New research shows how drinking sugary beverages early in life may lead to impaired memory in adulthood.
The study, published today in Translational Psychiatry, also is the first to show how a specific change to the gut microbiome — the bacteria and other microorganisms growing in the stomach and intestines — can alter the function of a particular region of the brain.
According to the Centers for Disease Control and Prevention, sugar-sweetened beverages are a leading source of added sugars in Americans’ diets. Nearly two-thirds of young people in the United States consume at least one sugary drink each day.
Neuroscientist Scott Kanoski, associate professor of biological sciences at the USC Dornsife College of Letters, Arts and Sciences, has studied the link between diet and brain function for years. His research has shown that consumption of sugary beverages impairs memory function in rats and that those same drinks change the gut microbiome.
In the current study, Kanoski and researchers at UCLA and the University of Georgia, Athens, sought to find out if a direct link exists between changes to the microbiome and memory function.
When the rats grew to be adults after about a month, the researchers tested their memories using two different methods. One method tested memory associated with a region of the brain called the hippocampus. The other method tested memory function controlled by a region called the perirhinal cortex.
The researchers found that, compared to rats that drank just water, the rats that consumed high levels of sugary drink had more difficulty with memory that uses the hippocampus. Sugar consumption did not affect memories made by the perirhinal cortex.
The scientists then checked the rats’ gut microbiomes and found differences between those that drank the sweet beverage and those that drank water. The sugar drinkers had larger populations of two particular species of gut bacteria: Parabacteroides distasonis and Parabacteroides johnsonii.
The researchers then asked if the Parabacteroides bacteria could, without the help of sugar, affect the rats’ memory function. They transplanted Parabacteroides bacteria that were grown in the lab into the guts of adolescent rats that drank just water. The rats receiving the bacteria showed memory impairment in the hippocampus when they grew to adulthood much the same as the sugar-drinking rats.
The scientists also found that, unlike the sugar-drinking rats, the transplanted rats also showed memory impairment in the perirhinal cortex. This difference provides further evidence that altered brain function associated with diet may actually be rooted in changes to the gut microbiome.
Previous studies have transplanted the entire gut microbiome from one group of animals to another, producing similar changes to brain function. However, this study is among the first to do so with just two specific species.
“It was surprising to us that we were able to essentially replicate the memory impairments associated with sugar consumption not by transferring the whole microbiome, but simply by enriching a single bacterial population in the gut,” said Kanoski, who is a corresponding author on the study.
Finally, the scientists examined the activity of genes in the hippocampus, comparing rats that drank the sugary beverage to those that drank just water and comparing water drinkers to those transplanted with Parabacteroides.
Gene activity did, in fact, change in both the rats that consumed the sugar-sweetened beverages and the rats transplanted with Parabacteroides. The genes that were affected control how nerve cells transmit electrical signals to other nerve cells and how they send molecular signals internally.
The results of this study confirm a direct link, on a molecular level, between the gut microbiome and brain function.
In future studies, Kanoski and the researchers hope to determine if changing habits, such as eating a healthier diet or increasing exercise, can reverse the harm to memory caused by elevated sugar consumption earlier in life.
In addition to Kanoski and Noble, study authors include Elizabeth Davis, Linda Tsan, Clarissa Liu, Andrea Suarez and Roshonda Jones from USC Dornsife; Christine Olson, Yen-Wei Chen, Xia Yang and Elaine Y. Hsiao UCLA; and Claire de La Serre and Ruth Schade from UGA.
Funding: The research was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants DK116942, DK104897, DK118000, DK111158, DK116558, DK 118944 and DK104363; National Institute on Aging award F31 AG064844; and Department of Defense ARO MURI award W911NF-17-1-0402.
The gut microbiome is increasingly implicated in modulating neurocognitive development and consequent functioning 1,2. Early life developmental periods represent critical windows for the impact of indigenous gut microbes on the brain, as evidenced by the reversal of behavioral and neurochemical abnormalities in germ free rodents when inoculated with conventional microbiota during early life, but not during adulthood 3–5.
Dietary factors are a critical determinant of gut microbiota diversity and can alter gut bacterial communities, as evident from the microbial plasticity observed in response to pre- and probiotic treatment, as well as the “dysbiosis” resulting from consuming unhealthy, yet palatable foods that are associated with obesity and metabolic disorders (e.g., “Western diet”; foods high in saturated fatty acids and added sugar) 6.
In addition to altering the gut microbiota, consumption of these dietary factors yields long-lasting memory impairments, and these effects are more pronounced when consumed during early life developmental periods vs. during adulthood 7–9. Whether diet-induced changes in specific bacterial populations are functionally related to altered early life neurocognitive outcomes, however, is poorly understood.
The hippocampus, which is well known for its role in spatial and episodic memory and more recently for regulating learned and social aspects of food intake control 10–15, is particularly vulnerable to the deleterious effects of Western dietary factors 16–18.
During the juvenile and adolescent stages of development, a time when the brain is rapidly developing, consumption of diets high in saturated fat and sugar 19–21 or sugar alone 22–25 impairs hippocampal function while in some cases preserving memory processes that do not rely on the hippocampus.
While several putative underlying mechanisms have been investigated, the precise biological pathways linking dietary factors to neurocognitive dysfunction remain largely undetermined 9. Here we aimed to determine whether sugar-induced alterations in gut microbiota during early life are causally related to hippocampal-dependent memory impairments observed during adulthood.
Early-life sugar consumption impairs hippocampal-dependent memory function without affecting other neurocognitive domains
Results from the Novel Object in Context (NOIC) task, which measures hippocampal-dependent episodic contextual memory function 26, reveal that while there were no differences in total exploration time of the combined objects on days 1 or 3 of the task (Fig. 1A,B), animals fed sugar solutions in early life beginning at PN 28 had a reduced capacity to discriminate an object that was novel to a specific context when animals were tested during adulthood (PN 60), indicating impaired hippocampal function (Fig. 1C, D).
Conversely, when tested in the novel object recognition task (NOR), which tests object recognition memory independent of context and is primarily dependent on the perirhinal cortex 26–28, animals fed sugar solutions in early life performed similarly to those in the control group (Fig. 1E).
Elevated anxiety and altered general activity levels may influence novelty exploration independent of memory effects and may therefore confound the interpretation of behavioral results. Thus, we next tested whether early life sugar consumption affects anxiety-like behavior using two different tasks designed to measure anxiety in the rat: the elevated zero maze and the open field task, that latter of which also assesses levels of general activity 29.
Early life sugar had no effect on time spent in the open area or in the number of open area entries in the zero maze (Fig. 1F, G). Similarly, early life sugar had no effect on distance travelled or time spent in the center zone in the open field task (Fig. 1H, I). Together these data suggest that habitual early life sugar consumption did not increase anxiety-like behavior or general activity levels in the rats.
Early life sugar consumption impairs glucose tolerance without affecting total caloric intake, body weight, or adiposity
Given that excessive sugar consumption is associated with weight gain and metabolic deficits 30, we tested whether access to a sugar solution during the adolescent phase of development would affect food intake, body weight gain, adiposity, and glucose tolerance in the rat.
Early life sugar consumption had no effect on body weight or total kcal intake (Fig. 1J, K), which is in agreement with previous findings 22,31,32. Animals steadily increased their intake of the 11% sugar solution throughout the study but compensated for the calories consumed in the sugar solutions by reducing their intake of dietary chow (Supplemental Fig. 1A, B).
There were no differences in body fat percentage during adulthood (Fig. 1L) or in total grams of body fat or lean mass. However, animals that were fed sugar solutions during early life showed impaired peripheral glucose metabolism in an intraperitoneal glucose tolerance test (IP GTT) (Fig. 1L, Supplemental Fig 1C-E).
Gut microbiota are impacted by early life sugar consumption
Principal component analyses of 16s rRNA gene sequencing data of fecal samples revealed a separation between the fecal microbiota of rats fed early life sugar and controls (Fig. 2A). Results from LEfSe analysis identified differentially abundant bacterial taxa in fecal samples that were elevated by sugar consumption.
These include the family Clostridiaceae and the genus 02d06 within Clostridiaceae, the family Mogibacteriaceae, the family Enterobacteriaceae, the order Enterobacteriales, the class of Gammaproteobacteria, and the genus Parabacteroides within the family Porphyromonadaceae (Fig. 2B,C). In addition to an elevated % relative abundance of the genus Parabacteroides in animals fed early life sugar (Fig 2D), log transformed counts of the Parabacteroides negatively correlated with performance scores in the NOIC memory task (R2=.64, P<.0001; Fig. 2E).
Within Parabacteroides, levels of three operational taxonomic units (OTUs) that were elevated by sugar significantly correlated negatively with performance in the NOIC task, two of which were identified as taxonomically related to P. johnsonii and P. distasonis (Fig. 2F, G). The significant negative correlation between NOIC performance and each of these OTUs was also present within the sugar groups alone (not shown). Abundance of other bacterial populations that were affected by sugar consumption were not significantly related to memory task performance.
There was a similar separation between groups in bacteria analyzed from cecal samples (Supplemental Fig. 2A). LEfSe results from cecal samples show elevated Bacilli, Actinobacteria, Erysipelotrichia, and Gammaproteobacteria in rats fed early life sugar, and elevated Clostridia in the controls (Supplemental Fig. 2B). Abundances at the different taxonomic levels in fecal and cecal samples are shown in (Supplemental Fig. 3, 4). Regression analyses did not identify these altered cecal bacterial populations as being significantly correlated to NOIC memory performance.
Early life Parabacteroides enrichment impairs memory function
To determine whether neurocognitive outcomes due to early life sugar consumption could be attributable to elevated levels of Parabacteroides in the gut, we experimentally enriched the gut microbiota of naïve juvenile rats with two Parabacteroides species that exhibited high 16S rRNA sequencing alignment with OTUs that were increased by sugar consumption and were negatively correlated with behavioral outcomes in rats fed early life sugar. P. johnsonii and P. distasoni species were cultured individually under anaerobic conditions and transferred to a group of antibiotic-treated young rats in a 1:1 ratio via oral gavage using the experimental design described in Methods and outlined in Supplemental Fig. 5A, and from 33. All rats treated with antibiotics showed a reduction in food intake and body weight during the initial stages of antibiotic treatment, however, there were no differences in body weight between the two groups of antibiotic treated animals by PN50, at the time of testing (Supplemental Fig. 5B, C).
Results from the hippocampal-dependent NOIC memory task showed that while there were no differences in total exploration time of the combined objects on days 1 or 3 of the task, indicating similar exploratory behavior, animals treated with Parabacteroides showed a significantly reduced discrimination index in the NOIC task (Fig 3A-D), indicating impaired performance in hippocampal-dependent memory function. When tested in the perirhinal cortex-dependent NOR task 26, animals treated with Parabacteroides showed impaired object recognition memory as indicated by a reduced novel object exploration index, with no differences in total exploration time (Fig 3E). These findings show that unlike sugar-fed animals, Parabacteroides enrichment impaired perirhinal cortex-dependent memory processes in addition to hippocampal-dependent memory.
Results from the zero maze showed a non-significant trend toward reduced time spent in the open arms and a reduced number of open arm entries for the Parabacteroides treated rats (Fig 3F, G), which is indicative of increased anxiety-like behavior. However, there were no differences in distance travelled or time spent in the center arena in the open field test, which is a measure of both anxiety-like behavior and general activity in rodents (Fig. 3H, I). Together these data suggest that Parabacteroides treatment negatively impacted both hippocampal-dependent perirhinal cortex-dependent memory function without significantly affecting general activity or anxiety-like behavior.
Similar to a recent report 34, Parabacteroides enrichment in the present study impacted body weight. Animals who received P. johnsonii and P. distasonis treatment showed reduced body weight 40 days after the transfer, with significantly lower lean mass and a trend toward reduced fat mass (Fig 3J-L). There were no differences in percent body fat between groups, nor were there significant group differences in glucose metabolism in the IPGTT (Supplemental Fig. 5D, E).
Early life sugar consumption and Parabacteroides enrichment alter hippocampal gene expression profiles
To further investigate how sugar and Parabacteroides affect cognitive behaviors, we conducted transcriptome analysis of the hippocampus samples.
Supplemental Fig 6A shows the results of principal component analysis revealing moderate separation based on RNA sequencing data from the dorsal hippocampus of rats fed sugar in early life compared with controls. Gene pathway enrichment analyses from RNA sequencing data revealed multiple pathways significantly affected by early life sugar consumption, including four pathways involved in neurotransmitter synaptic signaling: dopaminergic, glutamatergic, cholinergic, and serotonergic signaling pathways. Additionally, several gene pathways that also varied by sugar were those involved in kinase-mediated intracellular signaling: cGMP-PKG, RAS, cAMP, and MAPK signaling pathways (Fig. 4A, Supplemental Table 1).
Analyses of individual genes across the entire transcriptome using a stringent false-discovery rate criterion further identified 21 genes that were differentially expressed in rats fed early life sugar compared with controls, with 11 genes elevated and 10 genes decreased in rats fed sugar compared to controls (Fig 4B).
Among the genes impacted, several genes that regulate cell survival, migration, differentiation, and DNA repair were elevated by early life sugar access, including Faap100, which encodes an FA core complex member of the DNA damage response pathway 35, and Eepd1, which transcribes an endonuclease involved in repairing stalled DNA replication forks, stressed from DNA damage 36. Other genes associated with ER stress and synaptogenesis were also significantly increased by sugar consumption, including Klf9, Dgkh, Neurod2, Ppl, and Kirrel1 37,38,39,40.
Several genes were reduced by dietary sugar, including Tns2, which encodes tensin 2, important for cell migration 41, RelA, which encodes a NF/kB complex protein that regulates activity dependent neuronal function and synaptic plasticity 42, and Grm8, the gene for the metabotropic glutamate receptor 8 (mGluR8). Notably, reduced expression of mGluR8 receptor may contribute to the impaired neurocognitive functioning in animals fed sugar, as mGluR8 knockout mice show impaired hippocampal-dependent learning and memory 43.
Supplemental Fig 6B shows the results of principal component analysis of dorsal hippocampus RNA sequencing data indicating moderate separation between rats enriched with Parabacteroides and controls. Gene pathway analyses revealed that early life Parabacteroides treatment, similar to effects associated with sugar consumption, significantly altered the genetic signature of dopaminergic synaptic signaling pathways, though differentially expressed genes were commonly affected in opposite directions between the two experimental conditions (Supplemental Fig 7).
Parabacteroides treatment also impacted gene pathways associated with metabolic signaling. Specifically, pathways regulating fatty acid oxidation, rRNA metabolic processes, mitochondrial inner membrane, and valine, leucine, and isoleucine degradation were significantly affected by Parabacteroides enrichment. Other pathways that were influenced were those involved in neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s disease, though most of the genes affected in these pathways were mitochondrial genes (Fig. 4D, Supplemental Table 2).
At the level of individual genes, dorsal hippocampal RNA sequencing data revealed that 15 genes were differentially expressed in rats enriched with Parabacteroides compared with controls, with 13 genes elevated and two genes decreased in the Parabacteroides group compared with controls (Fig 4C).
Consistent with results from gene pathway analyses, several individual genes involved in metabolic processes were elevated by Parabacteroides enrichment, such as Hmgcs2, which is a mitochondrial regulator of ketogenesis and provides energy to the brain under metabolically taxing conditions or when glucose availability is low 44, and Cox6b1, a mitochondrial regulator of energy metabolism that improves hippocampal cellular viability following ischemia/reperfusion injury 45. Parabacteroides enrichment was also associated with incased expression of Slc27A1 and Mfrp, which are each critical for the transport of fatty acids into the brain across capillary endothelial cells 46,47.
reference link: https://www.biorxiv.org/content/10.1101/2020.06.16.153809v1.full
Original Research: The study will appear in Translational Psychiatry
