Many studies have been performed to explore the mechanisms underlying nicotine-induced rewarding effects, but molecular mechanisms still remain unclear. Metabonomics not only is involved within the system biology framework, but also provides a promising opportunity to explore the molecular mechanisms of diseases
[24, 25]. The current experiment was designed to study the metabolic profling of nicotine-induced CPP mice by applying 1H NMR-based metabonomic in four brain regions including hippocampus, PFC, NAc and striatum. Interestingly, we found that certain metabolites were markedly altered by nicotine. The modified metabolites are correlated with neurotransmitter disturbance, oxidative stress alteration, mitochondria dysregulation, membrane disruption, energy metabolism imbalance as well as amino acid disorder. We consider that the rewarding effect of nicotine in mice may be closely associated with these modified metabolites. In other words, changes in CPP behavior may contribute to differences between controls and nicotine-exposed animals in the metabolomic analysis.
Disturbance in neurotransmitters
Nicotine, a biologically active substance, has widespread pharmacological properties in the central and peripheral nervous systems. It has been shown that nicotine regulates release of multiple neurotransmitters, including dopamine, norepinephrine, serotonin, acetylcholine and amino acids in various regions of rat brain such as the cortex, hippocampus, NAc, striatum
[26, 27]. Repeated administration of nicotine induces both behavioral sensitization and alterations in dopamine and glutamate transmission as well as glutamatergic synaptic plasticity
[28, 29]. Considerable researches into the neurobiology of cocaine addiction have displayed that glutamate plays a crucial role in both the initiation and expression of addiction related behaviors
[30, 31]. In nicotine-induced CPP mice, glutamate showed a low level in hippocampus and striatum, but displayed a high level in NAc and PFC. Considering that glutamate not only affects glutamatergic synaptic plasticity but also modulates initiation and expression of addiction behaviors, we infer that altered glutamate equilibrium in neurotransmitter signaling may participate in nicotine-induced CPP in mice
Acetylcholine, an endogenous agonist of nAChR, is thought to modulate learning and memory by altering the oscillatory rhythms that result from the interaction of various hippocampal subregions
[33, 34]. In our study, nicotine enhanced acetylcholine level in striatum; moreover, an increase of tryptamine, a precursor of 5-HT, was displayed in NAc. Several preclinical studies suggest that acetylcholine exerts a myriad of effects on the addictive process and that persistent changes to the acetylcholine system induced by chronic drug use may enhance the risk of relapse. We speculate that nicotine can modify synaptic plasticity through altering neurotransmitter release, such as glutamate, acetylcholine and tryptamine.
It is reported that nicotine is closely bound up with oxidative stresss
. Glutathione, creatine and taurine, which are ubiquitous and important antioxidant, protect mitochondria against endogenous oxygen radicals
. In our present study, these substances in brain were significantly altered by nicotine.
Glutathione, one of the most abundant intracellular antioxidants in the brain, can modulate the activity of NMDARs
. In our study, glutathione in hippocampus displayed a low level in nicotine-induced CPP mice, implying the weakened antioxidative capability due to the decrease in glutathione.
Creatine is thought to have a multifaceted role in the brain. In addition to being involved in brain osmoregulation, it has recently been related with energy homeostasis and direct antioxidant effects
[38, 39]. We found that creatine was significantly decreased in hippocampus but increased in striatum by nicotine. As creatine can store energy for the cell, the alterations of creatine in hippocampus and striatum suggest an energetic shift in different brain regions.
Taurine, a neuroprotective amino acid, has been reported to be a neuroprotective agent in numerous investigations
. It acts as a free radical scavenger, possesses cytoprotective abilities and prevents the damage from oxidative stress and apoptosis induced by toxicants in various cells and tissues
. In our study, taurine in hippocampus and striatum showed a remarkable decrease after nicotine treatment, indicating the disturbances of anti-oxidative stress in brain.
Taken above, we speculate that nicotine affects brain antioxidative and energy storage capacity by modulating glutathione, taurine and creatine levels. The central nervous system may take adaptive measures to prevent the damage from oxidation by consuming taurine and glutathione, thus resulting in stressful declines of glutathione and taurine in hippocampus and striatum.
Membrane disruption and mitochondria dysregulation
It has been known that glycerol, phosphocholine and myo-inositol, acting as precursors for the synthesis of membrane phospholipids in the cell, exert a critical part on lipid metabolism
. Glycerol has been applied to investigate membrane phospholipid degradation in brain homogenates after cerebral ischemia and seizures
. The damage of membrane results in release of phospholipids and choline compounds, which are the major head of phospholipids
. Interestingly, we found increases of glycerol in NAc and phosphocholine in PFC in nicotine-induced CPP mice. The elevated glycerol and phosphocholine may reflect an enhanced degradation of cellular membranes in brain, hinting the disruption in membrane transportation or barrier function.
NAA is regarded as a biomarker of neuronal viability. Numerous evidences have indicated that NAA may be linked with multiple roles in neurons, such as neuronal metabolic function in mitochondria, myelinogenesis, osmoregulation
. Depletion of NAA is considered to be a reflection of neuronal loss or dysfunction
. In our present study, NAA in striatum showed an increased level in nicotine-induced CPP mice, suggesting that nicotine may be related to neuronal activity and modulate mitochondria function
The brain regulates energy homeostasis by balancing energy intake, expenditure and storage
. Neuronal activity is extremely energy demanding
. In our study, several metabolites related to energy metabolism were markedly modified by nicotine, including glucose, Lac, creatine, glycine and a-ketoglutaric acid.
NAD has been considered to be a key regulator of metabolism, stress resistance and longevity
. Moreover, NAD and NADH are the components of the central redox pair within cells and substrates of many dehydrogenases involved in brain energy metabolism
. In nicotine-induce CPP mice, NAD displayed a significant reduction in PFC. Addiction is a progress of energy consumption, so the decreased NAD in brain may reflect an enhanced energy demand induced by nicotine.
Glucose, a primary energy substrate for brain metabolism, plays an important role in energy homeostasis
[53–55]. We found that glucose was decreased in striatum, hippocampus and PFC,but increased in NAc by nicotine CPP paradigm. Nicotine stimulates brain metabolism, which leads to a significant increase in glucose transporter densities and local cerebral glucose utilization; moreover, nicotine can effectively improve learning and cognitive functions
[56, 57]. It is reported that cognition is highly energy dependent and glucose acts as main energy substrate of neurons
. Thinking above, our findings suggest that nicotine could modulate energy metabolism in various brain regions.
Ketone bodies (D-3-hydroxybutyrate (3OHB) and acetoacetate) serve as an energy source alternative to glucose for the brain and enter the blood brain barrier by the monocarboxylate transporter 1
. The proliferation and metabolic activity of neuroglial cells can be significantly enhanced by 3-HB and derivatives
. In our study, 3-HB and acetone were significantly decreased in PFC, but 3-HB was increased in NAc in nicotine-treated mice.
It is known that memory formation can increase synaptic transmission and morphological alterations at the synapse, both of which consume more energy in the neuron
[61, 62]. Taken above, it is reasonable to infer that nicotine-induced changes in these metabolites may reflect an energetic shift and alteration of energy storage capacity.
Changes in amino acid metabolism
Amino acid metabolism is extremely complex because large numbers of metabolites are involved. The disorder of amino acid metabolism is probably induced by gluconeogenesis, proteolysis and oxidative catabolism
. Amino acids as substrates are extremely equired for energy production during infection
. It is reported that a certain of metabolites, including sarcosine, uracil, kynurenine, glycerol-3-phosphate, leucine and proline, display a high level in metastatic prostate cancer and can potentially be regarded as biomarkers for progressive disease
. Similarly, we also found that several amino acids were significantly altered, including cystine, arginine, isoleucine, glutamate, lysine and phenylalanine. Because these amino acids belong to essential amino acids, non-essential amino acids or amino acid with putative neurotransmitter function, we speculate that protein metabolism in brain could be influenced by nicotine due to the alteration of amino acids.