats. Northern blot analysis demonstrated that the expression of neuronal nNOS mRNA was significantly reduced in the colonic tissues in aged rats. Basal and veratridine-induced release of L-[(3)H]citrulline were significantly decreased in colonic tissues from aged rats, compared to young rats. It is suggested that advanced age is associated with diminished gene expression of nNOS, nNOS synthesis and catalytic activity of NOS. This may explain the mechanism of delayed colonic transit observed in advanced age.

Sera from patients with type 2 Diabetes and Neuropathy Induce Autophagy and Colocalization with Mitochondria in SY5Y cells

The etiology of diabetic neuropathy is multifactorial and not fully elucidated, although oxidative stress and mitochondrial dysfunction are major factors. We reported previously that complement-inactivated sera from type 2 diabetic patients with neuropathy induce apoptosis in cultured neuronal cells, possibly through an autoimmune immunoglobulin-mediated pathway. Recent evidence supports an emerging role for autophagy in a variety of diseases. Here we report that exposure of human neuroblastoma SH-SY5Y cells to sera from type 2 diabetic patients with neuropathy is associated with increased levels of autophagosomes that is likely mediated by increased titers of IgM or IgG autoimmune immunoglobulins. The increased presence of macroautophagic vesicles was monitored using a specific immunohistochemical marker for autophagosomes, anti-LC3-II immunoreactivity, as well as the immunohistochemical signal for beclin-1, and was associated with increased co-localization with mitochondria in the cells exposed to diabetic neuropathic sera. We also report that dorsal root ganglia removed from streptozotocin-induced diabetic rats exhibit increased levels of autophagosomes and co-localization with mitochondria in neuronal soma, concurrent with enhanced binding of IgG and IgM autoimmune immunoglobulins. To our knowledge, this is the first evidence that the presence of autophagosomes is increased by a serum factor, likely autoantibody(ies) in a pathological condition. Stimulation of autophagy by an autoantibody-mediated pathway can provide a critical link between the immune system and the loss of function and eventual demise of neuronal tissue in type 2 diabetes.

Epigenetic Regulation of Genes That Modulate Chronic Stress-Induced Visceral Pain in the Peripheral Nervous System

BACKGROUND & AIMS Chronic stress alters the hypothalamic-pituitary-adrenal axis, increases gut motility, and increases the perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats. METHODS Male rats were subjected to 1 hour of water avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4-L5 and L6-S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention. RESULTS Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6-S2, but not L4-L5, DRGs. Stress also was associated with up-regulation in DNMT1-associated methylation of the Cnr1 promoter and down-regulation of glucocorticoid-receptor-mediated expression of CNR1 in L6-S2, but not L4-L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6-S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6-S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. CONCLUSIONS Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.