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

Alzheimer's Disease (AD), obesity, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH), which includes metabolic syndrome, have sharply increased in prevalence over the past several decades [1–6]. In addition, we have noted striking increases in age-specific AD mortality rates over the same time interval, and correlated these findings with sharply increased consumption of processed foods, use of preservatives, and demand for nitrogen-containing fertilizers [7]. Such rapid shifts in age-adjusted AD morbidity and mortality are more consistent with exposure-related rather than genetic etiologies. A common theme resonating from these lifestyle trends is that we have increased our exposures to nitrosamines (R1N(-R2)-N = O) and related compounds.

Nitrosamines form by chemical reactions between nitrites and secondary amines or proteins, and they exert toxic or mutagenic effects by promoting DNA damage, oxidative stress and reactive oxygen species generation [8]. The end result is increased lipid peroxidation, adduct formation, and pro-inflammatory cytokine activation [9], which also happen to be major mediators of human insulin-resistance diseases, including T2DM, NASH, and AD [10–16]. The notion that chronic injury caused by alkylating agents could lead to malignant transformation and/or tissue degeneration is consistent with the findings that: 1) chronic exposure to tobacco nitrosamines causes both lung cancer and emphysema with chronic obstructive pulmonary disease; and 2) treatment with streptozotocin (STZ), a nitrosamine-related compound, causes hepatocellular carcinoma, pancreatic carcinoma, malignant brain tumors, T2DM, neurodegeneration, and/or hepatic steatosis, depending on dose and route of administration [17–25]. Therefore, better characterization of the non-neoplastic and degenerative effects of nitrosamines is warranted.

STZ, like other N-nitroso compounds, functions as: 1) an alkylating agent and potent mutagen [17]; 2) an inducer of DNA adducts leading to apoptosis [26]; 3) a mediator of unscheduled DNA synthesis, triggering cell death [17]; 4) an inducer of single-strand DNA breaks and stimulus for nitric oxide (NO) formation following breakdown of its nitrosamine group [19]; and 5) an enhancer of the xanthine oxidase system leading to increased production of superoxide anion, H₂O₂, and OH^- radicals [27]. Ultimately, STZ-induced injury and DNA damage promote mitochondrial dysfunction [19], ATP deficits [28], and cell death. The structural similarities between STZ and nitrosamines, including N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) [29], together with experimental evidence that high doses of STZ cause cancer while lower doses cause diabetes or AD-type neurodegeneration with cognitive impairment [18, 19, 25], led us to hypothesize that while high doses of nitrosamine exposures cause cancer, lower, sub-mutagenic doses may promote insulin resistance-mediated degenerative diseases.

There is growing evidence that obesity, T2DM, and cognitive impairment are inter-related based on findings that: 1) the risk of developing mild cognitive impairment (MCI), dementia, or AD is increased in individuals with T2DM [10, 30] or obesity/dyslipidemic disorders [31]; 2) brain insulin resistance and insulin deficiency progress with severity of AD [32–35]; 3) experimental models of T2DM and/or obesity exhibit MCI [36, 37] and neurodegeneration with brain insulin resistance [38, 39]; 4) experimentally induced brain insulin resistance and insulin deficiency cause AD-type neurodegeneration and cognitive impairment [24, 25, 40–42]; 5) insulin sensitizer agents or intranasal insulin [43–49] can improve cognitive performance in experimental models of AD [22] or in humans with AD or MCI; and 6) similar molecular, biochemical, and mechanistic abnormalities in T2DM, NASH, and AD [10–14, 16, 50]. However, chronic high fat diet (HFD)-induced obesity that leads to NASH and T2DM is not sufficient to cause AD; instead, the associated neurodegeneration is relatively mild, despite brain insulin resistance [38, 39]. Correspondingly, most individuals with T2DM do not develop AD, and the vast majority of individuals with AD are neither obese nor diabetic. Therefore, some additional co-factors must determine who among the at-risk population will likely develop AD. Herein, we examined the hypothesis that early sub-mutagenic exposure to NDEA and chronic HFD feeding additively or synergistically mediate the triad of major insulin resistances diseases, including T2DM, NASH, and AD-type neurodegeneration with cognitive impairment.

We examined the degree to which limited early life NDEA exposure exacerbates the effects of later chronic HFD feeding on subsequent development of T2DM, NASH, and neurodegeneration. The relevance of this work to human disease is that, morbidity and mortality from major insulin-resistance diseases have escalated over the past several decades. Such trends more likely correspond with exposure rather than genetic etiologies. Our principal hypothesis is that shifts in lifestyles have led modern societies to chronically consume excess fats, and also to increase our exposures to pathogenic agents that cause insulin resistance. We focused our investigations on nitrosamines, particularly NDEA, because in previous studies, STZ, a nitrosamine-related chemical, was demonstrated to be mutagenic in high doses [17], and to cause insulin-resistance diseases at lower levels or after limited durations of exposure [18–25, 76]. However, since STZ is not generally available in the environment or foodstuffs, whereas nitrosamines and related compounds are widely present in our environment and contaminate our foods, we hypothesize that limited or chronic low-level exposures to nitrosamines account for the current insulin resistance disease epidemic in the United States. Moreover, given the clear role of high dietary fat intake as a mediator of obesity, T2DM, NAFLD/NASH, and cognitive impairment, we propose that the combined effects of HFD and NDEA exposure additively promote development of insulin resistance diseases.

The knowledge that: 1) human nitrosamine exposures occur through many sources including, processed and preserved foods, tobacco smoke (direct or second hand), and nitrate-containing fertilizers; and 2) nitrosamines are mutagenic and cause tissue injury in manners similar to STZ [17, 19, 27, 29], prompted us to consider whether nitrosamines could have pathogenic roles in our insulin resistance diseases epidemic. Although epidemiologic studies have correlated obesity and high dietary fat intake with rising rates of T2DM, NASH, and cognitive impairment [1–3, 5, 6, 77], experimental data are somewhat varied depending on the model, and no studies have demonstrated that obesity/T2DM is sufficient to cause significant AD-type neurodegeneration with cognitive impairment. In fact, the evidence convincingly informs us that these factors alone are not sufficient, and instead serve as co-factors in the pathogenesis of neurodegeneration, including AD. Therefore, we propose that high dietary fat intake exacerbates the adverse effects of limited NDEA or other nitrosamine exposures to cause insulin resistance diseases.

We generated an experimental in vivo model in which rat pups were treated with NDEA at doses that were 5- to 500-fold lower than the cumulative doses used to cause cancer [78–81], and beginning in early adolescence, we pair-fed the rats with high (60%) or low (10%) fat content diets. The use of young rats in these studies was inspired in part by longitudinal studies of nuns demonstrating that neuro-cognitive abnormalities occur decades earlier than the onset of dementia, indicating that very early life exposures may predispose individuals to develop AD, as well as other neurodegenerative diseases [52, 53], perhaps through epigenetic events such as DNA methylation or gene imprinting. Correspondingly, there is evidence that DNA methylation and other epigenetic changes in DNA increase with aging [82], and likely contribute to the pathogenesis of diseases such as diabetes and neurodegeneration [82, 83]. Moreover, there is experimental evidence that nitrosamines, as well as other adduct forming toxins that contaminate foods, can mediate DNA methylation [84, 85]. Since it could take years for epigenetic modifications to cause disease, and the findings in the "Nun Study" suggest that early events in life predispose individuals to develop AD [53], we utilized an experimental animal model in which low-dose NDEA exposures were administered early in life. The subsequent chronic HFD feeding during adolescence, also fits with the human disease model. Therefore, our experimental approach enabled us to examine effects of early life NDEA exposure on later cognitive function and neurodegeneration in the context of excess caloric intake, which is one of the major modifying factors correlated with insulin resistance diseases in our society.

Although the HFD feeding and NDEA treatments significantly increased body weight relative to control, and caused T2DM, characterized by fasting hyperglycemia, hyperinsulinemia, and pancreatic islet hypertrophy [86, 87], the rats were not obese and they did not have hyperlipidemia. On the other hand, the NDEA ± HFD groups had steatohepatitis with hepatic insulin and/or IGF-1 resistance which were more pronounced in rats that had the combined versus individual NDEA or HFD exposures. This suggests that HFD and NDEA function additively to promote NASH and T2DM. In contrast to the C57BL/6 mouse model of HFD feeding in which NASH was associated with obesity, T2DM, and hyperlipidemia [38, 39], the serological profile in the present model provided minimal evidence of hepatic insulin/IGF resistance. Since the serum ALT levels were increased in the HFD groups, perhaps biomarkers of hepatic injury in individuals with T2DM should be regarded as a potential indicator of steatohepatitis.

The NDEA treatments and HFD feedings independently caused significant deficits in spatial learning, and the combined exposures had the added effect of impairing learning and memory, again suggesting that the adverse effects were additive. Therefore, curbing either or both exposures could help prevent subsequent development of cognitive impairment. A second point is that, although the NDEA was delivered within a brief window early in life, its impact on cognitive function was sustained in the adults, similar to the effects of ic-STZ treatment [22, 25]. The mechanism underlying these prolonged and possibly progressive deficits may reside in the fact that nitrosamines promote the formation of adducts that can serve as persistent sources of oxidative stress, DNA damage, and protein mal-folding or dysfunction [8, 9, 26, 88], and ultimately lead to epigenetic changes in gene expression.

Reduced levels of ChAT expression were observed in brains of HFD, NDEA, and NDEA+HFD treated rats. In addition, NDEA exposure without HFD feeding reduced GFAP, but increased HNE and Aβ PP-Aβ, while NDEA+HFD reduced GFAP, GAPDH, Tau, pGSK-3β, AβPP-Aβ, and HNE immunoreactivities, and Tau and IL-1β mRNA, but increased pTau and the pTau/Tau ratio. Since HFD feeding alone had minimal effects on these biomarkers of neurodegeneration, the differences in reaction to NDEA versus NDEA+HFD could be attributed to interactive or amplifying effects of chronic HFD feeding/T2DM on early life, low-level NDEA exposure. This phenomenon was particularly noticeable with respect to the reductions in ChAT, which were modest in brains of rats exposed to NDEA or the HFD, but striking in brains exposed to NDEA plus HFD.

The NDEA-associated reductions in GFAP, Tau, and ChAT expression in brain are of interest because similar observations were made in humans with AD, and in the ic-STZ experimental animal model of AD-type neurodegeneration [22, 25, 51]. The reduced levels of GFAP suggest that glia (astrocytes) are targets of neurodegeneration. The reductions in Tau and ChAT expression are noteworthy because both genes are regulated by insulin/IGF stimulation, their expression levels are reduced in AD [51], and insulin/IGF resistance was demonstrated to be a prominent adverse effect of limited and low-level NDEA exposure resulting in neurodegeneration with cognitive dysfunction, as also occurs in AD [51]. Similarly, the relative increases in pTau, and reductions in pGSK-3β (inactive), GAPDH, and β-actin in the NDEA+HFD group reflect adverse effects of impaired insulin/IGF signaling with cytoskeletal collapse, increased oxidative stress, and reduced energy metabolism, similar to the effects of both i.c. STZ treatment in rats and sporadic AD in humans [51].

The effects of HFD, NDEA, or both exposures on AβPP and AβPP-Aβ were varied, but the most striking findings were significantly increased levels of AβPP-Aβ in NDEA-treated rats, and paradoxically decreased levels of AβPP-Aβ in brains of NDEA+HFD treated rats. Similarly, HNE immunoreactivity was also increased in brain by limited peripheral NDEA exposure, but these adverse effects of NDEA were abolished by chronic HFD feeding. The findings with respect to NDEA on AβPP-Aβ and HNE are consistent with previous observations that oxidative stress promotes AβPP-Aβ accumulation and lipid peroxidation in the CNS [89]. On the other hand, it appears that HFD feeding may have been somewhat protective, perhaps due to alterations in membrane lipid composition leading to enhanced intracellular signaling [90]. The fact that ChAT expression and cognitive function were most impaired in the NDEA+HFD group relative to control vis-à-vis low levels of AβPP-Aβ highlights the controversial role of AβPP-Aβ accumulation in relation to cognitive impairment in AD.

NDEA exposure and HFD feeding independently impaired insulin and IGF-1 signaling mechanisms in liver and brain, and in general, the combined exposures further reduced both hepatic and brain insulin and IGF-1 receptor binding compared with HFD feeding alone. Therefore, brain and hepatic insulin/IGF-1 resistance can be effectuated by either insult. Although impairments in binding to the insulin and IGF-1 receptors in brain could be explained in part by reduced expression of those receptors or receptor-bearing cells in rats treated with NDEA+HFD, generally, this was not the case for the NDEA-treated or HFD-fed groups in which the receptor expression was either elevated or similar to control. Most likely, the impaired receptor binding with attendant reduced expression of IRS signaling molecules mediated the insulin/IGF resistance. Moreover, the reductions in IGF-1 expression could account for progressive loss of IGF-1 receptor bearing cells in vivo. In some instances, ligand expression was increased, suggesting that compensatory responses had occurred due to insulin/IGF-1 resistance as occurs in T2DM.

The mechanisms of sustained brain and liver insulin- and IGF-1 resistance in the context of NDEA exposure ± HFD feeding are not well understood. The fact that NDEA ± HFD feeding caused NASH, led us to investigate whether toxic lipids stemming from NASH-related injury could contribute to NDEA-mediated neurodegeneration. Since pro-ceramide genes are increased in experimental models of NASH [68, 71, 74, 91], and ceramides cause neurodegeneration, pro-inflammatory cytokine activation, and insulin resistance [4, 38, 68, 71–74, 92, 93], we measured mRNA levels of pro-ceramide genes in liver and brain. Those studies revealed strikingly increased levels of several genes involved in ceramide generation via both biosynthesis and degradation pathways in livers of NDEA-treated rats, with virtually no additional impact of HFD feeding. Since both NDEA and ceramides are lipid soluble [94, 95], and therefore likely to readily cross the blood-brain barrier, NDEA exposure could cause CNS insulin resistance and chronic injury by dual mechanisms: 1) direct neurotoxic injury with locally increased production of adducts and pro-ceramide gene expression; and 2) increased hepatic ceramide synthesis leading to the establishment of a liver-brain axis of neurodegeneration. Correspondingly, the qRT-PCR results suggest that hepatic-origin ceramide is generated by both synthetic and degradative pathways in NDEA-treated rats, whereas in the brain, ceramide gene expression was strikingly increased via degradative pathway mechanisms, and inhibited via the synthetic pathways. Preliminary studies suggest that in vivo intraperitoneal administration of toxic ceramides is sufficient to cause brain insulin resistance, neurodegeneration, and cognitive impairment (Tong, et al, unpublished).

We demonstrated that limited, low-level exposure to sub-mutagenic doses of NDEA, together with chronic HFD feeding, act additively in causing peripheral, hepatic and brain insulin and IGF-1 resistance associated with T2DM, NAFLD/NASH, and neurodegeneration. The neurodegeneration had features in common with AD, including impairments in learning and memory, cholinergic function, and neuronal cytoskeletal gene and protein expression. These abnormalities were associated with increased pro-ceramide gene expression, and since ceramides promote insulin resistance, neuro-inflammation, and neurodegeneration, characterization and measurement of ceramides and related lipotoxins in blood may prove useful as biomarkers of insulin-resistance disease progression toward T2DM, NASH, or AD. The molecular and biochemical abnormalities detected are quite reminiscent of the findings in the ic-STZ model [51]. Altogether, these results raise concerns about the double insult of chronic, low-level nitrosamine exposure and high dietary fat consumption as major mediators of our insulin resistance disease epidemic.