Aspartyl-(asparaginyl) beta-hydroxylase, hypoxia-inducible factor-alpha and Notch cross-talk in regulating neuronal motility.

Aspartyl-(Asparginyl)-ß-Hydroxylase (AAH) promotes cell motility by hydroxylating Notch. Insulin and insulin-like growth factor, type 1 (IGF-I) stimulate AAH through Erk MAPK and phosphoinositol-3-kinase-Akt (PI3K-Akt). However, hypoxia/oxidative stress may also regulate AAH. Hypoxia inducible factor-1 alpha (HIF-1α) regulates cell migration, signals through Notch, and is regulated by hypoxia/oxidative stress, insulin/IGF signaling, and factor inhibiting HIF-1α (FIH) hydroxylation. To examine cross-talk between HIF-1α and AAH, we measured AAH, Notch-1, Jagged-1, FIH, HIF-1α, HIF-1ß, and the hairy and enhancer of split 1 (HES-1) transcription factor expression and directional motility in primitive neuroectodermal tumor 2 (PNET2) human neuronal cells that were exposed to H2O2, or transfected with short interfering RNA duplexes (siRNA) targeting AAH, Notch-1, or HIF-1α. We found that: 1) AAH, HIF-1α, and neuronal migration were stimulated by H2O2; 2) si-HIF-1α reduced AAH expression and cell motility; 3) si-AAH inhibited Notch and cell migration, but not HIF-1α; and 4) si-Notch-1 increased FIH and inhibited HIF-1α. These findings suggest that AAH and HIF-1α cross-talk within a hydroxylation-regulated signaling pathway that may be transiently driven by oxidative stress, and chronically regulated by insulin/IGF signaling.

Rat strain differences in susceptibility to alcohol-induced chronic liver injury and hepatic insulin resistance.

The finding of more severe steatohepatitis in alcohol fed Long Evans (LE) compared with Sprague Dawley (SD) and Fisher 344 (FS) rats prompted us to determine whether host factors related to alcohol metabolism, inflammation, and insulin/IGF signaling predict proneness to alcohol-mediated liver injury. Adult FS, SD, and LE rats were fed liquid diets containing 0% or 37% (calories) ethanol for 8 weeks. Among controls, LE rats had significantly higher ALT and reduced GAPDH relative to SD and FS rats. Among ethanol-fed rats, despite similar blood alcohol levels, LE rats had more pronounced steatohepatitis and fibrosis, higher levels of ALT, DNA damage, pro-inflammatory cytokines, ADH, ALDH, catalase, GFAP, desmin, and collagen expression, and reduced insulin receptor binding relative to FS rats. Ethanol-exposed SD rats had intermediate degrees of steatohepatitis, increased ALT, ADH and profibrogenesis gene expression, and suppressed insulin receptor binding and GAPDH expression, while pro-inflammatory cytokines were similarly increased as in LE rats. Ethanol feeding in FS rats only reduced IL-6, ALDH1-3, CYP2E1, and GAPDH expression in liver. In conclusion, susceptibility to chronic steatohepatitis may be driven by factors related to efficiency of ethanol metabolism and degree to which ethanol exposure causes hepatic insulin resistance and cytokine activation.

Nitrosamine exposure exacerbates high fat diet-mediated type 2 diabetes mellitus, non-alcoholic steatohepatitis, and neurodegeneration with cognitive impairment.

BACKGROUND: The current epidemics of type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NASH), and Alzheimer's disease (AD) all represent insulin-resistance diseases. Previous studies linked insulin resistance diseases to high fat diets or exposure to streptozotocin, a nitrosamine-related compound that causes T2DM, NASH, and AD-type neurodegeneration. We hypothesize that low-level exposure to nitrosamines that are widely present in processed foods, amplifies the deleterious effects of high fat intake in promoting T2DM, NASH, and neurodegeneration. METHODS: Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA) by i.p. Injection, and upon weaning, they were fed with high fat (60%; HFD) or low fat (5%; LFD) chow for 6 weeks. Rats were evaluated for cognitive impairment, insulin resistance, and neurodegeneration using behavioral, biochemical, molecular, and histological methods. RESULTS: NDEA and HFD +/- NDEA caused T2DM, NASH, deficits in spatial learning, and neurodegeneration with hepatic and brain insulin and/or IGF resistance, and reductions in tau and choline acetyltransferase levels in the temporal lobe. In addition, pro-ceramide genes, which promote insulin resistance, were increased in livers and brains of rats exposed to NDEA, HFD, or both. In nearly all assays, the adverse effects of HFD+NDEA were worse than either treatment alone. CONCLUSIONS: Environmental and food contaminant exposures to low, sub-mutagenic levels of nitrosamines, together with chronic HFD feeding, function synergistically to promote major insulin resistance diseases including T2DM, NASH, and AD-type neurodegeneration. Steps to minimize human exposure to nitrosamines and consumption of high-fat content foods are needed to quell these costly and devastating epidemics.

Hepatic ceramide may mediate brain insulin resistance and neurodegeneration in type 2 diabetes and non-alcoholic steatohepatitis.

Obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic steatohepatitis (NASH) can be complicated by cognitive impairment and neurodegeneration. Experimentally, high fat diet (HFD)-induced obesity with T2DM causes mild neurodegeneration with brain insulin resistance. Since ceramides are neurotoxic, cause insulin resistance, and are increased in T2DM, we investigated the potential role of ceramides as mediators of neurodegeneration in the HFD obesity/T2DM model. We pair-fed C57BL/6 mice with a HFD or control diet for 4-20 weeks and examined pro-ceramide gene expression in liver and brain and neurodegeneration in the temporal lobe. HFD feeding gradually increased body weight, but after 16 weeks, liver weight surged (P<0.001) due to lipid (triglyceride) accumulation (P<0.001), and brain weight declined (P<0.0001-Trend analysis). HFD feeding increased ceramide synthase, serine palmitoyl transferase, and sphingomyelinase expression in liver (P<0.05-P<0.001), but not brain. In HFD fed mice, temporal lobe levels of ubiquitin (P<0.001) and 4-hydroxynonenal (P<0.05 or P<0.01) increased, and tau, beta-actin, and choline acetyltransferase levels decreased (P<0.05-P<0.001) with development of NASH. In obesity, T2DM, or NASH, neurodegeneration with brain insulin resistance may be mediated by excess hepatic production of neurotoxic ceramides that readily cross the blood-brain barrier.

Epidemilogical trends strongly suggest exposures as etiologic agents in the pathogenesis of sporadic Alzheimer's disease, diabetes mellitus, and non-alcoholic steatohepatitis.

Nitrosamines mediate their mutagenic effects by causing DNA damage, oxidative stress, lipid peroxidation, and pro-inflammatory cytokine activation, which lead to increased cellular degeneration and death. However, the very same pathophysiological processes comprise the "unbuilding" blocks of aging and insulin-resistance diseases including, neurodegeneration, diabetes mellitus (DM), and non-alcoholic steatohepatitis (NASH). Previous studies demonstrated that experimental exposure to streptozotocin, a nitrosamine-related compound, causes NASH, and diabetes mellitus Types 1, 2 and 3 (Alzheimer (AD)-type neurodegeneration). Herein, we review evidence that the upwardly spiraling trends in mortality rates due to DM, AD, and Parkinson's disease typify exposure rather than genetic-based disease models, and parallel the progressive increases in human exposure to nitrates, nitrites, and nitrosamines via processed/preserved foods. We propose that such chronic exposures have critical roles in the pathogenesis of our insulin resistance disease pandemic. Potential solutions include: 1) eliminating the use of nitrites in food; 2) reducing nitrate levels in fertilizer and water used to irrigate crops; and 3) employing safe and effective measures to detoxify food and water prior to human consumption. Future research efforts should focus on refining our ability to detect and monitor human exposures to nitrosamines and assess early evidence of nitrosamine-mediated tissue injury and insulin resistance.

Nitrosamine exposure causes insulin resistance diseases: relevance to type 2 diabetes mellitus, non-alcoholic steatohepatitis, and Alzheimer's disease.

The current epidemics of type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NASH), and Alzheimer's disease (AD) all represent insulin-resistance diseases. Previous studies showed that streptozotocin, a nitrosamine-related com-pound, causes insulin resistance diseases including, T2DM, NASH, and AD-type neurodegeneration. We hypothesize that chronic human exposure to nitrosamine compounds, which are widely present in processed foods, contributes to the pathogenesis of T2DM, NASH, and AD. Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA) by i.p. (x3) or i.c. (x1) injection, and 2-4 weeks later, they were evaluated for cognitive-motor dysfunction, insulin resistance, and neurodegeneration using behavioral, biochemical, and molecular approaches. NDEA treatment caused T2DM, NASH, deficits in motor function and spatial learning, and neurodegeneration characterized by insulin resistance and deficiency, lipid peroxidation, cell loss, increased levels of amyloid-beta protein precursor/amyloid-beta, phospho-tau, and ubiquitin immunoreactivities, and upregulated expression of pro-inflammatory cytokine and pro-ceramide genes, which together promote insulin resistance. In conclusion, environmental and food contaminant exposures to nitrosamines play critical roles in the pathogenesis of major insulin resistance diseases including T2DM, NASH, and AD. Improved detection and prevention of human exposures to nitrosamines will lead to earlier treatments and eventual quelling of these costly and devastating epidemics.