Introduction

The epidemic of insulin resistance associated with obesity, metabolic syndrome, type 2 diabetes (T2DM), and cardiovascular diseases (CVD) is sweeping both developed and emerging countries. It is estimated that 20-30% of the adult population in most countries has the metabolic syndrome with some segments of the population even higher [1]. While insulin resistance is a key feature of the metabolic syndrome, it is likely that a similar number of people as those with the metabolic syndrome also have insulin resistance but do not have three of the four features of the metabolic syndrome including central obesity, dyslipidemia, hypertension and elevated blood sugar. The presence of the metabolic syndrome further raises the risk for developing type 2 diabetes by about 5-fold and persons with the metabolic syndrome are at roughly twice the risk for developing cardiovascular diseases compared with those without the syndrome [1]. Diabetes and cardiovascular complications of insulin resistance are only part of the problem since the insulin resistance syndrome also has consequences on brain function. Increased cognitive alterations are observed in subjects with T2DM and insulin resistance [2-4].

There is a large body of evidence linking impaired insulin function and glucose metabolism to the risk of developing Alzheimer’s Disease (AD)-type neurodegeneration [5-8]. Insulin resistance has been implicated in the pathogenesis of AD and the term “type 3 diabetes” has been used to describe AD [9]. Experimental brain diabetes produced by intracerebral administration of streptozotocin shares many features with AD, including cognitive impairment and disturbances in acetylcholine homeostasis. Experimental brain diabetes is also treatable with insulin sensitizer agents that are used to treat T2DM [9]. AD represents a form of diabetes that selectively involves the brain and has molecular and biochemical features that overlap with both type 1 and T2DM and helps to explain the term type 3 diabetes [9].

Presently there are no known treatments that can stop AD, present treatments can only slow the progression of the disease. Due to the link of insulin resistance and AD, factors that slow the progression of insulin resistance should have consequences on AD incidence and rate of progression.

Accumulating studies demonstrating that insulin and insulin signaling mechanisms are important for neuronal survival [5,7,9] and studies demonstrating reduced expression of the insulin receptor and related members of the insulin signaling pathway in patients and animals suffering from impaired brain function and Alzheimer’s disease (AD) illustrate the urgent need for further studies involving insulin resistance and the brain.

Diets high in fat and (or) fructose contribute prominently to insulin resistance and impaired cognition. Simple sugars and saturated fats are major components of the diet that promote obesity and insulin resistance. The effects of such diets on the brain are poorly understood but insulin resistance may play an important role by acting on brain energy metabolism and neuroprotective mechanisms. Addition of fructose to the water of rats fed a high-fat, high-glucose diet led to insulin resistance and impaired hippocampal synaptic plasticity and cognition in middle-aged rats [10]. We have shown in in vitro, animal, and human studies that CN, aqueous extracts of CN, and CN polyphenols not only improve insulin function but also act as antioxidant and anti-inflammatory compounds to counteract the negative effects of insulin resistance and obesity [11-19]. We have also shown that rats fed a high fat/high fructose diet develop insulin resistance that is prevented by addition of CN to the diet [20].

In this study, we postulated that since CN improves peripheral insulin sensitivity in animals fed a high fat/high fructose diet [20] and CN also improves insulin signaling in the liver and muscle associated with glycogen synthesis [21], that brain insulin signaling and behavior should also improve. Studies in animal models of impaired insulin sensitivity are needed to further understand the interactions among insulin, neurobiological processes, short-term memory, and cognition.

Materials and Methods

Results

In an associated study, we demonstrated that the animals fed the HF/HFr diet had decreased insulin sensitivity based upon the hyperinsulinemic euglycemic clamp procedure and that CN prevented the negative effects of the HF/HFr diet on insulin sensitivity [20].

mRNA effects

Consistent with the negative effects of the HF/HFr diet on insulin sensitivity, and behavior, there were also decreases in mRNA for glucose transporters in the hippocampus and cortex. Animals consuming the HF/HFr diet had a decrease in mRNA coding for GLUT1, the principal glucose transporter of the blood-brain barrier, in both the hippocampus and cortex that was prevented by the CN (Figure 1, left panel). mRNA coding for GLUT3, the main facilitative glucose transporter in neurons, was also decreased by the HF/HFr diet in the cortex with a return to the level of the control diet in the animals consuming the control diet plus CN (Figure 1, right panel).

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Figure 1. High Fat/High Fructose (HF/HFr) diet decreases Glut 1 and 3 in hippocampus and cortex.

Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.

https://doi.org/10.1371/journal.pone.0083243.g001

Contrary to that observed in the muscle [21], consumption of the HF/HFr diet led to increases in insulin receptor (Ir), Irs1 and 2 in the hippocampus and cortex that were not reversed by CN (Figure 2) demonstrating that changes in these variables in the hippocampus and cortex were not associated with similar changes in the periphery. Associated negative effects on these variables related to insulin resistance requires further study.

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Figure 2. High Fat/High Fructose diet increases Insr, Irs1 and Irs2 in hippocampus and cortex.

Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.

https://doi.org/10.1371/journal.pone.0083243.g002

There were significant effects due to CN on the mRNA coding for glycogen synthase, Gys1, in animals consuming both the control diet as well as those consuming the HF/HFr diet in both the hippocampus and cortex (Figure 3, left panel). Changes in the mRNA coding for glycogen synthase kinase (GSK-3β), which is involved in the control of the activity of glycogen synthase, were increased in animals consuming the HF/HFr diet and not altered by CN in animals consuming either the control or HF/HFr diet in either the hippocampus or cortex (Figure 3, middle panel). Increases in mRNA coding for protein kinase B (AKT1), a key control step in insulin signaling, were significant in animals consuming the HF/HFr diet plus CN in the hippocampus but not the cortex (Figure 3, right panel).

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Figure 3. High Fat/High Fructose diet effects on Gys1, Gsk3β and Akt1 in hippocampus and cortex.

Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.

https://doi.org/10.1371/journal.pone.0083243.g003

The negative effects of the HF/HFr diet on insulin sensitivity were associated with negative effects on mRNA coding for proteins associated with Alzheimer’s disease and memory loss including phosphatase and tensin homolog (Pten), Tau and amyloid precursor protein (App) (Figure 4). The HF/HFr diet led to increases in Pten (Figure 4, left panel), Tau (Figure 4, middle panel) and App (Figure 4, right panel) that were reversed by CN. Increases in these variables are associated with decreased insulin sensitivity . Cinnamon had no detectable significant effects on these variables in animals consuming the control diet. There were no detectable effects of CN on the animals consuming the control diet plus CN.

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Figure 4. High Fat/High Fructose diet increases Pten, Tau and App in hippocampus and cortex.

Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.

https://doi.org/10.1371/journal.pone.0083243.g004

Discussion

Cinnamon is often used to refer to Ceylon cinnamon or “true cinnamon” (Cinnamomum verum) however, related species of cassia (C. aromaticum and C. burmannii) are often sold as CN. Cinnamon was first imported to Egypt in 2000 BC and it is often referred to in the bible [19]. While the taste and coumarin content often differ substantially among cinnamons, many of the properties and effects of the true CN and related species are similar. Cinnamon has a long history of uses as a spice, flavoring agent, preservative, and pharmacological agent. More recent studies have documented the role of CN in the prevention of insulin resistance, metabolic syndrome and type 2 diabetes (see review) [19]. In addition, CN has been shown to alleviate factors associated with Alzheimer’s disease and memory loss by blocking and reversing TAU formation and blocking the effects of amyloid precursor protein [28]. The common link between Alzheimer-like changes and type 2 diabetes may be insulin resistance. The peripheral insulin-sensitizer drug, metformin, ameliorates neuronal insulin resistance and Alzheimer-like changes [5]. Evidence that insulin signaling mechanisms are important for neuronal survival [9,29] and studies demonstrating reduced expression of the insulin receptor in the brain of patients suffering from AD have generated increased interest in determining the role of impaired insulin actions associated with Alzheimer-like changes [5]. The fact that insulin receptors are widely expressed in the brain, combined with the observation that insulin crosses the blood-brain barrier by a saturable, receptor mediated transport mechanism, further highlights the observations that insulin likely plays a key role in memory and cognition [30,31]. Defective insulin signaling is associated with decreased cognitive ability and development of dementia. Neurons are insulin-dependent metabolically active tissues, like fat and muscle, that develop insulin resistance and thus cannot respond properly to the neurotrophic properties of insulin, resulting in neuronal injury, subsequent dysfunction and ultimately Alzheimer’s and related diseases [32].

Impaired insulin sensitivity, measured by HOMA, is associated with deficits in verbal fluency [33] and it is not necessary to wait to have overt signs of AD to detect changes in behavior, memory and cognition. Short-term memory alteration has been found in the early phases of AD [34] and in diets high in fat [35]. In addition, HbA1c is associated with executive functioning and working memory in people with insulin resistance [36] and CN also improves HbA1c [37-39].

In this study, we measured the effects of early changes in insulin sensitivity and the counteracting insulin sensitizing effects of CN, on changes in behavior and mRNA associated with insulin signaling and Alzheimer’s. The HF/HFr diet is known to induce changes in behavior [40,41], memory and cognition [42,43]. In our study, the HF/HFr did not induce changes in short-term memory as detected using the Y-maze. Animals fed with HF/HFr diet exhibited the same probability as rats fed with the control chow diet of spontaneous choice of unexplored arm in the second session of Y-maze. Further, they showed the same level of habituation to testing conditions as suggested by the decrease in defecation number and the enhanced locomotion in the second session compared to the first one.

In the elevated plus maze, the HF/HFr fed rats exhibited decreased exploration of the open arm. The reduced exploration of open arms cannot be explained by a decrease in locomotion since the explorations of closed arms were not different among groups. It would be better explained by an enhanced anxiety level [44]. The effect of HF/HFr on anxiety is congruent to the anxiogenic effect of a diet rich in sucrose [45] and with increased stress reactivity induced by a HF diet [46]. However, the latter effect is not always observed, especially in case of a short term HF diet [47]. The anxiety level of HF/HFr rats seems context-dependent as it was observed in EPM but not in Y-maze when HF/HFr rats coped with the Unknown arm during the second session. The differences between both behaviors may be related to the level of aggressiveness as Y-maze Unknown arm is more secure than the open arm of EPM due to the wall and the dim light.

The HF/HFr diet used in this study has been shown to induce an impairment of insulin-sensitivity in muscle and liver [21]. We also observed brain mRNA expression alterations indicative of insulin resistance in this study (Figures 1-4). Such alterations might have behavioral counterparts. Since insulin crosses the blood-brain barrier by a saturable, receptor mediated transport mechanism and acts on receptors widely expressed in the brain [48], it may modulate brain functions such as memory and cognition [30,31,49]. Impaired insulin sensitivity would be one relevant hypothesis to explain HF/HFr diet-induced behavioral alterations. In humans, impaired insulin sensitivity, as measured by HOMA score, is associated with deficits in verbal fluency [33] and intranasal insulin increases verbal memory in humans [50]. HbA1c is associated with executive functioning and working memory in people with insulin resistance [36]. In animals, impaired insulin-sensitivity is also associated with behavioral perturbations [40,42,45] while intracerebroventricular insulin increases memory in rodents [51]. The hippocampus is one of the potential targeted brain areas, as insulin receptors are found at high densities [31]. Furthermore, the hippocampus inhibits stress reaction and supports explicit and spatial memory [33]; all functions being modulated by insulin effects. One hypothesis would be that insulin resistance in hippocampus might explain the enhanced anxiety. Alternatively, the frontal cortex would be also questioned as insulin receptors are also highly expressed [48] and working memory is impaired in case of insulin resistance [36]. This is in agreement with the fact that insulin sensitivity is also impaired in frontal cortex area.

The mechanisms by which insulin sensitivity may act on brain deserve discussion. Brain is an insulin-dependent metabolically active tissue, like fat and muscle. Insulin may therefore act through its metabolic effects. The exposure to a challenging environment is associated with brain activation during which an increase in breakdown of glycogen occurs [52]. The rapid increase in glycogen turnover appears important for memory formation as supported by the memory loss after inhibition of glycogen turnover [53]. Insulin, which favors glycogen turnover [54] and improves memory [48,51], may therefore act on brain function through its metabolic effects. The same can be concluded for frontal cortex as insulin increases working memory [55]. Decreases in insulin sensitivity were associated with the anticipated decreases in Glut 1, in both the hippocampus and cortex. GLUT1 is highly expressed in endothelial cells of blood brain barrier and is responsible for transporting glucose from the blood to extracellular space of the brain [56]. Alternatively, insulin may act through its effect on neurotrophicity [7,48]. There is evidence that insulin signaling mechanisms are important for neuronal survival [9,29]. In the case of insulin resistance, the brain cannot respond properly to the neurotrophic properties of insulin, resulting in neuronal injury, subsequent dysfunction and ultimately Alzheimer’s and related diseases [32].

We observed several mRNA expression changes supporting the hypothesis that insulin resistance might be associated with the occurrence of AD brain markers. The increase in TAU filaments is associated with extensive deposition of amyloid β and a strong association with AD. The amyloid β hypothesis states that amyloid β deposition directly affects neurons, including neurofibrillary tangles and neuronal death, leading to memory impairment which is one of the key clinical signs of AD-related brain dysfunction. Mice engineered to over express mutant APP exhibit increased APP deposition and memory impairment [57]. However, reducing amyloid β generation or removing deposits failed to halt the progression of dementia [58,59]. Consistent with these studies, we observed an increase in mRNA coding for TAU and APP in both hippocampus and frontal cortex. GSK-3β intervenes before neurofibrillary tangles are formed since GSK-3β phosphorylates TAU protein, a step leading to granular TAU oligomers, then neuronal dysfunction and synaptic loss [58].

Recent studies have documented the role of CN in the prevention of insulin resistance, metabolic syndrome and type 2 diabetes (see review) [19]. CN also inhibits the misfolding of human islet amyloid polypeptide which is regarded as a causative factor for type 2 diabetes mellitus [60]. CN supplementation reverses some, but not all, the deleterious effects of the HF/HFr diet in liver and muscle [21]. In muscle, there were no significant effects of CN on Insr, Irs1 and Irs2 in animals consuming the control diet whereas all three were reduced in the animals consuming the HF/HFr diet. Negative effects of the HF/HFr diet were prevented in the animals consuming the HF/HFr diet plus CN. Contrary to the effects observed for muscle, in the hippocampus and cortex, consumption of the HF/HFr diet led to increases in Ir, Irs1 and Irs2 that were not prevented by the consumption of CN (Figure 2). This apparent anomaly is consistent with the study demonstrating that IRS 2 is a negative regulator of memory function and restricts dendritic spine generation [61]. Deletion of Irs2 reduces amyloid deposition and rescues behavioral deficits in APP transgenic mice [62] and increases in Irs2 in this study are consistent with negative effects on learning and memory. Although in a nonsignificative manner, cinnamon blunted the HF/HFr diet effects on anxiety in EPM.

Cinnamon also prevented the decreases in Glut1 in animals consuming the HF/HFr diet plus CN. Similar effects were observed for Glut3, also named the “brain glucose transporter” because it is expressed at high levels in nerves and neural tissue [63]. Cinnamon increased the expression of Gys1 not only in the control animals but also reversed the decreases in Gys1 expression in the cortex in the animals consuming the HF/HFr diet. This suggests that cinnamon may have corrected some metabolic effects of insulin resistance.

In addition, CN alleviated the negative effects of the HF/HFr diet on Alzheimer-related variables in the brain that we analyzed. Recent studies showed that CN alleviates factors associated with Alzheimer’s disease and memory loss by blocking and reversing TAU formation and blocking the effects of amyloid precursor protein [28]. An aqueous extract of Ceylon cinnamon (C. zeylanicum) was shown to inhibit hallmark signs of AD including TAU aggregation and the formation of TAU filaments [16]. TAU filaments removed from the brain of a person who died with AD were also disassociated by a CN extract. A type A-linked procyanidin trimer purified from the aqueous extract was shown to contain a significant portion of the inhibitory activity. This type A trimer has subsequently been purified and identified as cinnamtannin B-1, MW 864 (Richard Anderson, unpublished results). However, there are other active components in cinnamon and Lu et al. [64] demonstrated that both type A- and B-type proanthocyanidins may improve insulin sensitivity. Cinnamon proanthocyanidins were also shown to be the major anti-diabetic components of a cinnamon water extract in the prevention of the misfolding of human islet amyloid polypeptide, a proposed causative factor for type 2 diabetes [60].

A limitation of this study is that additional measurements involving protein levels and protein modification levels were not measured. This study was designed to demonstrate that changes in insulin sensitivity due to diet and cinnamon are associated with changes in behavior and changes in the brain related to insulin sensitivity. This objective was accomplished, however, future studies should include protein related changes.

Conclusion

In summary, this study demonstrated that short-term changes in diet led to not only changes in insulin sensitivity and behavior but also early changes in mRNA coding for proteins related to memory including glycogen synthase kinase 3β, glycogen synthase, PTEN, TAU, and APP that could be prevented or alleviated due to the intake of CN. The CN fed rats were more active in a Y maze test than rats fed the control and HF/HFr diets. The HF/HFr diet fed rats showed greater anxiety in an elevated plus maze test that was lessened by feeding CN. In conclusion, the negative effects of a HF/HFr diet on brain insulin signaling and behavior were alleviated by CN suggesting neuroprotective effects of CN associated with whole body improved insulin sensitivity and related changes in the brain. The consumption of foods or diets that lead to decreased insulin sensitivity may lead not only to changes in insulin related variables but also possible early changes in behavior that can be prevented or alleviated by CN. Additional studies are needed to further characterize the proteins and metabolites associated with these changes.

Acknowledgments

We are indebted to Renaud Maury and Nadine Fidier who participated in the conditioning of the animals.

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