Lisa Longato

Limited Alzheimer-Type Neurodegeneration in Experimental Obesity and Type 2 Diabetes Mellitus

Alzheimers disease (AD) is associated with brain insulin resistance and insulin deficiency, whereas Type 2 diabetes mellitus (T2DM) is associated with peripheral insulin resistance. This study assesses the degree to which T2DM causes AD-type neurodegeneration. In a C57BL/6 mouse model of obesity and T2DM, we characterized the histopathology, gene expression, and insulin and insulin-like growth factor (IGF)-receptor binding in temporal lobe. High fat diet (HFD) feeding for 16 weeks doubled mean body weight, caused T2DM, and marginally reduced mean brain weight. These effects were associated with significantly increased levels of tau, IGF-I receptor, insulin receptor substrate-1 (IRS-1), IRS-4, ubiquitin, glial fibrillary acidic protein, and 4-hydroxynonenol, and decreased expression of beta-actin. HFD feeding also caused brain insulin resistance manifested by reduced BMAX for insulin receptor binding, and modestly increased brain insulin gene expression. However, HFD-fed mouse brains did not exhibit AD histopathology, increases in amyloid-beta or phospho-tau, or impairments in IGF signaling or acetylcholine homeostasis. Obesity and T2DM cause brain atrophy with insulin resistance, oxidative stress, and cytoskeleton degradation, but the absence of many features that typify AD suggests that obesity and T2DM may contribute to, but are not sufficient to cause AD.

The liver-brain axis of alcohol-mediated neurodegeneration: role of toxic lipids.

Alcohol abuse causes progressive toxicity and degeneration in liver and brain due to insulin resistance, which exacerbates oxidative stress and pro-inflammatory cytokine activation. Alcohol-induced steatohepatitis promotes synthesis and accumulation of ceramides and other toxic lipids that cause insulin resistance. Ceramides can readily cross the blood-brain barrier, and ceramide exposure causes neurodegeneration with insulin resistance and oxidative stress, similar to the effects of alcohol. Therefore, in addition to its direct neurotoxic effects, alcohol misuse establishes a liver-brain axis of neurodegeneration mediated by toxic lipid trafficking across the blood-brain barrier, leading to progressive white matter degeneration and cognitive impairment.

PPARδ agonist attenuates alcohol-induced hepatic insulin resistance and improves liver injury and repair

BACKGROUND/AIMS Chronic ethanol exposure impairs liver regeneration due to inhibition of insulin signaling and oxidative injury. PPAR agonists function as insulin sensitizers and anti-inflammatory agents. We investigated whether treatment with a PPARdelta agonist could restore hepatic insulin sensitivity, survival signaling, and regenerative responses vis-a-vis chronic ethanol feeding. METHODS Adult rats were fed isocaloric liquid diets containing 0% or 37% ethanol, and administered a PPARdelta agonist by i.p. injection. We used liver tissue to examine histopathology, gene expression, oxidative stress, insulin signaling, and regenerative responses to 2/3 hepatectomy. RESULTS Chronic ethanol feeding caused insulin resistance, increased oxidative stress, lipid peroxidation, DNA damage, and hepatocellular injury in liver. These effects were associated with reduced insulin receptor binding and affinity, impaired survival signaling through PI3K/Akt/GSK3beta, and reduced expression of insulin responsive genes mediating energy metabolism and tissue remodeling. PPARdelta agonist treatment reduced ethanol-mediated hepatic injury, oxidative stress, lipid peroxidation, and insulin resistance, increased signaling through PI3K/Akt/GSK3beta, and enhanced the regenerative response to partial hepatectomy. CONCLUSIONS PPARdelta agonist administration may attenuate the severity of chronic ethanol-induced liver injury and ethanols adverse effects on the hepatic repair by restoring insulin responsiveness, even in the context of continued high-level ethanol consumption.

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.

Insulin resistance in experimental alcohol-induced liver disease

Background and Aim:  Chronic ethanol consumption impairs liver regeneration due, in part, to inhibition of insulin signaling. This study characterizes the mechanisms and consequences of ethanol‐impaired insulin signaling in relation to oxidative injury and altered gene expression.

Ceramide-Mediated Insulin Resistance and Impairment of Cognitive-Motor Functions

Obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic steatohepatitis (NASH) are associated with cognitive impairment, brain insulin resistance, and neurodegeneration. Recent studies linked these effects to increased pro-ceramide gene expression in liver and increased ceramide levels in serum. Since ceramides are neurotoxic and cause insulin resistance, we directly examined the role of ceramides as mediators of impaired signaling and central nervous system function using an in vivo model. Long Evans rat pups were administered C2Cer:N-acetylsphinganine or its inactive dihydroceramide analog (C2DCer) by i.p. injection. Rats were subjected to rotarod and Morris water maze tests of motor and cognitive function, and livers and brains were examined for histopathology and integrity of insulin/IGF signaling. C2Cer treatment caused hyperglycemia, hyperlipidemia, and mild steatohepatitis, reduced brain lipid content, and increased ceramide levels in liver, brain, and serum. Quantitative RT-PCR analysis revealed significant alterations in expression of several genes needed for insulin and IGF-I signaling, and multiplex ELISAs demonstrated inhibition of signaling through the insulin or IGF-1 receptors, IRS-1, and Akt in both liver and brain. Ultimately, the toxic ceramides generated in peripheral sources such as liver or adipose tissue caused sustained impairments in neuro-cognitive function and insulin/IGF signaling needed for neuronal survival, plasticity, and myelin maintenance in the brain. These findings support our hypothesis that a liver/peripheral tissue-brain axis of neurodegeneration, effectuated by increased toxic lipid/ceramide production and transport across the blood-brain barrier, could mediate cognitive impairment in T2DM and NASH.

Insulin Resistance, Ceramide Accumulation, and Endoplasmic Reticulum Stress in Human Chronic Alcohol-Related Liver Disease

Background. Chronic alcohol-related liver disease (ALD) is mediated by insulin resistance, mitochondrial dysfunction, inflammation, oxidative stress, and DNA damage. Recent studies suggest that dysregulated lipid metabolism with accumulation of ceramides, together with ER stress potentiate hepatic insulin resistance and may cause steatohepatitis to progress. Objective. We examined the degree to which hepatic insulin resistance in advanced human ALD is correlated with ER stress, dysregulated lipid metabolism, and ceramide accumulation. Methods. We assessed the integrity of insulin signaling through the Akt pathway and measured proceramide and ER stress gene expression, ER stress signaling proteins, and ceramide profiles in liver tissue. Results. Chronic ALD was associated with increased expression of insulin, IGF-1, and IGF-2 receptors, impaired signaling through IGF-1R and IRS1, increased expression of multiple proceramide and ER stress genes and proteins, and higher levels of the C14, C16, C18, and C20 ceramide species relative to control. Conclusions. In human chronic ALD, persistent hepatic insulin resistance is associated with dysregulated lipid metabolism, ceramide accumulation, and striking upregulation of multiple ER stress signaling molecules. Given the role of ceramides as mediators of ER stress and insulin resistance, treatment with ceramide enzyme inhibitors may help reverse or halt progression of chronic ALD.

Overexpression of insulin receptor substrate-1 and hepatitis Bx genes causes premalignant alterations in the liver†‡

Activation of the insulin (IN)/insulin receptor substrate‐1 (IRS‐1)/mitogen‐associated protein kinase (MAPK) and the Wnt/β‐catenin signaling cascades occurs frequently in hepatocellular carcinoma (HCC) associated with persistent viral infection. The aims of this study were to provide a chronic proliferative stimulus through IRS‐1 in the context of hepatitis Bx (HBx) protein expression in transgenic mice and determine if constitutive expression of these genes is sufficient to cause hepatocyte dysplasia and cellular transformation. We generated transgenic mice in which the HBx (ATX), IRS‐1, or both (ATX+/IRS‐1) genes were expressed under a liver‐specific promoter. We also assessed histology and oxidative damage as well as up‐regulation of molecules related to these signal transduction cascades in the liver by quantitative reverse‐transcriptase polymerase chain reaction. Whereas mice with a single transgene (ATX or IRS‐1) did not develop tumors, ATX+/IRS‐1+ double transgenic livers had increased frequency of hepatocellular dysplasia and developed HCC. All three transgenic lines had significantly increased insulin growth factor 1 (IGF‐1), Wnt 1 and Wnt 3 mRNA levels, and evidence of DNA damage and oxidative stress. The ATX+/IRS+ double transgenic mice were distinguished by having the highest level of activation of Wnt 3 and Frizzled 7 and selectively increased expression of IGF‐II, proliferating cell nuclear antigen, and aspartyl‐(asparaginyl)‐β‐hydroxylase, a gene associated with increased cell migration. Conclusion: These results suggest that continued expression of the ATX or IRS‐1 transgenes can contribute to hepatocyte transformation but are not sufficient to trigger neoplastic changes in the liver. However, dual expression that activates both the IN/IRS‐1/MAPK and Wnt/β‐catenin cascades is sufficient to cause dysplasia and HCC in a previously normal liver. (HEPATOLOGY 2009;49:1935‐1943.)

Acetaldehyde-Mediated Neurotoxicity: Relevance to Fetal Alcohol Spectrum Disorders

Ethanol-induced neuro-developmental abnormalities are associated with impaired insulin and IGF signaling, and increased oxidative stress in CNS neurons. We examined the roles of ethanol and its principal toxic metabolite, acetaldehyde, as mediators of impaired insulin/IGF signaling and oxidative injury in immature cerebellar neurons. Cultures were exposed to 3.5 mM acetaldehyde or 50 mM ethanol ± 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism, and viability, mitochondrial function, oxidative stress, DNA damage, and insulin responsiveness were measured 48 hours later. Acetaldehyde or ethanol increased neuronal death and levels of 8-OHdG and 4-HNE, and reduced mitochondrial function. Ethanol inhibited insulin responsiveness, whereas acetaldehyde did not. 4-MP abated ethanol-induced oxidative stress and mitochondrial dysfunction, but failed to restore insulin responsiveness. Furthermore, alcohol and aldehyde metabolizing enzyme genes were inhibited by prenatal ethanol exposure; this effect was mediated by acetaldehyde and not ethanol + 4MP. These findings suggest that brain insulin resistance in prenatal alcohol exposure is caused by direct effects of ethanol, whereas oxidative stress induced neuronal injury is likely mediated by ethanol and its toxic metabolites. Moreover, the adverse effects of prenatal ethanol exposure on brain development may be exacerbated by down-regulation of genes needed for metabolism and detoxification of alcohol in the brain.

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

BackgroundThe current epidemics of type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NASH), and Alzheimers 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.MethodsLong 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.ResultsNDEA 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.ConclusionsEnvironmental 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.