Enteric neuron degeneration has been observed during aging, and in individuals with metabolic dysfunction including obesity and diabetes. Honokiol, a naturally occurring compound, is an activator of Sirtuin-3 (SIRT3) that has antioxidant activity. Its role in modulating enteric neuron-specific neurodegeneration is unknown. We studied the effects of honokiol and its fluorinated analog, hexafluoro-honokiol, on enteric neuronal differentiation and survival. We used a previously established model of mouse primary enteric neuronal cells and an enteric neuronal cell line treated with palmitate (PA) and lipopolysaccharide (LPS) to induce mitochondrial dysfunction and enteric neuronal cell death. The effect of honokiol and hexafluoro-honokiol was assessed on neuronal phenotype, fiber density, differentiation, and pyroptosis. Honokiol and hexafluoro-honokiol significantly increased neuronal networks and fiber density in enteric neurons and increased levels of neuronal nitric oxide synthase and Choline acetyltransferase mRNA. Hexafluoro-honokiol and honokiol also significantly increased SIRT3 mRNA levels and suppressed palmitate and LPS-induced neuronal pyroptosis. SIRT3 knock-down prevented the hexafluoro-honokiol mediated suppression of mitochondrial superoxide release. Our data supports a neuroprotective effect of honokiol and its derivative and these could be used as prophylactic or therapeutic agents for treating enteric neurodegeneration and associated motility disorders.
Nonalcoholic fatty liver disease (NAFLD) is associated with increased oxidative stress that leads to hepatocyte and mitochondrial damage. In this study we investigated the mechanisms involved in the induction of oxidative stress and impairment of mitochondrial quality control and mitophagy in hepatocytes by the saturated fatty acid palmitate and Western diet feeding in mice and if their harmful effects could be reversed by the neurotrophic factor glial cell derived neurotrophic factor (GDNF). Western diet (WD)-feeding increased hepatic lipid peroxidation in control mice and, in vitro palmitate induced oxidative stress and impaired the mitophagic clearance of damaged mitochondria in hepatocytes. This was accompanied by reductions in hepatocyte sirtuin 3 (SIRT3) deacetylase activity, gene expression and protein levels as well as in superoxide dismutase enzyme activity. These reductions were reversed in the liver of Western diet fed GDNF transgenic mice and in hepatocytes exposed to palmitate in the presence of GDNF. We demonstrate an important role for Western diet and palmitate in inducing oxidative stress and impairing mitophagy in hepatocytes and an ability of GDNF to prevent this. These findings suggest that GDNF or its agonists may be a potential therapy for the prevention or treatment of NAFLD.
Background: Diarrhea is present in up to 30–50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte–enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. Methods: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. Conclusions: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.
The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-β) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and β-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP+) compared with those cultured with MPP+ alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-β signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.
Neurodegeneration of the central and enteric nervous systems is a common feature of aging and aging-related diseases, and is accelerated in individuals with metabolic dysfunction including obesity and diabetes. The molecular mechanisms of neurodegeneration in both the CNS and ENS are overlapping. Sirtuins are an important family of histone deacetylases that are important for genome stability, cellular response to stress, and nutrient and hormone sensing. They are activated by calorie restriction (CR) and by the coenzyme, nicotinamide adenine dinucleotide (NAD+). Sirtuins, specifically the nuclear SIRT1 and mitochondrial SIRT3, have been shown to have predominantly neuroprotective roles in the CNS while the cytoplasmic sirtuin, SIRT2 is largely associated with neurodegeneration. A systematic study of sirtuins in the ENS and their effect on enteric neuronal growth and survival has not been conducted. Recent studies, however, also link sirtuins with important hormones such as leptin, ghrelin, melatonin, and serotonin which influence many important processes including satiety, mood, circadian rhythm, and gut homeostasis. In this review, we address emerging roles of sirtuins in modulating the metabolic challenges from aging, obesity, and diabetes that lead to neurodegeneration in the ENS and CNS. We also highlight a novel role for sirtuins along the microbiota-gut-brain axis in modulating neurodegeneration.
Background
Development of pre-transplantation islet culture strategies that preserve or enhance β-cell viability would eliminate the requirement for the large numbers of islets needed to restore insulin independence in type 1 diabetes patients. We investigated whether glial cell line-derived neurotrophic factor (GDNF) could improve human islet survival and post-transplantation function in diabetic mice.
Methods
Human islets were cultured in medium supplemented with or without GDNF (100 ng/ml) and in vitro islet survival and function assessed by analyzing β-cell apoptosis and glucose stimulated insulin release. In vivo effects of GDNF were assessed in streptozotocin-induced diabetic nude mice transplanted under the kidney capsule with 2000 islet equivalents of human islets pre-cultured in medium supplemented with or without GDNF.
Results
In vitro, human islets cultured for 2–10 days in medium supplemented with GDNF showed lower β-cell death, increased Akt phosphorylation and higher glucose-induced insulin secretion than islets cultured in vehicle. Human islets pre-cultured in medium supplemented with GDNF restored more diabetic mice to normoglycemia and for a longer period after transplantation than islets cultured in vehicle.
Conclusions
Our study shows that GDNF has beneficial effects on human islet survival and could be used to improve islet post-transplantation survival.
Glial cell line-derived neurotrophic factor (GDNF) is a factor produced by glial cells that is required for the development of the enteric nervous system. In transgenic mice that overexpress GDNF in the pancreas, GDNF has been shown to enhance β-cell mass and improve glucose control, but the transcriptional and cellular processes involved are not known. In this study we examined the influence of GDNF on the expression of neurogenin3 (Ngn3) and other transcription factors implicated in early β-cell development, as well as on β-cell proliferation during embryonic and early postnatal mouse pancreas development. Embryonic day 15.5 (E15.5) mouse pancreatic tissue when exposed to GDNF for 24 h showed higher Ngn3, pancreatic and duodenal homeobox gene 1 (Pdx1), neuroD1/β2, paired homeobox gene 4 (Pax4), and insulin mRNA expression than tissue exposed to vehicle only. Transgenic expression of GDNF in mouse pancreata was associated with increased numbers of Ngn3-expressing pancreatic cells and higher β-cell mass at embryonic day 18 (E18), as well as higher β-cell proliferation and Pdx1 expression in β-cells at E18 and postnatal day 1. In the HIT-T15 β-cell line, GDNF enhanced the expression of Pax6. This response was, however, blocked in the presence of Pdx1 small interfering RNA (siRNA). Chromatin immunoprecipitation studies using the HIT-T15 β-cell line demonstrated that GDNF can influence Pdx1 gene expression by enhancing the binding of Sox9 and neuroD1/β2 to the Pdx1 promoter. Our data provide evidence of a mechanism by which GDNF influences β-cell development. GDNF could be a potential therapeutic target for the treatment and prevention of diabetes.
Glial cell line–derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid β-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding.
In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid β-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid–induced hepatic lipotoxicity.
Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic β-cells. Although islet transplantation has shown great potential in the treatment of hyperglycemia in type 1 diabetes patients, availability of donor islets remains a major obstacle. Cell replacement strategies aimed at generating functional insulin-producing cells through differentiation of adult pancreatic stem cells/progenitors have been hindered by a lack of suitable markers for their identification.
Background & Aims: High-fat diet (HFD) feeding is associated with gastrointestinal motility disorders. We recently reported delayed colonic motility in mice fed a HFD for 11 weeks. In this study, we investigated the contributing role of gut microbiota in HFD-induced gut dysmotility. Methods: Male C57BL/6 mice were fed a HFD (60% kcal fat) or a regular/control diet (RD) (18% kcal fat) for 13 weeks. Serum and fecal endotoxin levels were measured, and relative amounts of specific gut bacteria in the feces were assessed by real-time polymerase chain reaction. Intestinal transit was measured by fluorescent-labeled marker and a bead expulsion test. Enteric neurons were assessed by immunostaining. Oligofructose (OFS) supplementation with RD or HFD for 5 weeks also was studied. In vitro studies were performed using primary enteric neurons and an enteric neuronal cell line. Results: HFD-fed mice had reduced numbers of enteric nitrergic neurons and showed delayed gastrointestinal transit compared with RD-fed mice. HFD-fed mice had higher fecal Firmicutes and Escherichia coli and lower Bacteroidetes compared with RD-fed mice. OFS supplementation protected against enteric nitrergic neuron loss in HFD-fed mice, and improved intestinal transit time. OFS supplementation resulted in a reduction in fecal Firmicutes and Escherichia coli and serum endotoxin levels. In vitro, palmitate activation of TLR4 induced enteric neuronal apoptosis in a Phospho-c-Jun N-terminal kinase-dependent pathway. This apoptosis was prevented by a c-Jun N-terminal kinase inhibitor and in neurons from TLR4-/- mice. Conclusions: Together our data suggest that intestinal dysbiosis in HFD-fed mice contribute to the delayed intestinal motility by inducing a TLR4-dependent neuronal loss. Manipulation of gut microbiota with OFS improved intestinal motility in HFD mice.