Tobacco Smoke Exposure Impairs Brain Insulin/IGF Signaling: Potential Co-Factor Role in Neurodegeneration

Abstract

Background:

Human studies suggest tobacco smoking is a risk factor for cognitive impairment and neurodegeneration, including Alzheimer’s disease (AD). However, experimental data linking tobacco smoke exposures to underlying mediators of neurodegeneration, including impairments in brain insulin and insulin-like growth factor (IGF) signaling in AD are lacking.

Objective:

This study tests the hypothesis that cigarette smoke (CS) exposures can impair brain insulin/IGF signaling and alter expression of AD-associated proteins.

Methods:

Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 or 8 weeks (CS4, CS8), or CS8 followed by 2 weeks recovery (CS8+R). Gene expression was measured by qRT-PCR analysis and proteins were measured by multiplex bead-based or direct binding duplex ELISAs.

Results:

CS exposure effects on insulin/IGF and insulin receptor substrate (IRS) proteins and phosphorylated proteins were striking compared with the mRNA. The main consequences of CS4 or CS8 exposures were to significantly reduce insulin R, IGF-1R, IRS-1, and tyrosine phosphorylated insulin R and IGF-1R proteins. Paradoxically, these effects were even greater in the CS8+R group. In addition, relative levels of ^S312-IRS-1, which inhibits downstream signaling, were increased in the CS4, CS8, and CS8+R groups. Correspondingly, CS and CS8+R exposures inhibited expression of proteins and phosphoproteins required for signaling through Akt, PRAS40, and/or p70S6K, increased AβPP-Aβ, and reduced ASPH protein, which is a target of insulin/IGF-1 signaling.

Conclusion:

Secondhand CS exposures caused molecular and biochemical abnormalities in brain that overlap with the findings in AD, and many of these effects were sustained or worsened despite short-term CS withdrawal.

Keywords

Alzheimer’s disease cigarette smoke insulin resistance mouse model neurodegeneration

INTRODUCTION

The sharply increased prevalence rates of Alzheimer’s disease (AD) within all age groups from 45 to 85 over the past several decades, i.e., even after correcting for age, suggest that exposure-related co-factors contribute to its pathogenesis [1]. Growing evidence supports the concept that AD is largely a brain metabolic disorder with molecular and biochemical features shared in common with diabetes mellitus [2 –5]. Therefore, it is critical that we identify environmental agents that disrupt insulin and/or insulin-like growth factor (IGF) signaling networks, particularly in the brain. Furthermore, the frequent co-occurrence of AD with subclinical or preclinical systemic insulin resistance states, obesity, diabetes, or non-alcoholic fatty liver disease suggests that these disease clusters share underlying mechanisms [6 –13]. This concept is reinforced by data showing parallel epidemics of various insulin resistance diseases that have emerged across a broad age range and within the past 4-5 decades [1 , 14–17]. Such phenomena cannot be explained by increased longevity or strong genetic risk factors. Finally, data linking AD-associated familial gene mutations and risk factors to progressive brain insulin resistance or impaired insulin signaling [3 , 18–23] demonstrate convergence toward the metabolic hypothesis, helping to explain why familial AD takes decades to develop vis-à-vis abnormal genotypes from the moment of conception.

Streptozotocin (STZ) is a well-characterized toxin that is used to generate models of Type 1 or Type 2 diabetes mellitus [24, 25]. The findings that low-dose intracerebral (i.c.) administration of STZ causes cognitive impairment and AD-type neurodegeneration with deficits in brain insulin and insulin-like growth factor (IGF) signaling and energy metabolism [26 –28], and that human brains with sporadic AD have molecular, biochemical, and metabolic abnormalities that are virtually identical to those in the i.c. STZ model that worsen with disease stage advancement had two main effects. It gained interest in the use of i.c. STZ as an experimental model of sporadic AD, and it led to the concept that AD is a metabolic degenerative disease driven by progressive impairment of brain insulin/IGF signaling and attendant neuroinflammation, oxidative stress, and activation of unfolded protein response pathways [5 , 29–37], prompting the term, ‘type 3 diabetes’ [35]. However, STZ is actually a nitrosamine [24] that has genotoxic effects [38], which when administered systemically at high doses causes cancer [24, 38], and at low doses, peripheral insulin resistance with Type 2 diabetes and steatohepatitis, i.e., fatty liver disease [39, 40]. Since streptozotocin is not available to the general population, we hypothesized that environmental and lifestyle exposures to other nitrosamines may be responsible for various forms of insulin resistance-type diseases, including AD [41 –43]. Furthermore, the aggregate observations led us to the current concept that like STZ, nitrosamines in general have two faces of evil: the ‘high-dose face’ causes cancer, while the ‘low-dose face’ causes insulin resistance-mediated degenerative diseases. Interestingly, this concept is supported by epidemiologic studies linking betel nut (Areca) chewing to Type 2 diabetes [44, 45], metabolic syndrome [46, 47], and cancer [48 –51]. Betel nut contains specific nitrosamines, i.e., N-nitrosoguvacoline and N-nitrososoguvacine [52]. However, betel nut consumption mainly occurs in Southeast Asia and China. In Western societies, the two main nitrosamine exposures linked to lifestyles and habits are N-nitrosodiethylamine (NDEA) and tobacco-specific nitrosamines.

NDEA exposure occurs via consumption of in cured meats, cheeses, beer, and processed foods that contain nitrate or nitrite preservatives [1 , 54]. With high heat, chemical reactions of sodium nitrite or nitrate with proteins lead to the formation of nitrosamines, e.g., NDEA. High doses of NDEA are carcinogenic [55 –57], whereas chronic low-level exposures cause a spectrum of insulin resistance diseases, including steatohepatitis, type 2 diabetes, and AD-type neurodegeneration with cognitive impairment [41]. All of these effects are exacerbated by chronic high-fat diet feeding [58, 59].

The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is highly abundant carcinogen present tobacco smoke [60 –63]. However, recently we demonstrated that like other nitrosamines, low doses of NNK are not carcinogenic, and instead lead to hyperglycemia, hyperlipidemia, steatohepatitis with impairments in insulin and IGF-1 signaling through PI3K-Akt pathways, and increased DNA damage, lipid peroxidation, and pro-inflammatory cytokine activation [64]. Further studies demonstrated that NNK exposures cause neurodegeneration and impair spatial learning and memory and brain insulin/IGF-1 signaling [65 –67]. With due urgency from a public health perspective, we extended our efforts by investigating the adverse effects of tobacco smoke exposures on the integrity of insulin/IGF signaling mechanisms in the brain.

A few studies have already linked chronic cigarette smoking to increased rates of neurocognitive dysfunction [68 –70] and structural abnormalities in the brain including volumetric changes in white matter [70 –72], and gray matter atrophy in the temporal and parietal lobes [70 , 73] as revealed by neuroimaging [72 , 74–82]. Furthermore, meta-analysis demonstrated significant correlations between smoking and atrophy of gray matter structures in the anterior cingulate, prefrontal cortex, and cerebellum [83]. Epidemiological investigations have shown higher rates of cigarette smoking among patients with AD compared with normal aged controls [81 , 84–90]. The present study examines the effects of secondhand (sidestream) cigarette smoke (CS) exposures and a brief period of CS withdrawal on brain insulin/IGF signaling mechanisms and several indices of AD-type neurodegeneration in adult male A/J mice.

METHODS

Experimental model

These studies utilized an A/J mouse model similar to the one developed in 2002 [91]. The A/J strain was used because of their high susceptibility to lung defects after tobacco smoke exposure [92]. Furthermore, the A/J model replicates the human experience in that following chronic (5 months) tobacco smoke exposure, plasma cotinine levels were found to be comparable to those in active human smokers, and the mice develop emphysema and lung tumors [91, 93]. The shorter-term exposures that we employed do not produce these end-point diseases [93 –95]. Adult (8 weeks old) A/J male mice (n = 5–6/group) were exposed to CS or air as follows: 1) 8 weeks of room air only (A8); 2) 4 weeks of CS (CS4); 3) CS8; 4) CS8 followed by 2 weeks recovery (CS8+R) as described previously [94, 95]. Group sizes were limited by the chamber volume. CS was generated from research grade Kentucky 3R4F cigarettes (Tobacco Research Institute, University of Kentucky, Lexington, KY) using an industry standard Teague Enterprises TE-10 Smoking Machine (Davis, CA). Cigarettes contained 11 mg of total particulate matter (TPM) and 0.73 mg of nicotine. Sidestream and mainstream smoke were mixed in a ratio of 89% to 11% , which is similar to environmental tobacco smoke exposures. Six cigarettes were puffed simultaneously, one time per minute for 9 puffs. The cigarettes were burned for 6 hours/day, 5 days/week and for 4 or 8 weeks duration.

Mice were adapted to CS by ramping up concentration and exposure period in the first week. The chamber atmosphere was monitored for total suspended particles. Under these conditions, the chamber atmosphere had 21% oxygen and approximately 24 ppm of carbon monoxide. Before use, the cigarettes were kept for 48 h in a standardized atmosphere humidified with 70% glycerol-30% water. Throughout the experiment, mice were housed under humane conditions and kept on a 12-h light/dark cycle with free access to food. At the end of the experiment, freshly harvested brains were micro-dissected and portions were snap frozen and stored at –80°C for biochemical and molecular studies. All experiments were performed in accordance with protocols approved by the University of Southern California’s Institutional Animal Care and Use Committee, and conformed to guidelines established by the National Institutes of Health.

Gene expression studies

Total RNA was extracted from the frontal lobes using the RNeasy Mini Kit. The cDNA templates were generated with the AMV 1st Strand cDNA Synthesis Kit. Duplex probe hydrolysis-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays [96] were used to measure mRNA transcripts encoding insulin, IGF-1, and IGF-2 polypeptides, their corresponding receptors, and insulin receptor substrates (IRS) 1 and 2. Gene expression probes were labeled with FAM and the internal control probe for β-actin was labeled with Y555. PCR amplifications were performed in a LightCycler 480 PCR machine and results were analyzed using the LightCycler Software 4.0. See Supplementary Table 1 for primer sequences and probes.

Insulin/IGF-1 signaling networks

We used bead-based multiplex Enzyme-linked immunosorbent assays (ELISAs) to measure immunoreactivity to the insulin receptor (InsulinR), IGF-1 receptor (IGF-1R), IRS-1, Akt, proline-rich Akt substrate of 40 kDa (PRAS40), ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3β (GSK-3β), and ^{pYpY1162/1163}-InsulinR, ^{pYpY1135/1136}-IGF-1R, ^pS312-IRS-1, ^pS473-Akt, ^pT246-PRAS40, ^pTpS421/424-p70S6K, and ^pS9-GSK-3β. Samples (50μg protein) were incubated with magnetic beads coated with capture antibodies and immunoreactivity was detected with another epitope-specific primary antibody conjugated to phycoerythrin. Plates were read in a MAGPIX (Bio-Rad, Hercules, CA).

ELISAs for detecting other cellular proteins

Direct binding duplex ELISAs were used to measure immunoreactivity to tau, phospho-tau (S396 and T205), amyloid-beta protein precursor-amyloid-beta (AβPP-Aβ), ubiquitin, 8-hydroxy-deoxy-guanine (8-OHdG), and aspartyl-asparaginyl-β-hydroxylase (ASPH). Target immunoreactivity was measured by ELISA and normalized to large ribosomal protein (RPLPO) directly measured in the same wells [97]. To do this, frozen brain tissue was homogenized in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA acid pH 8.0, 50 mM NaF, 0.1% triton x-100) in the presence of protease and phosphatase inhibitors [98 –101]. Homogenates were centrifuged to obtain supernatant fractions for analysis. Protein concentration was measured by the Bicinchoninic assay (BCA) assay. Protein homogenates (100 ng/50μl) were adsorbed onto the bottom surfaces of maxisorp 96-well plates by overnight incubation on a rotating platform. Non-specific binding was prevented by incubation with 1% bovine serum albumin in TRIS buffered saline. The samples were incubated with primary antibodies (0.5–1μg/ml) overnight at 4°C and immunoreactivity was detected with species-specific secondary antibodies conjugated to horseradish peroxidase and Amplex UltraRed soluble fluorophore (Ex 530 nm/Em 590 nm). Subsequently, the samples were incubated with biotinylated RPLPO (Proteintech Group Inc, Chicago, IL) and immunoreactivity was detected with streptavidin-conjugated alkaline phosphatase (1 : 1000) and the 4-Methylumbelliferyl phosphate (4-MUP) substrate (Ex 360 nm/Em 450 nm). Fluorescence was measured in a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Positive and negative controls for non-specific binding (primary or secondary antibody omissions) were incorporated into all studies. Calculated target protein/RPLPO ratios were used for inter-group comparisons.

Sources of reagents

Antibodies to tau (ab64193), phospho S396-tau (ab109390), phospho T205-tau (ab4841), ubiquitin (Ubi-1), and 8-OHdG (ab62623), and AβPP-Aβ 1-42 (ab10148) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibody to RPLPO (RPL23 16086-1-AP) was purchased from Proteintech Inc (Chicago, IL). The A85G6 ASPH monoclonal antibody was generated in our laboratory as described elsewhere [102]. The RNeasy Mini Kit was from Qiagen (Valencia, CA). The AMV 1st Strand cDNA Synthesis Kit was purchased from Hoffman-LaRoche Ltd (Nutley, NJ). Multiplex Akt and phospho-Akt pathway bead based ELISA kits, Amplex UltraRed soluble fluorophore HRP-substrate, and 4-MUP alkaline phosphatase substrate were purchased from Invitrogen (Carlsbad, CA). The MAGPIX machine was purchased from Bio-Rad (Hercules, CA).

Statistics

Data depicted in graphs reflect group means±S.E.M. Data were analyzed by repeated measures one-way analysis of variance (ANOVA) with the Tukey post-hoc multiple comparison test using GraphPad Prism 6 software (San Diego, CA).

RESULTS

CS modulation of insulin/IGF/IRS related mRNA transcripts

The mean frontal lobe mRNA levels of insulin and IGF-1 polypeptides, InsulinR, IRS-1, and IRS-2 did not vary significantly with CS exposure or recovery (Fig. 1A, B, D, G, H). IGF-2 mRNA levels were modestly elevated in the CS4 and CS8 groups relative to control (Fig. 1C). In the CS8+R group, the mean frontal lobe IGF-2 mRNA level was similar to control and significantly lower than in the CS8 group. CS exposures had significant effects on IGF-1R (F = 11.11, p = 0.0003) and IGF-2R (F = 3.32, p = 0.04) expression. Post hoc tests revealed significantly reduced levels of IGF-1R in the CS8 and CS8+R groups relative to A8 and CS4 (Fig. 1E), and reduced levels of IGF-2R in the CS8 relative to the other three groups (Fig. 1F). In essence, CS withdrawal normalized IGF-2 and IGF-2R gene expression, but failed to abate the CS8 suppression of IGF-1R.

CS effects on InsulinR, IGF-1R, and IRS-1 protein expression and phosphorylation

In contrast to the modest or negligible effects on insulin/IGF-1/IRS-1 gene expression, CS exposures and/or withdrawal significantly altered the corresponding protein and phosphorylated protein levels including, InsulinR (F = 27.15, p < 0.0001), IGF-1R (F = 14.42, p < 0.0001), IRS-1 (F = 14.23, p < 0.0001), ^pYpY-InsulinR (F = 27.15, p < 0.0001), ^pYpY-IGF-1 R (F = 8.83, p = 0.001), and ^S312-IRS-1 (F = 10.17, p = 0.0005) (Fig. 2). Except for ^S312-IRS-1, the mean levels of each of these proteins and phospho-proteins were similarly reduced by the CS4 and CS8 exposures, and further reduced in the CS8+R group relative to A8 (Fig. 2A-E). The mean levels of ^S312-IRS-1 were similar in the A8, CS4, and CS8 groups; ^S312-IRS-1 expression was significantly reduced in CS8+R relative to the other three groups (Fig. 2F). The calculated ratios of phosphorylated/total protein provide indices of relative activation or inhibition of specific components of the signaling pathway. Parallel CS-associated reductions in total and phosphorylated levels of InsulinR and IGF-1R rendered the mean ^pYpY-InsulinR/total and ^pYpY-IGF-1R/total IGF-1R ratios similar across all groups (Fig. 2G, H). This suggests that any impairment in insulin and IGF-1 signaling would have been driven in large measure by decreased expression of the receptor proteins and lower availability of substrate for tyrosine phosphorylation/kinase activation. In contrast, the ^S312-IRS-1/total IRS-1 ratios were significantly modulated by CS exposures (F = 11.03, p = 0.0004), as manifested by the higher levels in CS4, CS8, and CS8+R relative to A8 (Fig. 2I). Since ^S312-IRS-1 is inhibitory, the increased relative levels of S312 phosphorylation indicate that the CS exposures impaired signaling through IRS-1, and that short-term CS withdrawal did not abrogate this effect.

CS effects on the Akt pathway

The CS exposures significantly altered the mean levels of total Akt (F = 9.57, p = 0.0007), ^pS473-AKT (F = 14.69, p < 0.0001), and ^pS473-AKT/total Akt (F = 3.48, p = 0.04). The mean levels of Akt, ^pS473-AKT, and ^pS473-AKT/total Akt were similarly reduced in the CS4 and CS8 groups relative to A8, and further reduced by CS8+R relative to A8, CS4, and CS8 (Fig. 3A, E, I). As occurred with respect to the InsulinR and IGF-1R, the inhibitory effects of CS and CS+R on Akt signaling were mainly mediated by reduced Akt protein expression and to a lesser extent by disproportionate reductions in ^S473-Akt.

Like Akt, frontal lobe levels of total GSK-3β (F = 3.70, p = 0.03), ^pS9-GSK-3β (F = 14.69, p < 0.0001), and ^pS9-GSK-3β/total GSK-3β (F = 9.69, p = 0.0007) were significantly modulated by CS exposures or withdrawal (Fig. 3B, F, J). GSK-3β levels were significantly reduced by CS4 and CS8 exposures, and further reduced in the CS8+R group relative to A8 (Fig. 3B). In contrast, ^pS9-GSK-3β and ^pS9-GSK-3β/total GSK-3β were similar among the A8, CS4, and CS8 groups, but significantly reduced by CS8+R relative to the other 3 groups (Fig. 3F, J). Since S9 phosphorylation of GSK-3β is inhibitory, the findings indicate that CS did not increase GSK-3β activation, despite pronounced inhibition of Akt signaling. Conceivably, the CS-associated reductions in GSK-3β protein could represent an adaptive compensatory response to preserve cellular homeostasis and survival. On the other hand, the striking inhibition of S9 phosphorylation in CS8+R brains suggests that CS withdrawal may activate GSK-3β vis-à-vis low levels of Akt activation. Such a response would likely promote cell death, and inhibit metabolism, myelin maintenance, and neuronal plasticity.

CS exposures and/or withdrawal significantly modulated frontal lobe expression of ^pT246-PRAS40 (F = 8.5, p = 0.001), but not total PRAS40 or ^pT246-PRAS40/total PRAS40 (Fig. 3C, G, K). CS exposure and withdrawal effects on PRAS40 signaling were complex. PRAS40 protein levels were reduced in CS4 relative to A8 frontal lobes. In addition, CS4 had trend reduction effects on ^pT246-PRAS40 (p = 0.06), CS8 had no effect, and CS8+R significantly reduced ^pT246-PRAS40 relative to all other groups (Fig. 3G). No significant CS exposure or withdrawal effects occurred with respect to ^pT246-PRAS40/total PRAS40 (Fig. 3K).

CS exposure or withdrawal modulated p70S6K (F = 8.06, p = 0.002), ^pTpS421/424-p70S6K (F = 23.96, p < 0.0001), and ^pTpS421/424-p70S6K/total p70S6K (F = 10.25, p = 0.0005) levels in the frontal lobe (Fig. 3D, H, L). CS4 and CS8+R exposures significantly reduced p70S6K protein expression, while CS8 had no effect (Fig. 3D). CS4 and CS8 caused similarly significant reductions in ^pTpS421/424-p70S6K expression, and CS8+R caused further significant reductions in ^pTpS421/424-p70S6K relative to the other 3 groups (Fig. 3H). The mean levels of ^pTpS421/424-p70S6K/total p70S6K were significantly reduced in the CS8 and CS8+R groups relative to A8 and CS4 (Fig. 3L), reflecting selective or proportionally greater inhibition of p70S6K phosphorylation than protein expression with longer periods of CS exposure, irrespective of short-term withdrawal.

Effects of CS on markers of neurodegeneration

We used duplex ELISAs to measure tau, pTau, AβPP-Aβ (1-42), ubiquitin, 8-OHdG, and ASPH. Tau protein expression was not significantly altered by CS exposure, although the levels were lowest in the CS8 and CS8+R groups (Fig. 4A). pTau expression was modestly increased by CS4 and CS8+R, but significantly reduced in the CS8 relative to CS4 and CS8+R groups (Fig. 4B). CS exposures significantly altered AβPP-Aβ expression (F = 9.79; p < 0.0001), as was evidenced by the progressively higher levels in CS8 followed by CS8+R relative to A8 and CS4 (Fig. 4C). Ubiquitin (Fig. 4D) and 8-OHdG (Fig. 4) were not significantly altered by CS exposures or withdrawal. ASPH, a marker of neuronal cell adhesion and motility as needed for plasticity [103 –106], was significantly modulated by CS exposure (F = 3.95; p = 0.016), as shown by the significantly lower levels in the CS4 and CS8+R groups relative to A8 (Fig. 4F).

In summary, 8 but not 4 weeks of CS exposure inhibited IGF-1R and IGF-2R gene expression and subsequent CS withdrawal inhibited IGF-2, normalized IGF-2R, and had no effect on IGF-1R expression. CS exposures had no detectable effects of insulin, IGF-1, insulin, IRS-1, or IRS-2 expression. In essence, CS exposures had limited effects on gene expression pertaining to the upstream components of the insulin/IGF signaling pathways. In contrast, CS exposures had broad inhibitory effects on proteins and phosphoproteins regulating signaling through the insulin and IGF-1 receptors and IRS-1. Correspondingly, proteins and phospho-proteins that mediate signaling through Akt, PRAS40 and p70S6K were also inhibited by CS exposures. CS withdrawal generally did not reverse these effects and in most instances exacerbated the adverse trends relative to CS4 and/or CS8. With regard to neuronal and stress markers, CS8 and CS8+R significantly increased expression of AβPP-Aβ, and CS4 and CS8+R significantly reduced ASPH expression. AβPP-Aβ accumulation is a feature of AD, and ASPH has a functional role in mediating cell growth and remodeling, which are needed for neuronal plasticity.

DISCUSSION

Tobacco smoke contains hundreds of toxins. Although our main focus is on tobacco-specific nitrosamines, carbon monoxide exposures have the potential to cause harm. The smoke exposure system was such that cigarettes were burned at one location and delivered to the exposure chambers housing the animals. In the system where the cigarettes were burned, CO was 24 ppm, which is well above that present in natural air (less than 0.5 ppm) but comparable to the amounts exhaled by regular tobacco smokers (25–30 ppm) [107], and well below what is regarded as tolerable in the environment (up to 70 ppm) and toxic to the central nervous system (150–200 ppm) [108]. However, the maximum levels of CO in the chambers were much lower, close to 3 ppm, and comparable to human cigarette smoking exposure. CO neurotoxicity is manifested by delirium, loss of consciousness, or death, due to hypoxic-ischemic encephalopathy which leads to progressive white matter degeneration [109] and death of iron-rich neurons in the globus pallidus and substantia nigra. Under the experimental conditions employed, the risk of CO neurotoxicity was nil and correspondingly, the histopathological effects on the brain were not characteristic of CO-mediated neurodegeneration (data not shown).

Our working hypothesis is that tobacco nitrosamine exposures mediate neurotoxic and neurodegenerative effects of smoking. The most potent and abundant tobacco-specific nitrosamines in CS are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) [63 , 111]. A single cigarette releases up to 2μg of nitrosamine products into the air [112], and therefore can cause disease via first- or secondhand smoke exposures. Although most research on smoking- and nitrosamine-induced diseases has focused on their cancer-promoting effects, emerging data support the concept that low-level nitrosamine exposures also threaten health by causing progressive degenerative diseases linked to impairments in insulin/IGF signaling through cell survival and metabolic pathways [41 , 59]. In this regard, we have shown that low-level nitrosamine exposures cause liver and brain degenerative pathology with impairments in insulin and IGF signaling, as well as diabetes [59, 64]. In the brain, the adverse effects of low-dose nitrosamine exposures mimic molecular, biochemical, and pathological abnormalities observed in AD. The present work circles back to the main clinical and epidemiological concerns regarding the potential contributions of CS exposures in the pathogenesis of neurodegeneration. It is noteworthy that the experimental model utilized mimics effects of sidestream CS, i.e., secondhand exposures.

To investigate the effects of CS exposures and short-term withdrawal on the integrity of insulin/IGF signaling through IRS and Akt pathways in the brain, we measured gene expression and immunoreactivity corresponding to different components of the pathways, as well as proteins frequently abnormally expressed or accumulated at different stages of AD and other neurodegenerative diseases. Since the CS exposures had no detectable effects on insulin or insulin gene expression, any impairment in brain insulin signaling that occur with CS exposure would likely be mediated by post-translational mechanisms. The CS8-associated suppression of IGF-1R and IGF-2R mRNAs indicates that impairments in signaling through these receptors are at least partly mediated at the level of transcription. The failure of short-term CS withdrawal to normalize IGF-1R expression suggests that either this adverse effect may be stubbornly sustained or possibly irreversible. In contrast, the IGF-2 and IGF-2R responses to CS withdrawal appeared to be plastic as both the increased levels of IGF-2 and reduced levels of IGF-2R were normalized. Although IGF-2 signaling utilizes different and IRS-independent pathways compared with insulin and IGF-1, it can be recruited to activate PI3K-Akt [113, 114] and thereby potentially rescue adverse effects of impaired insulin and IGF-1 signaling.

In contrast to the gene expression studies, the multiplex ELISA results demonstrated pronounced inhibitory effects of CS4 and CS8 on insulin receptor, IGF-1 receptor, and IRS-1 proteins and tyrosine phosphorylated insulinR and IGF-1R, and further suppression of these proteins after CS withdrawal. The parallel shifts in the levels of total and phosphorylated insulin and IGF-1 receptor proteins indicate that the main abnormalities in signaling would have been due to reduced expression of the receptor proteins rather than disproportionate inhibition of tyrosine phosphorylation. With regard to the IGF-1R, the mechanism is at least partly due to downregulation of gene expression. However, regarding the insulinR and IRS-1, an alternative explanation is required. One possibility is that CS exposures activate cellular cascades that lead to increased degradation of these molecules. Further research is needed to examine this novel mechanism of brain insulin/IGF resistance.

Although the inhibitory effects of CS and CS+R on tyrosine phosphorylated insulin and IGF-1 receptors could be attributed to reduced levels of receptor protein expression, the relative increases in S312-phosphorylation of IRS-1 in the three experimental groups requires a different interpretation. S312 phosphorylation of IRS-1 is inhibitory [115], indicating that crosstalk or downstream signaling through various pathways, including Akt also would have been inhibited. Conceivably, inhibition of signaling through the insulin and IGF-1 receptors could have contributed to this response. Alternatively, the enhanced S312 phosphorylation of IRS-1 could represent a toxic effect of CS exposure.

The CS-associated inhibitory effects on Akt expression and phosphorylation were likely mediated by impairments in upstream signaling through the insulin/IGF-1 receptors and IRS-1. In addition, the reductions in total Akt as well as GSK-3β protein could have been caused by direct effects of the CS exposures via the same unknown mechanisms that decreased the expression levels of the insulin and IGF-1 receptors and IRS-1. Reduced levels of Akt phosphorylation could account for the decreased levels of ^S9-GSK-3β since Akt activation normally inhibits GSK-3β via phosphorylation. Inhibition of Akt vis-à-vis activation of GSK-3β in the CS8+R groups suggests that the brief period of CS withdrawal may have worsened brain functions that are regulated by Akt signaling, including metabolism, growth and neuronal plasticity.

The effects of CS exposures on downstream signaling through PRAS40 and p70S6K were varied in that PRAS40 protein, ^pT246-PRAS40, and p70S6K levels were reduced in the CS4 and CS8+R groups but not the CS8 group. The apparent recovery of the CS8 exposed brains is not readily explained. In contrast, the effects of CS4, CS8, and CS8+R on ^pT421/pS424-p70S6K paralleled those of ^pYpY-Insulin-R and ^pS473-Akt, suggesting that the responses were directly consequential to the inhibition of insulin and Akt signaling, which regulate p70S6K activation. Since p70S6K regulates cell motility, cell growth, cell survival, and protein translation, CS exposure may mediate its adverse effects on the brain by impairing these functions, and could possibly account for the lower protein expression levels of insulinR, IGF-1R, IRS-1, and Akt.

To begin examining the consequences of CS exposure and withdrawal on proteins altered with neurodegeneration, we measured immunoreactivity to tau, phospho-tau, Aβ, ubiquitin, 8-OHdG, and ASPH. CS8 reduced frontal lobe levels of phospho-tau. Otherwise, there were no meaningful effects of CS on tau or phospho-tau expression. The absence of CS exposure effects on tau contrasts with a recent study showing elevated levels of tau following CO exposure [116], and argues against a role for CO neurotoxicity as a mediator of neurodegeneration in our model. Since insulin and IGF-1 signaling regulate tau phosphorylation and tau transport into growing neurites [117], the reduced levels of phospho-tau in CS8 brains could reflect the longer periods of impaired insulin/IGF-1 signaling, whereas CS withdrawal likely reversed this response. Surprisingly, there were no significant CS-associated changes in ubiquitin or 8-OHdG levels. However, we did observe significantly increased frontal lobe levels of AβPP-Aβ in both the CS8 and CS8+R groups, suggesting that CS exposures promote Aβ accumulation in the brain over time. However, frontal lobe Aβ was further increased in the CS8+R group in which the mice were maintained 2 weeks longer than the CS8 mice. Since insulin/IGF-1 resistance and AβPP-Aβ accumulation are early abnormalities in AD [34], the findings suggest that CS exposures may serve as cofactors driving the neurodegeneration cascade but once established, short-term CS withdrawal may not be sufficient to halt progression of disease. It would be of interest to examine the direct effects of longer-term secondhand and firsthand CS exposures and variable periods of CS withdrawal on various parameters of neurodegeneration including cognitive-behavioral function.

Finally, we measured ASPH protein expression and found the levels to be significantly lower in the CS4 and CS8+R groups relative to control. ASPH protein expression is increased by insulin and IGF-1 stimulated signaling through insulinR, IGF-1R, IRS-1, and Akt [103 , 118]. Therefore, the inhibitory effects of the CS on these pathways correspond with the reductions in ASPH protein. The relatively modest effects observed in the CS8 group mimics the trends regarding PRAS40 and ^pT246-PRAS40 expression, suggesting that CS impairment of Akt signaling through PRAS40 mediated the reductions in ASPH protein expression. ASPH mediates cell motility and adhesion, which are needed for remodeling and repair [105 , 120]. ASPH signals through NOTCH [104 , 120], and NOTCH regulates diverse cellular functions at the level of transcription [121, 122]. However, AD-associated impairments in NOTCH signaling are linked to reduced neuronal plasticity, AβPP-Aβ accumulation, and neurodegeneration [123 –126]. Therefore, inhibition of ASPH expression could represent another mechanism by which CS exposures promote neurodegeneration.

Footnotes

ACKNOWLEDGMENTS

Supported by AA-11431 and AA-12908 from the National Institutes of Health and the Tobacco-Related Disease Research Program Grant 17RT-0171.

Authors’ disclosures available online ().

Appendix

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