Early limited nitrosamine exposures exacerbate high fat diet-mediated type 2 diabetes and neurodegeneration

The prevalence rates of Alzheimer's Disease (AD), Parkinson's disease (PD), obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome have increased exponentially over the past several decades, and thus far show few hints of plateau [1–4]. The relatively short time interval flanking dramatic shifts in age-adjusted AD and PD mortality is consistent with exposure-related rather than genetic etiologies of these diseases. We have noted that the striking increases in AD and PD mortality rates followed sharply increased consumption of processed foods, use of preservatives, and demand for nitrogen-containing fertilizers [4]. A common theme resonating from these unnecessary lifestyle trends is that we have inadvertently increased our chronic exposures to nitrosamines (R1N(-R2)-N = O) and related compounds.

Nitrosamines form by chemical reactions between nitrites and secondary amines or proteins. Nitrosamines exert their toxic and mutagenic effects by alkylating N-7 of guanine, leading to increased DNA damage [5] and generation of reactive oxygen species such as superoxide (O₂-) and hydrogen peroxide (H₂O₂). Consequences include increased lipid peroxidation, protein adduct formation, and pro-inflammatory cytokine activation [6]. Of note is that these very same molecular and biochemical pathogenic cascades are activated in major human insulin-resistance diseases, including T2DM, non-alcoholic steatohepatitis (NASH), and AD [7–13]. The concept that chronic injury caused by exposure to alkylating agents could result in malignancy and/or tissue degeneration is not far-fetched given the facts that: 1) chronic exposures to tobacco nitrosamines cause both lung cancer and emphysema; and 2) treatment with streptozotocin (STZ), a nitrosamine-related compound, causes hepatocellular carcinoma, pancreatic carcinoma, malignant brain tumors, T2DM, AD-type neurodegeneration, and/or hepatic steatosis, depending on dose and route of administration [14–22]. Therefore, although research on nitrosamine-related compounds has been focused on their mutagenic properties, thorough characterization of their non-neoplastic and degenerative effects is clearly warranted. In this regard, guidance may be obtained from what is already known about STZ-induced diseases.

STZ, like other N-nitroso compounds, causes cellular injury and disease by functioning as: 1) an alkylating agent and potent mutagen [14]; 2) an inducer of DNA adducts leading to increased apoptosis [23]; 3) a mediator of unscheduled DNA synthesis, triggering cell death [14]; 4) an inducer of single-strand DNA breaks and stimulus for nitric oxide (NO) formation following breakdown of its nitrosamine group [16]; and 5) an enhancer of the xanthine oxidase system leading to increased production of superoxide anion, H₂O₂, and OH^- radicals [24]. Ultimately, STZ-induced cellular injury, DNA damage, and oxidative stress cause mitochondrial dysfunction [16], ATP deficiency [25] and apoptosis. The structural similarities between STZ and nitrosamines, including N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) [26], together with experimental evidence that high doses of STZ cause cancer while lower doses cause diabetes or AD-type neurodegeneration with cognitive impairment [15, 16, 22] led us to hypothesize that while high doses of environmental and consumed nitrosamines cause cancer, lower, sub-mutagenic doses may promote insulin-resistance mediated degenerative diseases, including T2DM, AD, and possibly other neurological diseases.

Another key point is that obesity, T2DM, and cognitive impairment are likely inter-related, as suggested by the findings of: 1) increased risk for developing mild cognitive impairment (MCI), dementia, or AD in individuals with T2DM [7, 27] or obesity/dyslipidemic disorders [28]; 2) progressive brain insulin resistance and insulin deficiency in AD [29–32]; 3) cognitive impairment in experimental animal models of T2DM and/or obesity [33, 34]; 4) AD-type neurodegeneration and cognitive impairment in experimentally induced brain insulin resistance and insulin deficiency [21, 22, 35–37]; 5) improved cognitive performance in experimental models of AD [19] and in human subjects with AD or MCI after treatment with insulin sensitizer agents or intranasal insulin [38–44]; and 6) similar molecular, biochemical, and mechanistic abnormalities in T2DM and AD [7–13]. On the other hand, recent studies showed that HFD feeding causes obesity, T2DM, and cognitive impairment, but is not sufficient to cause AD [45, 46]. Therefore, it's likely that chronic HFD feeding which results in peripheral insulin resistance may provide a second-hit, and that combined with low-dose nitrosamine or other environmental exposures, it may increase the severity of neurodegeneration.

In the present study, we examined the hypothesis that chronic HFD feeding and limited exposure to sub-mutagenic doses of NDEA function additively to cause insulin resistance with T2DM and neurodegeneration. We focused our investigations on the cerebellum because, apart from subcortical neurodegenerative diseases such as multiple system atrophy (MSA) [47–51], progressive supranuclear palsy (PSP) [52], cerebellar ataxias [48, 53, 54], PD [52, 55], and motor neuron disease (MND) [56], in which cognitive impairment has been linked to cerebellar degeneration, there is growing evidence that cerebellar degeneration is also a feature of AD [57–59], Lewy body dementia (LBD) [60, 61], and fronto-temporal dementia [62]. Although cerebellar degeneration in AD can be caused by superimposed PSP [63], MSA [64], or MND [65], it often occurs independently. Cerebellar degeneration in AD is manifested clinically by gait instability and increased falling [66], and structurally, by significant atrophy of the posterior cerebellar lobes, which correlates with cognitive impairment [67]. The major histopathological features of AD cerebellar atrophy include, reductions in Purkinje cell population, atrophy of the molecular and granule cell layers [68], increased amyloid deposition and gliosis in the cortex [69]; increased ubiquitin-immunoreactivity in senile plaques and degenerating neurites [70]; extensive abnormalities in dendritic spine density and synaptic structure in vestibulocerebellar, visual, and auditory pathways [71, 72]; and degeneration of cerebellar white matter fibers with loss of climbing fibers and presynaptic varicosities [73]. In aggregate, synaptic pathology, rather than neurofibrillary tangles and plaques, represents the main correlate of cerebellar degeneration in AD.

In addition, cerebellar degeneration can be caused by systemic diseases such as diabetes mellitus [74], chronic alcoholism [75], or obesity with metabolic syndrome [45, 46]. These systemic diseases share in common with primary central nervous system (CNS) degenerative diseases, impairments in cognition, and deficits in insulin and IGF signaling mechanisms, insulin/IGF responsive gene expression, and energy metabolism (glucose utilization) in the brain, including cerebellum [29, 75–78]. Therefore, understanding the mechanisms by which lifestyle and environmental mediators promote neurodegeneration of the cerebellum has relevance to a broad spectrum of human CNS diseases.

Our studies examined the degree to which limited early life NDEA exposure exacerbates the effects of chronic HFD feeding on the development of T2DM and neurodegeneration. The importance of this work is that morbidity and mortality rates from T2DM and neurodegeneration have soared over the past several decades, suggesting that exposures rather than genetics dictate their etiologies. Our over-arching hypothesis is that shifts in lifestyles and economics have led us to chronically consume excess fat, and get exposed to agents that cause insulin resistance. Consideration given to potential pathogenic agents was focused by the experimental evidence showing that STZ, a nitrosamine-related chemical, is mutagenic in high doses [14], but causes insulin-resistance diseases at lower levels or more limited durations of exposure, including early in life [15–22, 92]. While STZ is generally not available to consumers, nitrosamines and related compounds are widely present in our environment and contaminate food sources. Therefore, we entertained the hypothesis that either limited or chronic low-level exposures to nitrosamines account for the observed shifts in morbidity and mortality from insulin resistance diseases. Moreover, given the clear role of high dietary fat intake as a mediator of obesity, T2DM, or cognitive impairment, we proposed that the combined effects of HFD and NDEA exposure may act additively or synergistically to cause insulin resistance diseases, including T2DM and neurodegeneration.

We generated an in vivo model in which rat pups were treated with NDEA at doses that were 5- to 500-fold lower than the cumulative doses needed to produce cancer in experimental animals [93–96], and beginning in early adolescence, we pair-fed the rats with either high (60%) or low (5%) fat containing diets. The NDEA doses were selected to be far below those needed for carcinogenesis and were based on empirical studies demonstrating absence of acute toxic effects in the rats. Longer durations of NDEA exposure were not used in order to avoid the possibility that the cumulative doses would lead to malignant transformation. The use of young rats enabled us to compare results with previous observations in the STZ model [81]. Moreover, longitudinal studies of nuns revealed that neuro-cognitive deficits precede the onset of dementia by decades [97, 98], suggesting that early life exposures may contribute to the pathogenesis of AD, perhaps through gene imprinting. Although chronic HFD feeding and limited NDEA exposure increased body weight and caused T2DM/peripheral insulin resistance with fasting hyperglycemia, hyper-insulinemia, and hyper-leptinemia [99, 100], the rats were not obese and they did not have hyper-lipidemia. Instead, the serum lipid profile was favorable, consistent with a previous report of a related experimental model [84].

We focused our investigations on the cerebellum because the cerebellum can be adversely affected in AD, as well as other neurodegenerative diseases [47, 50, 53, 56, 59, 60, 65, 68, 69, 71], and cerebellar degeneration causes cognitive impairment [49, 57–59, 62, 63, 66, 67, 72]. Previous studies demonstrated significant structural, functional, and metabolic abnormalities in AD cerebella [57–59, 82], including insulin and IGF resistance [30], similar to the findings in more traditional targets of AD, i.e. cortical and limbic structures. Finally, by focusing on one brain region, we were able to investigate a novel concept concerning potential additive or synergistic effects of double-hits, i.e. nitrosamine plus HFD exposures, on neurodegeneration in general.

Although both NDEA and HFD feeding caused structural injury and impaired gene expression in ways that could cause insulin/IGF resistance in the brain, their specific effects were not identical. The main effect of NDEA, with or without HFD feeding, was to reduce mRNA levels of insulin receptor, IGF-2 receptor, and IRS-2, which would have impaired signaling at the receptor level, and downstream through IRS-2, one of main docking proteins responsible for transmitting survival, growth, metabolism, and plasticity pathway signals [80, 81]. Correspondingly, NDEA treatment inhibited tau and ChAT, which are regulated by insulin and IGF [81], and are reduced in brains with AD [81]. Reductions in tau, a major structural protein in CNS neurons, could result in cytoskeletal collapse and synaptic disconnection. Alternatively, the finding could reflect neuronal loss associated with neurodegeneration. The reduced levels of ChAT reflect deficits in acetylcholine homeostasis that contribute to cognitive impairment with neurodegeneration [101, 102]. Correspondingly, in preliminary studies, we detected evidence of significant spatial learning and memory deficits in NDEA and NDEA+HFD treated rats by Morris Water Maze testing (Additional file 1, Table S1). The potential contribution of non-cerebral, specifically cerebellar degeneration to cognitive impairment was demonstrated in human subjects with PD, whereby mild cognitive impairment was found to be associated with reduced nicotinic acetylcholine receptor binding in the cerebellum and brainstem [103].

In previous studies using a mouse model of diet-induced obesity [45, 46], we showed that chronic HFD feeding causes brain insulin resistance [46]. Similarly, herein we demonstrated that the HFD-fed rats had reduced levels of brain IRS-1 mRNA, which would have been sufficient to cause brain insulin resistance due to impaired transmission of insulin receptor activated signals; correspondingly, ChAT expression was reduced. In addition, HFD feeding increased GFAP, HNE, and N-Tyr immunoreactivities, consistent with findings in previous studies [45, 46], and providing further evidence that HFD feeding contributes to neurodegeneration by increasing gliosis/astrocyte activation and oxidative stress with lipid peroxidation, as occur in AD. The finding that chronic HFD feeding did not significantly alter tau or AβPP expression also supports our previous conclusion that HFD feeding contributes to, but is not sufficient to cause AD-type neurodegeneration [45, 46].

The combined effect of early, limited NDEA exposure plus chronic HFD feeding significantly reduced insulin and ChAT mRNA levels, and ChAT, AChE, GSK-3β, and pGSK-3β immunoreactivities. In addition, tau and ChAT expression levels were lowest in the HFD+NDEA group. Since tau and ChAT are regulated by insulin/IGF signaling [81], the impairment in insulin gene expression was likely pivotal in mediating these effects of HFD+NDEA exposure. In the HFD+NDEA group, both GSK-3β and pGSK-3β levels were reduced, but relative reductions were greater for pGSK-3β compared with total GSK-3β, reflecting a net relative increased in GSK-3β activity, similar to the findings in AD and PD in humans [29, 30, 104]. Therefore, HFD+NDEA treated rats exhibited persistent brain oxidative stress associated with increased levels of HNE (lipid peroxidation), N-Tyr, and GSK-3β activity. Although the NDEA exposure was delivered within a brief window during the human equivalent of childhood or early adolescence, its adverse effects on the brain were sustained into adulthood, similar to the findings with respect to the i.c.-STZ model [19, 22]. Conceivably, such prolonged and possibly progressive deficits may occur because nitrosamines promote formation of DNA and protein adducts [105–107] that can serve as persistent sources of oxidative stress, and cause further DNA damage and protein dysfunction.

Recently, we demonstrated a role for ceramide-mediated neurodegeneration in a model of diet-induced obesity with T2DM [45], and showed that in vitro ceramide exposure causes neurodegeneration with impairments in neuronal viability, energy metabolism and insulin/IGF signaling mechanisms [83], consistent with previous reports [45, 83, 85, 88–90, 108–110]. Moreover, other investigators have demonstrated significantly increased levels of ceramides and other sphingolipids in brains with neurodegeneration [76, 90, 110–114]. Therefore, we measured pro-ceramide mRNA levels to determine if the HFD or NDEA-associated neurodegeneration and insulin/IGF resistance were likely mediated by increased brain ceramide levels. Those studies demonstrated strikingly increased expression of several genes regulating ceramide production via both de novo biosynthesis or sphingomyelin degradation pathways in NDEA-treated rats, irrespective of chronic HFD feeding. Since NDEA is lipid soluble [115, 116] and can cross the blood-brain barrier [117], it is probable that the neurodegeneration with insulin/IGF resistance associated with limited NDEA exposure was mediated by direct and persistent neurotoxic effects of compound.

In conclusion, we have demonstrated that limited early exposure to sub-mutagenic doses of NDEA causes T2DM and neurodegeneration with impairments in insulin/IGF signaling mechanisms, and deficits in cholinergic and neuronal cytoskeletal gene and protein expression in brain, whereas chronic HFD feeding alone produces more restrictive deficits in insulin/IGF signaling mechanisms with reduced ChAT expression and increased oxidative stress. The combined exposures caused overlapping structural and molecular abnormalities that were additive with regard to the breadth of insulin/IGF signaling molecules that were impaired, and synergistic with respect to the degrees in which ChAT and tau expression were reduced and pro-ceramide genes increased. Since experimentally, exposure to neurotoxic ceramides produces similar effects, their increased levels in cerebrospinal fluid could serve as biomarkers of insulin-resistance mediated neurodegeneration. Finally, the findings suggest that our insulin resistance disease epidemics are linked to sub-mutagenic exposures to nitrosamines and related compounds, combined with chronic consumption of high fat content foods, indicating that these diseases are preventable.