Metformin Cognitive Function and Changes in The Gu

1 Metformin, cognitive function, and changes in the gut microbiome
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3 Running title: Metformin, gut microbiome, and cognition.
5 Marisel Rosell-Díaz1,2,3 , José Manuel Fernández-Real1,2,3,4*
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Department of Diabetes, Endocrinology and Nutrition, Dr. Josep Trueta University Hospital, Girona, Spain.
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Nutrition, Eumetabolism and Health Group, Girona Biomedical Research Institute (IdibGi), Girona, Spain.
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CIBERobn Fisiopatología de la Obesidad y Nutrición, Girona, Spain.
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Department of Medical Sciences, School of Medicine, University of Girona, Girona, Spain.
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12 Keywords: metformin, type 2 diabetes, gut microbiota, cognition.

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14 Corresponding author:
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15 * José-Manuel Fernández-Real, M.D., PhD. e-mail: jmfreal@idibgi.org. ORCID ID: 0000-0002-7442-9323
16 Department of Diabetes, Endocrinology and Nutrition, Dr. Josep Trueta University Hospital, Girona, Spain.
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17 Nutrition, Eumetabolism and Health Group, Girona Biomedical Research Institute (IdibGi), Girona, Spain.
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18 CIBERobn Fisiopatología de la Obesidad y Nutrición, Girona, Spain.
19 Department of Medical Sciences, School of Medicine, University of Girona, Girona, Spain.

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20 Avinguda de França s/n, 17007, Girona, Spain. Phone: +34 972940200 / Fax: +34 97294027.
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22 Financial Support
23 This work was partially funded by Instituto de Salud Carlos III (Madrid, Spain) through the project PI15/01934,
24 PI18/01022, PI21/01361 (to J.M.F.-R.). M.R.D. is funded by Instituto de Investigación Biomédica de Girona Dr.
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25 Josep Trueta (Girona, Spain) through the Horizon 2020 Framework Programme of the European Union under the
26 Marie Skłodowska -Curie Innovative Training Network grant agreement No 859890.
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28 Disclosures
29 No conflicts of interest, financial or otherwise, are declared by the authors.
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© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. This is an
Open Access article distributed under the terms of the Creative Commons Attribution -NonCommercial-
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2 Abstract
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4 The decline in cognitive function and the prevalence of neurodegenerative disorders are among
5 the most serious threats to health in old age. The prevalence of dementia has reached 50 million
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6 people worldwide and has become one of the major public health problems. The causes of age-
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7 related cognitive impairment are multiple, complex, and difficult to determine. However, t ype
8 2 diabetes (T2D) is linked to an enhanced risk of cognitive impairment and dementia. Human
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9 studies have shown that patients with T2D exhibit dysbiosis of the gut microbiota. This
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10 dysbiosis may contribute to the development of insulin resistance and increased plasma
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lipopolysaccharide concentrations. Metformin medication mimics some of the benefits of
12 calorie restriction and physical activity, such as greater insulin sensitivity and decreased
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13 cholesterol levels, and hence may also have a positive impact on aging in humans. According
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14 to recent human investigations, metformin might partially restore gut dysbiosis related to T2D.
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15 Likewise, some studies showed that metformin reduced the risk of dementia and improved
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16 cognition, although not all studies are concordant. Therefore, this review focused on those
17 human studies describing the effects of metformin on the gut microbiome (specifically the
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18 changes in taxonomy, function, and circulating metabolomics), the changes in cognitive
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function and their possible bidirectional implications.
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1 1. Metformin-cognition-gut microbiota.
3 The decline in cognitive function and the prevalence of neurodegenerative disorders are among
4 the most serious threats to health in old age. Mild cognitive impairment occurs in about 40% of
5 the population aged ≥65 years and is associated with a lower quality of life, and increased
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6 incidence of associated pathology (1). The prevalence of dementia has reached 50 million
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7 people worldwide and has become one of the major public health problems. Around 2050 this
8 number will increase threefold (2). The causes of age-related cognitive impairment are multiple,
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9 complex, and difficult to determine. However, the risk of developing inflammation and
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10 cognitive impairment increases in the presence of metabolic diseases such as type 2 diabetes
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(T2D) and hypercholesterolemia. Low-grade systemic inflammation associated with metabolic
12 disorders has been reported to affect the brain as people age (3).
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14 Microbiome-gut-brain axis is a bidirectional communication system. This complex system

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15 involves the gut microbiota, the enteric nervous system, the autonomic nervous system, the
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16 neuroendocrine system, and the central nervous system. This relationship is facilitated by the
17 vagus nerve, the circulatory system, and the immune system (4). The influence of the gut
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18 microbiota on the development of the gut-brain axis is crucial at birth. A microbiome
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categorized as "pathogenic" can independently initiate disease even in individuals who do not
20 possess inherent susceptibility (5). Modifying the microbiome through dietary changes and
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21 other interventions could alter its function, whether it occurs within an individual or is inherited
22 by future generations(6). Therefore, phenotypes in response to a dysbiosis of the gut microbiota
23 could be expected in the next generation. This was demonstrated in mice, where fecal microbial
24 transplantation from patients with autism spectrum disorder was sufficient to promote autism-
25 like behavioral changes in the offspring (7).
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1 The gut microbiota can influence a wide range of physiological processes. Furthermore, it has
2 been identified as an important factor in modulating brain function and structure. In this sense,
3 fecal microbiota composition has been found to be associated with cognition in humans. For
4 instance, bacterial species from the Bacteroidetes (Bacteroides fragilis CAG:558, Bacteroides
5 caccae CAG:21) and the Proteobacteria phyla (Citrobacter freundii, Enterobacter cloacae,
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6 Salmonella enterica, and Klebsiella aerogenes) were negatively associated with immediate
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7 memory scores (5). Conversely, species of the Firmicutes phylum (Clostridium sp. 27_14 or
8 Clostridium sp. CAG:230) were positively associated with better scores in tests measuring
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9 executive function (working memory) and verbal and learning memory (5). On the other hand,
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10 species belonging to the Bacteroidetes phylum (Bacteroides plebeius, Bacteroides gallinarum,
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Bacteroides mediterranensis) have been negatively associated with inhibitory control
12 (executive function); whereas positive associations with executive performance were found
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13 with Eubacterium sp. CAG:603 and Firmicutes bacterium CAG:238 (8,9). In addition, fecal
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14 microbiota is involved in the development and progression of various psychiatric and

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15 neurological conditions, such as depression, autism, stroke, Parkinson's disease, and

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16 Alzheimer's disease (10).
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18 The biguanide metformin is a widely used drug for the treatment of T2D (11). Metformin lowers
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glucose levels by suppressing hepatic gluconeogenesis. It is widely accepted that the pleiotropic
20 effects of the drug are due to its action on the mitochondria causing a mild and specific
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21 inhibition of the respiratory chain complex 1. Inhibition of this complex leads to a decrease in
22 cellular energy charge. The resulting drop in cellular energy activates Adenosine
23 monophosphate-activated protein kinase (AMPK). Once activated, AMPK activates the
24 catabolic pathways that generate Adenosine triphosphate (ATP) and deactivates those cellular
25 processes that consume ATP. Although mitochondria play an important role in the mechanism
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1 of action of biguanides, not all their effects are mediated by this organelle. It has been proposed
2 that metformin may also activate AMPK through a mechanism involving a lysosomal protein
3 (mTOR activator 1) (12–14).
5 However, not all therapeutic effects of metformin are mediated by AMPK. Metformin could
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6 lead to inhibition (via AMP) of fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis
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7 (12–14). Additionally, metformin treatment increases glucagon-like peptide 1 (GLP-1)
8 concentrations, which appears to be another pathway by which it impacts glucose homeostasis
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9 (12–14). GLP-1 is a hormone synthesized and secreted by the brain and the L cells in the
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10 intestine in response to nutrients and bacterial factors (15). In diabetic rats with cerebral
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ischemic damage, recombinant GLP-1 reduced the neurological deficit and infarct area by
12 inhibiting oxidative stress and apoptosis. Treatment with SLAB51 (formulation of nine live
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13 bacterial strains: Streptococcus thermophilus, B. longum, B. breve, B. infantis, L. acidophilus,
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14 L. plantarum, L. paracasei, L. delbrueckii, and L. brevis) increased plasma GLP-1

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15 concentrations, leading to a reduction in brain amyloid load and resulting in modulation of

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16 neuronal functions such as learning and memory in Alzheimer's disease mice models (15).
17 Comparatively to T2D patients on a low-fat diet, T2D patients on a low-carbohydrate almond-

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18 based diet had higher GLP-1 levels and an increased relative abundance of bacteria that produce
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SCFAs, including Roseburia, Ruminococcus, and Eubacterium. At the conclusion of the trial,
20 this group of T2D patients displayed improved glycemic control and a lower depression test
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21 score (16) Although the results are promising, the role of GLP-1 is not as thoroughly studied
22 and characterized in humans as in animal models.
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24 On the other hand, human studies have shown that patients with T2D exhibit dysbiosis of the
25 fecal microbiota (17,18). This dysbiosis may contribute to the development of insulin resistance
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1 and increased plasma lipopolysaccharide concentrations (19). According to recent human
2 investigations, metformin might partially restore gut dysbiosis related to T2D (20–22).
3 Metformin medication mimics some of the benefits of calorie restriction and physical activity,
4 such as greater insulin sensitivity(23) and decreased cholesterol levels(24), and hence may also
5 have a positive impact on aging in humans (25,26). Besides, some studies showed that
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6 metformin reduced the risk of dementia (27,28). We aimed to review here those studies related
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7 to metformin and its relationship with the gut microbiome and cognition in humans.
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9 2. Impact of Metformin on cognition
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T2D is related to an enhanced risk of cognitive impairment and dementia (29). In fact, both
12 insulin resistance and increased circulating glucose levels are associated with poor attention
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13 and executive function (processing speed, sustained attention, and working memory) (30) and
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14 memory processes (31,32). Although the mechanisms by which metformin has neuroprotective
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15 effects are not fully elucidated, metformin primarily exerts its effects on energy homeostasis
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16 within neurons by targeting AMPK (33). In a study involving human neural stem cells (hNSCs),
17 it was observed that cells treated with amyloid -beta exhibited a substantial decrease in cell
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18 viability. This reduction was found to be associated with lowered expressions of AMPK,
19 neuroprotective genes (Bcl-2 and CREB), and mitochondria-associated genes (PGC1α, NRF-
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20 1, and Tfam), while also showing heightened activation of caspase 3/9 and increased cytosolic
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21 cytochrome c activity (33). Metformin treatment effectively eliminated the detrimental effects
22 induced by amyloid-beta in hNSCs through downregulation of caspase 3/9 activities and
23 cytosolic cytochrome c. Furthermore, metformin co-treatment was critical in restoring the
24 fragmented mitochondria in amyloid-beta-affected hNSCs, approaching them to normal
25 morphology(33). Besides, metformin has been described to alleviate Alzheimer's disease-
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1 associated neuropathological changes in differentiated mouse neuroblastoma cell line Neuro-
2 2a (34) and to reduce tau protein phosphorylation in cultured neurons and mouse brains(35).
3 Metformin has also been demonstrated to protect against apoptotic cell death in primary cortical
4 neurons(36). It can normalize the reduced cell proliferation and neuroblast differentiation
5 induced by T2D in the dentate gyrus of the rat hippocampus (37). Lastly, metformin-induced
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6 AMPK activation protected hNSCs against cytotoxicity induced by advanced glycation end
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7 products (38).
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9 Regarding cognition, the cognitive impairment induced by scopolamine was reversed by 100
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10 mg/kg/day of metformin in male Wistar rats. Metformin demonstrated a reduction in
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impairments related to spatial learning and memory and short-term working memory. This
12 beneficial effect was linked to a decrease in inflammation and oxidative stress via Akt activation
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13 (39). Other research examined the effects of chronic metformin treatment and a chronic high-
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14 fat diet on middle-aged male (12 months) C57BL/6 mice. The findings indicated enhanced
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15 spatial learning abilities, improved coordination during running tasks, and a decrease in
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16 memory impairments(40). However, this study had some limitations, such as the absence of a
17 control group receiving metformin treatment. Finally, in male Wistar rats, the administration of
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18 metformin at a dosage of 100 mg/kg/day reversed the cognitive deterioration linked to both a
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high-fat diet and chronic restraint stress(41). These two studies consider latency as the
20 parameter used for cognitive assessment in the Morris water maze testing. Escape latency can
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21 be affected by factors such as elevated body weight, diminished motor function, or slower
22 swimming speeds. The body weight of these rats was influenced by the high-fat diet, metformin
23 treatment, and chronic restraint stress, indicating that latency may not provide an accurate
24 estimate for cognition.
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1 Over the last decade, interest has grown in the possible effects of metformin on cognition in
2 human subjects. Several retrospective and prospective studies have been conducted to elucidate
3 whether metformin exerts (or not) benefits on cognition, as summarized below.
5 2.1 Positive effects of metformin on cognition
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7 Metformin treatment has been linked to a significantly lower risk of dementia (27,28,42,43),
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8 and neurodegenerative diseases (44,45) and with improvement in three cognitive domains,
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9 memory, semantic memory, and executive function (30,46–51) , mostly in subjects with T2D
10 (Table 1).
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12 One of the most populated studies evaluated Australian individuals aged 70 to 90 years over
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13 the course of six years (n=103,767 with T2D and metformin). Ninety-one cases of dementia
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14 during the six-year study were reported, of which 73 were subjects without T2D (8.2% of that
15 group), eight were T2D without metformin (14.5% of that group), and four were T2D with
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16 metformin (6% of that group). In T2D subjects without metformin, the rate of decline was
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17 accelerated specifically in executive function (p=0,006). Hence, an 81% reduction in incident

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18 dementia risk was reported in T2D subjects with metformin treatment (HR=0,19, IC 95 % 0,04–
19 0,85; p = 0,030) (42).

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21 Consistently, an observational study included 559,106 US veterans over 60 years of age with a
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22 diagnosis of T2D. The incidence rate of dementia was lower in the group of patients treated
23 with metformin (6.2 cases per 1000 person-years) compared to the groups treated with
24 sulfonylureas and thiazolidinedione (13.4 cases per 1000 person-years). Combination therapy
25 with metformin and thiazolidinedione reduced the risk of all-cause dementia. After two years
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1 of treatment, the combination of metformin and sulfonylureas became protective against all
2 causes of dementia (HR=0.91, 95% CI 0.88-0.95) (43).
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4 Likewise, an unmatched cohort (n=147 729 subjects who used metformin) and a matched
5 cohort (n=15, 676 subjects who used metformin and n=15,676 subjects who never took
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6 metformin) were investigated in Taiwan. This study informed a lower risk of dementia in
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7 patients taking metformin in both the unmatched cohort (HR=0.550, 95% CI 0.508-0.596,
8 p<0.0001) and the matched cohort (HR=0.707, 95% CI 0.632-0.791, p<0.0001) (28).
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9 A cohort study enrolled 17,200 subjects over 65 years of age from the United States (US) with
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10 a medical history of T2D. The investigation showed a lower risk of dementia in patients <75
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years old in metformin users compared to sulfonylurea users (Hazard ratio (HR)=0.67, 95%
12 confidence interval [CI] 0.61–0.73, p<0.001). The same result was noted in patients using
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13 metformin ≥ 75 years old (HR=0.78, 95% CI 0.72–0.83, p<0.001). After adjusting for
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14 confounding factors, metformin remained protective against the risk of dementia but only in
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15 <75 years old patients (HR=0,89, 95% CI 0.79–0.99, p<0,033) (27).

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17 A longitudinal cohort study examined 6,046 patients from the US with T2D (n=2,993 without
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18 metformin, n=932 patients with metformin ≤1 year, n=566 with metformin between 1-2 years,
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n=789 patients with metformin between 2-4 years, n=766 patients with metformin > 4 years).
20 The results revealed that the use of metformin for two to four years (HR=0.62, 95% CI 0.45 to
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21 0.85) and over four years (HR=0.19, 95% CI 0.12 to 0.31) was associated with a significant
22 reduction in the incidence of neurodegenerative disease (dementia, Parkinson’s disease,
23 Alzheimer’s disease, cognitive impairment). In general, the results for each neurodegenerative
24 disease subtype resembled the results obtained with the neurodegenerative disease (44).
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1 In another report, 527,138 middle-aged Europeans were evaluated (24,087 subjects with
2 Alzheimer’s disease, 47,793 subjects with Alzheimer’s disease-by-proxy, and 383,378 control
3 subjects). These authors used Mendelian randomization to investigate the effect and
4 mechanisms of metformin on Alzheimer's disease. Mitochondrial complex 1 demonstrated a
5 significant impact on Alzheimer's disease (OR=0.88, p=4.73×10−4 ). A lower risk of
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6 Alzheimer's disease was linked to reduced Mitochondrial complex 1-related (NDUFA2) gene
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7 expression (OR=0.95, p=4.64×10−4 ). In the non-diabetic population, genetically proxied
8 metformin use was linked to a 4% decreased risk of Alzheimer's disease (OR=0.96, 95% CI
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9 0.95-0.98, p=1.06×10−4 ) (45).
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Regarding to cognition, a survey in Canada included T2D subjects (1,192 subjects with normal
12 cognition, 671 subjects with mild cognitive impairment, and 807 subjects with AD dementia).
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13 Metformin treatment improved immediate memory (standardized coefficients (β)=0.069, 95%
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14 CI 0.01-0.12, p=0.0202) and delayed memory (β=0.089, 95% CI 0.032-0.146, p=0.0024)

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15 performance across time (48).

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17 Similarly, one study included 487 individuals without cognitive impairment from Spain, of
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18 whom 148 had a medical history of T2D. It was reported a better score in memory (Cohen's d =
19 0,38, 95 % CI -0,02-0,79; p=0,115), executive functions (d = 0.51, 95% CI −0.06-1.08; p =

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20 0.086) and global cognition (d = 0.48, 95% CI −0.01-1.04; p = 0.124) in metformin treated
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21 patients at baseline. Nevertheless, these results were not maintained after three years of follow -
22 up (47). Consistent with these findings, other research reported a higher performance in verbal
23 memory (p < 0.001), working memory (p < 0.01) and executive function (p < 0.01) in 23 T2D
24 patients using exclusively metformin treatment. The latter subjects studied were selected from
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1 an Australian population-based cohort study, which included 1,814 individuals aged 65-69
2 years (49).
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4 A 16-week placebo-controlled crossover study was conducted on 20 non-diabetic subjects with
5 mild cognitive impairment or mild dementia from the US. This research reported that metformin
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6 can pass through the blood-brain barrier after measuring the levels of metformin in
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7 cerebrospinal fluid (one measure at baseline and the other at week eight). Besides, metformin
8 was linked to better executive function (Trail Making Test Part B (TMT-B): p=0.03) and trends
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9 suggested improvement in learning/memory (Paired Associates Learning (PAL): p=0.06) and
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10 attention (Delayed Matching to Sample (DMS): p=0.07) (50). Additionally, two other studies
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showed an improvement in executive function after using metformin. The first was conducted
12 in Canada with 24 surviving pediatric brain tumor subjects, and the other in the US with 2925
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13 participants (30,51).
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15 Lastly, in another small study including 80 subjects with T2D and mild cognitive impairment
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16 in the US (40 metformin-treated and 40 placebo-treated subjects), after controlling for the
17 baseline Disease Assessment Scale-cognitive subscale, the metformin group showed a

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18 considerable improvement in the memory test (p=0.02) (46). In conclusion, the positive effects
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of metformin have been related to a lower incidence of dementia and other neurodegenerative
20 diseases, as well as improved cognition, primarily in subjects with T2D.

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22 2.2 No effects or negative effects of metformin on cognition
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24 On the other hand, one study conducted in the US on 2,280 participants of which 776 were in
25 the metformin intervention arm, reported no significant effects of metformin on cognition (52).
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1 Another study of 333 US women who were overweight or obese breast cancer survivors did not
2 demonstrate metformin's effect on cognition (53). A prospective study in 508 T2D patients did
3 not note a correlation between metformin and a higher score in cognitive tests during an average
4 follow-up of 3.7 years. Moreover, metformin was associated with an enhanced risk of mild
5 cognitive impairment (subhazard ratio= 2.75; 95% CI 1.64-4.63, p<0.001) (54).
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7 Further research included 732 participants from South Korea, of which 93 subjects had T2D
8 with 2.9 years of follow-up. This research outlined that metformin was related with fast decline
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9 of Mini-Mental State Examination (MMSE) scores (odds ratio=4.47, 95% CI 1.24-16.05, p=
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10 0.022) and Verbal Immediate Recall scores (odds ratio=7.37, 95% CI 1.19-45.56, p= 0.032)
11 (55).
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13 These mixed effects of metformin on cognition could be due to differences in sample size. Most
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14 studies showing positive associations between metformin and cognition have large sample
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15 sizes, with many including between 100,000 and 500,000 participants. However, the two
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16 studies where no results were found between metformin use and cognitive function had small
17 sample sizes (n=333 and n=776) and shorter follow-up periods compared to those where there
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18 were significant results.
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20 The study that reported an association between metformin and rapid mild cognitive impairment
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21 had only 200 metformin-treated participants. Additionally, metformin-treated subjects had
22 other associated comorbidities such as hypertension, stroke, and coronary artery bypass
23 grafting. These diseases have a detrimental impact on cognitive function and serve as significant
24 risk factors for dementia(56,57), making them major confounders in the analysis. Nevertheless,
25 metformin treatment has been shown to decrease serum vitamin B12 concentrations (58).
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1 Vitamin B12 depletion could increase the risk of cognitive impairment(59), thus, it could be a
2 parameter to monitor in patients with T2D treated with metformin.
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4 In general, none of the studies investigating the relationship between cognition and metformin
5 considered the results individually in men and women, despite the known sex disparities in
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6 neurobiology and cognitive function (60). Other important factors such as nutrition, physical
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7 activity, or episodes of hypoglycemia were not considered and could also explain the
8 inconsistencies between the studies. Therefore, further large-scale longitudinal studies are
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9 needed to elucidate the potential benefits of metformin on cognition in humans.
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3. Impact of Metformin on gut microbiome in human studies
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13 Some studies have indicated that the intestine, and not the liver, is a primary target of metformin
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14 action. Contrary to oral administration, intravenous metformin did not show glucose-lowering
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15 effects (61–63). Metformin reaches 30-300-fold higher concentrations in jejunal tissue

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16 compared to plasma concentrations (64–66). Caenorhabditis elegans engages in a beneficial
17 interaction with E. coli, a comparable but much simpler relationship than the vast bacterial
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18 communities with positive effects that reside in the human gut. Metformin slows the aging
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process in C. elegans only if gut bacteria are present (67). Thus, the effects of metformin on
20 host physiology seem to be possibly regulated by its interaction with the gut content, including
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21 the gut microbiota and dietary intake.
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23 3.1 Changes induced by metformin at the level of Bacterial Taxonomy.
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1 In addition to be the most used oral hypoglycemic agent in T2D, metformin also has potential
2 therapeutic uses (for instance, in interstitial lung disease (68), cardiovascular disease (69), in
3 the prevention of hyperlipidemia (70,71) and aging (72). This wide spectrum of diseases
4 suggests that the effects on gastrointestinal system and the gut microbiota might have a role in
5 the systemic actions of metformin (73,74). However, human studies on the effects of metformin
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6 on fecal microbiota are quite recent, with less than a decade of research. Insulin resistance and
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7 T2D have been uncovered to be associated with a dysbiosis of the gut microbiome and a
8 decrease in microbial genetic richness in the context of chronic low-grade inflammation (34).
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9
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10 Bacterial taxonomy, bacterial functions and metabolomics are affected by metformin. The most
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consistent findings across the studies related to changes in bacterial composition were the
12 increased abundance of bacteria belonging to the Enterobacteriaceae family (75–77),

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13 specifically the Escherichia/ Shigella genera, mainly Escherichia coli species (20,22,75,78–
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14 83), and the increased abundance of Akkermansia muciniphila species (22,83,84). In addition,
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15 another consistent finding was the decrease in the Intestinibacter bartlettii species
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16 (20,22,78,81,83). Metformin use was also associated with an increase in Ruminococcus torques
17 abundance, although only in one study (80). In contrast, metformin use was associated with a
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18 decrease in the abundance of genera such as Romboutsia (75,81), Clostridium (20,78,81,82),
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Roseburia (20,80), and Roseburia intestinalis species (80), among others (Table 2).
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21 Bacterial functionality analysis demonstrated that indirect effects of metformin treatment, such
22 as reduced gut lipid absorption and local inflammation triggered by lipopolysaccharide (LPS),
23 could provide a competitive advantage to Escherichia spp. This enrichment of Escherichia
24 species could trigger a positive feedback loop that further perpetuates the observed taxonomic
25 changes (20). Metformin also leads to a reduction of fecal microbiota diversity, creating the
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1 perfect niche for Escherichia / Shigella bacteria. Interestingly, some studies found a higher
2 initial presence of Escherichia/ Shigella spp. in the samples from participants who later
3 manifested adverse effects (75).
5 Dysbiosis of the gut microbiota, with the predominance of LPS-synthesizing bacteria, such as
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6 Escherichia/ Shigella, could enhance intestinal permeability and bacterial translocation to the
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7 systemic circulation and worsening insulin resistance (85). In fact, the increased abundance of
8 Escherichia/Shigella was positively correlated with the levels of these proinflammatory
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9 cytokines (IL-6, CXCL2, NLRP3, and IL-1β) (86–89). Importantly, the presence of bacterial
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10 endotoxins has been demonstrated in typical senile plaque lesions in Alzheimer's disease brains
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(90). Thus, it seems contradictory that the enrichment of Escherichia/ Shigella genera could
12 have beneficial effects. A detailed mechanistic investigation suggests agmatine as an E. coli-

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13 produced metabolite necessary for the effects of metformin on host lipid metabolism and
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14 lifespan in both C. elegans and Drosophila melanogaster (73). The study demonstrated a causal
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15 link between metformin supplementation and agmatine production by E. coli to increase host
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16 lifespan, with the phosphotransferase system (PTS)-Crp bacterial axis as a central regulator of
17 the effects of metformin on the host. Metformin treatment was also associated with a predicted
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18 increase in agmatine production capacity by E. coli and other Enterobacteriales in subjects with
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T2D(73). However, it is not possible to determine if the rise in agmatine in metformin-treated
20 T2D patients is linked with an increase in longevity when compared to T2D patients not taking
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21 the medication. Hence, the interaction of metformin with the gut microbiota (with dietary
22 nutrients as a critical factor) could contribute to the beneficial effects and negative side effects
23 observed with metformin use.
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1 Some authors demonstrated that metformin enhances the abundance of E. coli while impairing
2 Intestinibacter bartlettii growth (80). Functional analysis of I. bartlettii reveals that it can
3 degrade fucose, implying an indirect role in mucus degradation (80). I. bartlettii has also been
4 linked to changes in propionate metabolism (83), but the effects of the Intestinibacter genus on
5 human health are uncertain (20).
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7 In contrast, Akkermansia muciniphila is a bacterium specialized in mucin degradation. It also
8 strengthens the integrity of the intestinal epithelium, regulates intestinal barrier function, and is
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9 important in glucose homeostasis (91). Some human studies have reported an increase in the
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10 abundance in Akkermansia muciniphila after metformin treatment (22,83,84). Additionally,
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metformin increases the growth of this bacterium in pure cultures. A. muciniphila was
12 negatively associated with low-grade inflammation, T2D, and insulin resistance in mice (92).
A
13 Likewise, in mice treated with metformin, an increase in the proportion of A. muciniphila and
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14 a positive correlation with the number of mucin-producing goblet cells have been demonstrated.
D
15 Therefore, an increase in the mucus layer by goblet cells could provide a barrier to LPS (93–
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16 95). Besides, the administration of metformin exhibited a notable effect on the relative
17 abundance of A. muciniphila in aged mice. This, in turn, led to a significant reduction in the
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18 levels of the systemic inflammatory biomarker IL-6 (96). Excessive production of peripheral
19
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IL-6 has been observed to contribute to elevated IL-6 levels within the brain. Systemic
20 production of IL-6 can potentially cross the blood-brain barrier and, upon reaching the brain,
A
21 may have a detrimental impact on neurogenesis(97,98). Overproduction of IL-6 impairs
22 neurotransmission in brain regions that modulate cognition, such as the hippocampus and
23 prefrontal cortex (99,100).
24
16
1 Despite these results in animal models, there is no evidence that A. muciniphila is associated
2 with improved glycemic control in humans, furthermore, little or no evidence suggests a causal
3 role of A. muciniphila on the control of obesity/diabetes in humans. One study in humans did
4 not find associations between changes in A. muciniphila abundance and glycated hemoglobin
5 levels (22). Other studies in humans did not report increases in the A. muciniphila abundances
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6 related to metformin use (75,78,79). These differences may result from individual factors such
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7 as age (101,102), immune response (103,104) and fiber intake (105), or polyphenol availability
8 (106,107). Although the direct effect of metformin on A. muciniphila has not been fully
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9 characterized in humans, the increased abundance of A. muciniphila due to metformin treatment
U
10 could restore the increased intestinal permeability induced by T2D and high-fat diets.
11
N
12 On the other hand, the increase in Ruminococcus torques abundance induced using metformin
A
13 has been associated with an increase in short-chain fatty acids (SCFA) production (80). In
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14 contrast, metformin use was associated with decreases in the abundance of the Roseburia genus
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15 (20,80), and Roseburia intestinalis species (80). The genus Roseburia comprises five clearly
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16 characterized species, including Roseburia intestinalis, all of which produce SCFA.
17 Specifically, Roseburia intestinalis is one of the major producers of butyrate. The abundance
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18 of this genus has decreased in subjects with Parkinson’s disease. Contradictory results have
19
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been found with major depressive disorder, where both increases and decreases in abundance
20 have been seen.

A
21
22 The inconsistencies between studies could be determined because metformin dose, time of drug
23 use, study duration, presence, or absence of T2D, time of diagnosed T2D, racial differences,
24 and sample size. Notably, some of these studies employed 16S ribosomal (rRNA) sequencing
25 to analyze the composition of the gut microbiota. 16S rRNA introduces systematic biases, copy
17
1 number variations, and variability in PCR amplification. Hence, the shotgun metagenomics
2 approach is more accurate in functional characterization of the microbial community, according
3 to strains, species, and their biological role (108).
5 3.2 Metformin-induced changes in metagenomics functions and metabolomics
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6
7 Over the past decade, numerous studies have shown that the effects of bacterial composition
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8 and its function are reflected in the circulating metabolome. Regarding functional analysis, the
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9 major findings are related to acetate, lipopolysaccharide, and peptidoglycan synthesis
10 pathways, glucose metabolism pathways, and bile acids(20,22,80,81,109). In addition, the main
11
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findings concerning metabolomics are related to variable alterations in the concentrations of
N
12 short-chain fatty acids, hippuric acid, and amino acids (20–22,76,80,109,110). The two most
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13 investigated results in the literature are related to increased concentrations of SCFA and bile
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14 acids.
15
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16 3.2.1 Findings related to Short-Chain Fatty Acids

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17
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18 One of the best characterized functions of the gut microbiota is the production of SCFA through
19 the fermentation of non-digestible carbohydrates derived from the diet. The hypothesis that
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20 metformin partly exerts its beneficial effect on cognition is related to SCFA biological
21 functions. SCFA inhibit neuroinflammation and regulate enteric nervous system, and are
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22 involved in brain function, neuroplasticity, and behavior. Furthermore, SCFA regulate
23 neurotransmitter synthesis and the expression of receptors, such as dopamine and γ-
24 aminobutyric acid receptors (111,112). Therefore, disequilibrium in SCFA production could
25 play a determinant role in the development of cognitive impairment and neurodegenerative
26 diseases.
18
1 The SCFA (acetate, propionate, and butyrate) have a crucial role in controlling metabolism and
2 supply of energy, in addition to preserving the homeostasis of the gut environment. Acetate is
3 categorized as a dominantly obesogenic SCFA because it acts as a substrate for the synthesis
4 of cholesterol and fatty acids in the liver and other tissues. Propionate has been described to
5 protect from diet-induced obesity by upregulating the expression of the gene that encodes leptin
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6 synthesis. Propionate has gluconeogenic effects in the liver, whereas acetate and butyrate are
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7 lipogenic. Butyrate is involved in the regulation of colonic mucosal homeostasis, cell
8 proliferation, and differentiation, enhancing insulin sensitivity. Butyrate has been also shown
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9 to increase energy expenditure and protect colonocyte membrane function by limiting oxidative
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10 stress (4,113,114).
11
N
12 A multi-country metagenome dataset in subjects from Denmark, Swedish and China (199 T2D
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13 subjects, 31 Type 1 Diabetes subjects, and 554 nondiabetic subjects) showed that T2D patients
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14 without metformin treatment showed a decrease in the abundance of genera containing

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15 butyrate-producing species such as Roseburia and Subdoligranulum. In contrast, in metformin-

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16 treated subjects, a notable increase in the Subdoligranulum genus was observed. The final
17 analysis reported a rise in butyrate and propionate production in those treated with metformin
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18 (20).
19
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20 Likewise, SCFA production in 24 Dutch T2D patients (9 without metformin and 15 using
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21 metformin) was compared. SCFA levels were shown to be higher in T2D patients who were
22 not taking metformin, particularly butyrate (115). In another study, a group of 18 subjects with
23 newly diagnosed T2D treated with placebo was compared with another of 22 subjects with
24 newly diagnosed T2D treated with metformin recruited in Spain. In the metformin-treated
25 group, elevated fecal concentrations of butyrate and propionate were observed in men.
19
1 However, no significant results were obtained when men and women were analyzed together
2 (22).
3 Lastly, 121 overweight/obese subjects were studied in the US. Those individuals taking
4 metformin displayed higher acetate and butyrate levels at six months, but these results were not
5 sustained at 12 months. In the same way, metformin raised SCFA-producing Ruminococcus
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6 torques and SCFA-production pathways (80).
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7
8 Thus, metformin appears to exert beneficial effects on neuroinflammation and glucose
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9 homeostasis, either by a direct effect on SCFA-producing pathways or by increasing the SCFA-
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10 producing gut microbiota. However, the exact mechanism whereby metformin modulates both
11
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gut microbiota and metabolic homeostasis remains unclear.
12
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13 3.2.2 Metformin and microbiota functions related to bile acid metabolism.
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14
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15 Bile acid biosynthesis comprises two mechanisms and anatomical sites. The first is the de novo
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16 synthesis of primary bile acids from cholesterol in the liver, and the second is the formation of
17 secondary bile acids because of bacterial enzyme modification of primary bile acids in the
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18 gut(116). Primary bile acids can be deconjugated, oxidized and epimerized, or dehydroxylated,
19
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by the gut microbiota to create secondary bile acids. Because deconjugated primary bile acids
20 can operate as signaling molecules that alter the overall bile acid pool, the microbiota could
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21 have evolved the deconjugation mechanism to further modulate bile synthesis. Increased
22 concentrations of antimicrobial bile acids, cholic acid, and chenodeoxycholic acid also result
23 from deconjugation, which may cause modifications in microbiome composition(116,117). All
24 major bacterial phyla such as contain enzymes (bile salt hydrolases) capable of performing the
25 deconjugation process, implying that the genes encoding these enzymes (bsh genes) are
20
1 horizontally transferable(116–118). Bile salt hydrolases can deconjugate glycine- and taurine-
2 bound primary bile acids. The gut microbiota can also employ the released residues of glycine
3 and taurine as nutrient resources, thus, deconjugation is a gut microbiome critical function
4 (116).
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6 In contrast, epimerization requires the actions of α-and β-bile acid hydroxysteroid
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7 dehydrogenases (HSDHs)(117). 3α- and 3β-HSDHs have been found in the gut microbiota of
8 various Firmicutes phylum, although intraspecies 3-hydroxy epimerization has only been
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9 identified in Peptostreptococcus productus, Clostridium. perfringens, and Eggerthella
lenta(116,119). 7α-HSDHs are identified among members of the genera Clostridium,
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10
Eubacterium, Bacteroides, and Escherichia, while 7 β-HSDHs are exclusively found in

11
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12 Firmicutes. Species belonging to Clostridium, Eubacterium, and Ruminococcus genera are the
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13 only ones capable of intraspecies 7-epimerization (117,120). Lastly, due to the inaccessibility
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14 of the hydroxyl group, dehydroxylation can only occur after deconjugation. Species with 7α-
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15 dehydroxylation activity belong to the Firmicutes phylum (Clostridium (C. scindens, and C.
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16 hylemonae), and Eubacterium genera)(117).
17
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18 Bile acids play a key role in glucose, energy, and lipid homeostasis. The modulating effects of
19
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bile acids on various metabolic pathways occur primarily through intracellular nuclear receptors
20 binding, among them FXR. FXR is a ligand-activated nuclear receptor that modulates hepatic
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21 bile acid metabolism and is implicated in several metabolic diseases (121,122).
22
23 Glycoursodeoxycholic acid (GUDCA) is a bile acid obtained by the conjugation of
24 ursodeoxycholic acid (secondary bile acid) and glycine. GUDCA has been linked to a
25 neuroprotective function due to its antiapoptosis, anti-inflammatory, and antioxidant effects.
21
1 Its role in metabolic disorders is starting to be examined because the interaction of GUDCA
2 and intestinal FXR could reduce body weight gain and improve glucose intolerance as well as
3 insulin resistance (121,122). In this regard, metformin treatment raised bile acid GUDCA levels
4 in the gut in 12 T2D patients. Therefore, metformin might inhibit bile acid reabsorption, leading
5 to a longer exposure time of the intestine to bile acids and an increase in the concentration of
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6 bile acids in the feces. Prolonged exposure to bile acids could result in the binding of bile acids
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7 to intestinal FXR (21).
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9 Consistently, a study conducted in China with 22 T2D patients, metformin therapy raised
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10 GUDCA levels by decreasing the abundance of B. fragilis and the activity of its bile salt
11
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hydrolase. Metformin restrained the growth of B. fragilis by affecting folate and methionine
12 metabolism. This study demonstrated that enhancements of glucose metabolism in metformin-

A
13 treated mice were transposed by B. fragilis colonization (109).
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14
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15 Consequently, one survey reported a raise in the plasma bile acid (primary, secondary, and
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16 unconjugated) concentration after four months of metformin treatment in 22 T2D patients.
17 Also, an increase in the abundance of bsh genes was described after two months of metformin
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18 treatment. Hence, an increase in bsh genes could lead to an increment in the concentration of
19
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unconjugated bile acids. Furthermore, this study found a negative correlation between glycated
20 hemoglobin and unconjugated bile acids concentrations (22). Therefore, the effects of
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21 metformin on glucose metabolism could be mediated by an AMPK-independent pathway
22 through the intestinal FXR, B. fragilis, GUDCA, and bile acids.
23
24 4. Clinical implications of the Metformin-induced changes in the gut-brain axis.
25
22
1 Neurodegenerative diseases and cognitive impairment pose a therapeutic and diagnostic
2 challenge. The risk of developing these pathologies increases the longer a person lives with
3 T2D, especially if there has not been adequate metabolic control (123). To date, metformin has
4 been proposed to show neuroprotective, anti-inflammatory, and antioxidant effects and its use
5 has been associated with a lower risk of dementia in subjects with T2D (27,28,42,44). Likewise,
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6 improved memory has been demonstrated in patients with mild cognitive impairment and T2D
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7 who regularly use metformin (46,48). Also, a lower incidence of neurodegenerative diseases
8 has been described in older adults with T2D and metformin treatment(44). Specifically,
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9 metformin has been associated with better cognition, hence, better executive
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10 function(30,42,47,49), memory(47,49), and semantic memory(49) in T2D subjects. In non-
11
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diabetic subjects with mild cognitive impairment, metformin use has also been associated with
12 improved attention and executive function (50). Even better cognitive performance has been
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13 observed after the use of metformin in pediatric brain tumor survivors(51). Despite these
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14 encouraging results, an association between metformin and cognitive impairment has also been
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15 reported (55). Part of the metformin's beneficial effects could be modulated by the gut
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16 microbiota composition and functionality, with an increase in the production of SCFA and bile
17 acids.
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18
19
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Despite the encouraging results, most studies investigating the metformin-gut-microbiota-brain
20 axis in humans are mainly associative in nature, except for one study that employed
A
21 transplantation of human gut microbiota into germ-free mice and a gut simulator. In this study,
22 the transfer of metformin-altered fecal microbiota to germ-free mice demonstrated that gut
23 microbiota-metformin interaction is required to improve glucose metabolism. Moreover, when
24 feces were cultured in vitro with metformin in a simulated gut environment, transcriptome
23
1 analyses revealed direct effects of metformin on the microbiota. These effects included the
2 regulation of gene expression in gut bacteria encoding metalloproteins (22).
3
4 Human research is not progressing as fast as animal research. Nevertheless, human research on
5 the gut microbiota-gut-brain-metformin axis has reported interesting and consistent
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6 associations. Clinical research has demonstrated important differences in cognition as well as
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7 gut microbiota composition and metabolomics when comparing individuals with and without
8 metformin. However, many of the studies are case- control comparisons that do not consider
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9 critical confounding factors such as diet.
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10
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While animal studies have been instrumental in generating hypotheses and establishing causal
12 relationships, there is still much ground to cover to fully translate these findings to human
A
13 research. To understand the level of contribution of metformin on gut microbiota and cognition,
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14 research on genetic information and risk alleles associated with the diseases of interest will be
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15 necessary. Studies with large human cohorts and follow-up over time will be necessary. This
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16 will permit the definition of the directionality of cause and effect to generate hypotheses that
17 can be tested in humans and experimental systems. Across cohorts, replication will also be
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18 critical. Longitudinal correlation of blood and urinary biomarkers with microbial products
19
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involved in cognition and inflammation will be essential to determine cellular and molecular
20 mechanisms. Lastly, intervention studies aimed at directly changing the gut microbiota will also
A
21 be worth exploring. Understanding the complex metformin-gut-brain axis interactions could
22 allow us to individualize and target the beneficial actions of metformin as well as diminish its
23 adverse effects.
24
25 5. Conclusions
24
1
2 Metformin is a well-tolerated drug that has been used for T2D treatment since the 1950s. It is
3 widely accepted that the pleiotropic effects of the drug are due to its action on the mitochondria
4 causing a mild and specific inhibition of the respiratory-chain complex 1. However, the exact
5 mechanism of action of this drug remains elusive. Metformin's primary target of action has been
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6 identified as the gut, and intriguingly, its ability to slow down the aging process in C. elegans
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7 is dependent on the presence of gut microbiota. This review highlighted the existing
8 understanding from human studies regarding the effects of metformin on the gut microbiome,
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9 the associated alterations in cognitive function, and the potential bidirectional implications of
U
10 these interactions.
11
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12 T2D has been related to impaired memory, executive functions, and an increased risk of
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13 dementia. Metformin could have benefits on cognition through several pathways, decreasing
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14 glycation end products, inflammation, and preventing the development of the metabolic
D
15 syndrome. Metformin treatment has been reported to have mixed effects on cognition. Despite
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16 there being studies where metformin shows a positive impact on memory, attention, and
17 executive functions, some studies showed no significant effects of metformin on cognition or

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18 even an enhanced risk of mild cognitive impairment. However, the precise mechanisms
19
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underlying the neuroprotective effects of metformin or its potential to enhance cognitive
20 performance across various cognitive domains remain elusive.

A
21
22 Several studies have demonstrated that metformin exerts changes in the composition of the gut
23 microbiota taxonomy, function and in the metabolome. Metformin seems to fundamentally
24 affect the bacterial composition of patients with T2D, resembling the composition of the gut
25 microbiota of patients without T2D. In addition, it has effects on several metabolic synthesis
25
1 pathways, the concentration of bile acids, and SCFA. In parallel to metformin, there are other
2 factors that influence the gut microbiota such as medications, sex, diet, body mass index, and
3 other diseases. It is vital to control for confounding factors to ensure that the observed results
4 in studies evaluating metformin effects are in fact attributable to metformin.
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6 Despite its cost, the study of the fecal microbiota by means of shotgun metagenomics is
RI
7 increasingly important. To elucidate the effects of metformin on gut microbiota, it is essential
8 to understand the functional characterization of the microbial community (strains, species) as
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9 well as to infer its biological role and metabolic phenotype. Transplantation of fecal microbiota
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10 from humans to mice could also be necessary to validate metformin's mechanism of action and
11
N
findings. To our knowledge, no studies have investigated the interrelationship among
12 metformin, gut microbiota, and cognition. Thus, it will be of interest to explore whether the
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13 effects of metformin on cognition could be mediated or regulated by the gut microbiota.
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14
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15 Acknowledgments
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16 Figures 1 and 2 were created with BioRender.com.
17
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18 Author Contributions
19 J.M.F.-R. conceptualized and designed research; M.R.D. drafted manuscript and prepared
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20 figures; J.M.F.-R. edited and revised manuscript; all authors approved the final version.
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26
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33 from normal human intestinal flora but not lactic acid bacteria exhibit 7α- and 7β-
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34 hydroxysteroid dehydrogenase activities. Microb Ecol Health Dis 2004;16(4):195–201.

35 121. Huang K, Liu C, Peng M, Su Q, Liu R, Guo Z, Chen S, Li Z, Chang G.
36 Glycoursodeoxycholic Acid Ameliorates Atherosclerosis and Alters Gut Microbiota in
37 Apolipoprotein E-Deficient Mice. J Am Heart Assoc 2021;10(7):e019820–e019820.
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38 122. Ticho AL, Malhotra P, Dudeja PK, Gill RK, Alrefai WA. Intestinal Absorption of
39 Bile Acids in Health and Disease. In: Comprehensive Physiology.; 2019:21–56.
40 123. Simó R, Ciudin A, Simó-Servat O, Hernández C. Cognitive impairment and
dementia: a new emerging complication of type 2 diabetes—The diabetologist’s perspective.
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41
42 Acta Diabetol 2017;54(5):417–424.
43 124. Elbere I, Silamikelis I, Dindune II, Kalnina I, Ustinova M, Zaharenko L,
44 Silamikele L, Rovite V, Gudra D, Konrade I, Sokolovska J, Pirags V, Klovins J. Baseline
45 gut microbiome composition predicts metformin therapy short-term efficacy in newly
46 diagnosed type 2 diabetes patients. PLoS One 2020;15(10):e0241338–e0241338.
47 125. de la Cuesta-Zuluaga J, Mueller NT, Corrales-Agudelo V, Velásquez-Mejía EP,
48 Carmona JA, Abad JM, Escobar JS. Metformin Is Associated With Higher Relative
49 Abundance of Mucin-Degrading <em>Akkermansia muciniphila</em> and
35
36
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Several Short-Chain Fatty Acid–Producing Microbiota in the Gut. Diabetes Care
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Table 1 Human studies focused on metformin and cognition.
Reference and Country Study design Findings related to cognition
N
Clinical
Trials.gov
A
identifier
(51) Canada 24 surviving pediatric brain In the metformin-treated group compared with the placebo-treated group:
M
(NCT02040376) tumor subjects.
Greater total numbers of correct responses on List Sorting Working Memory (p = 0,05)
were observed. Also, a higher information processing speed was noted (decreased
D average latency on the Cambridge Neuropsychological Test Automated Battery)

(p=0,03). The highest total number of words recalled for immediate recall on the
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Children’s Auditory Verbal Learning Test-2/ Rey Auditory Verbal Learning Test
(p=0,01) and a significant increase in the axonal water fraction within the corpus
callosum (p=0,04) were observed.
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(54) US 508 T2D subjects. There was no significant association between metformin and cognitive performance on
cognitive tests. Metformin was associated with an enhanced risk of mild cognitive
impairment (subhazard ratio= 2.75; 95% CI 1.64-4.63, p<0.001).
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(42) Australia 67 T2D patients with No differences in baseline cognitive performance were found between the groups. In T2D
metformin (mean age subjects with metformin, a significantly lower rate of decline over six years was reported
A
78,25±4,6), 56 T2D patients (p=0,032). In addition, in T2D subjects without metformin, the rate of decline was
without metformin (mean accelerated for executive function (p=0,006), memory, language, and
age 80,0±4,7), 903 attention/processing speed (without reaching statistical significance). Hence, an 81%
nondiabetic individuals reduction in incident dementia risk was reported in T2D subjects with metformin
(mean age 78,8±4,8). treatment (HR=0,19, 95 % CI 0,04–0,85, p = 0,030)
(55) South 93 T2D patients with Metformin was related with fast decline of MMSE scores (odds ratio (OR)=4.47, 95%
Korea metformin (mean age CI 1.24-16.05, p= 0.022) and Verbal Immediate Recall scores (OR=7.37, 95% CI 1.19-
75,8±6,5), 64 T2D patients 45.56, p= 0.032).
37
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without metformin (mean
age 77,2±7,4), 575
N
nondiabetic individuals
(mean age 76,8 ± 6,7).
A
(49) Australia 74 T2D patients with diet In 23 patients with T2D on metformin-only treatment, significantly better performance
control (mean age 66.3±1.5), was observed on the Immediate Recall (verbal memory, p < 0.001), Digit Span Backward
M
24 T2D patients with insulin (working memory, p < 0.01), and Trail Making Test Part B (executive function, p < 0.01)
treatment (mean age tests at baseline. It was reported in the longitudinal analysis that participants with
67.3±0.3), 113 T2D patients exclusive metformin (n=76) had a significant protective effect on performance for choice
D with oral hypoglycemic

agents (mean age 66.7 ±
reaction time (p < 0.01).
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1.6), 1603 nondiabetic
individuals (mean age 66,6
±1,5).
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(48) Canada 1192 subjects with normal In patients with T2D who received metformin-only treatment, improvements in the
cognition (mean age performance of immediate memory (β=0.069, 95% CI 0.01-0.12, p=0.0202) and delayed
72.25±8.28), 671 subjects memory (β=0.089, 95% CI 0.032-0.146, p=0.0024) was observed across time.
with mild cognitive Metformin did not display a significant association with memory changes in patients with
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impairment (mean age mild cognitive impairment and AD dementia.

74.37±8.2), 807 subjects
with Alzheimer disease
A
dementia (mean age

76.11±7.93).
(30) US 2925 participants. In the metformin-treated group compared with subjects with other anti-diabetic drugs
(n=311): significantly higher scores on Digit Symbol Substitution Test (DSST) and
Animal Fluency Test (AFT) were found; hence, increased linguistic fluency as assessed
with AFT, and enhanced executive function, as assessed by the DSST.
(44) US 6046 patients with T2D In metformin-treated group compared with no treated group:
(mean age 63.20±10.90 ≤1 year of metformin treatment was associated with an increased risk of
years old), 2993 without neurodegenerative disease (HR=1.16, 95% CI 0.89-1.51)
38
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metformin treatment (mean 1-2 years of metformin therapy reduced neurodegenerative disease risk without statistical
age 65.82±11.66 years old), significance (HR=0.80, 95% CI 0.56-1.13).
N
932 patients with metformin 2–4 years (HR=0.62, 95% CI 0.45-0.85) and ≥4 years (HR=0.19, 95% CI 0.12-0.31) of
treatment ≤1 year, (mean metformin use were linked to a significative lower risk of neurodegenerative disease.
A
age 61.25±10.74 years old),
566 with metformin
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treatment between 1-2 years
(mean age 60.68±9.16 years
old), 789 patients with
D metformin treatment
between 2-4 years (mean age
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60.23±8.98 years old), 766
patients with metformin
treatment > 4 years (mean
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age 60.21±8.24
years old).
(50) US 20 subjects (mean age Metformin was found to be associated with enhanced executive functioning (TMT-B:
(NCT01965756) 70.1±6.89 years old) p=0.03), and trends suggested improvement in learning/memory (PAL: p=0.06) and
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attention (DMS: p=0.07). In addition, during the eight weeks of metformin treatment,
was found a significant increase in superior and middle orbitofrontal cerebral blood flow
(p <0,05).
A
(27) US 17 200 subjects using In metformin users compared to sulfonylurea users was noted a lower risk of dementia in
metformin, 11440 subjects patients <75 years old (HR=0.67, 95% CI 0.61–0.73, p<0.001). The same result was
using sulfonylureas (mean obtained in patients ≥ 75 years (HR=0.78, 95% CI 0.72–0.83, p<0.001). After adjusting
age 73.5±5.9 years old) for the inverse probability of treatment weighting (IPTW) method, metformin remained
protective against the risk of dementia but only in <75 years old patients (HR=0.89; 95%
CI 0.79–0.99, p<0,033).
39
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(28) Taiwan An unmatched cohort of 147 Metformin consumption was related to a lower risk of dementia in the unmatched cohort
729 metformin-treated (HR=0.550, 95% CI 0.508-0.596, p<0.0001) and in the matched cohort (HR=0.707, 95%
N
patients and 15 676 non- CI 0.632-0.791, p<0.0001).
treated subjects. Another
A
matched cohort of 15,676
people with metformin and
M
15 676 people without
metformin.
(46) US 40 subjects takingIn the metformin-treated group compared with the placebo-treated group:
(NCT00620191)
D metformin (mean
65,3±7,0 years old), 40
ageIn crude analysis, higher improvement in Selective Reminding Test (SRT) was found.
However, after controlling for baseline Disease Assessment Scale-cognitive subscale
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subjects taking placebo (ADAS-Cog), the metformin group showed a considerable improvement in SRT total
(mean age 64,1±7,9 recall (p=0.02). The highest dose of metformin was correlated to a statistically significant
years old). increase in total SRT recall words.
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(47) Spain 148 subjects with T2D At baseline, subjects taking metformin had better performance in executive functions (d =
(mean age 65.9±4.7 years 0.51, 95% CI −0.06-1.08; p = 0.086), memory (d = 0,38, 95 % CI -0,02-0,79; p = 0,115),
old), 339 nondiabeticand global cognition (d = 0.48, 95% CI −0.01-1.04; p = 0.124). In contrast, those without
individuals (mean agemetformin had a better performance in executive functions (mean change of 0.36 vs.
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64.9±4.7 years old). 0.02, p = 0.005), and global cognition (mean change of 0.29 vs. −0.02, p = 0.001) after
three years.
Non-diabetic individuals exhibited greater increases in memory (p < 0.001) and global
A
cognition (p= 0,003) after one and three years compared to subjects on T2D with
metformin. T2D subjects without metformin exhibited a better mean rate of change in
executive functions compared to non-diabetic individuals (p=0.032).
(52) US 2280 participants (749 with There were no cognitive differences between the intervention groups. Metformin
lifestyle intervention, 776 exposure over time had no effect on cognition.
with metformin
intervention, 755 with
placebo intervention) (mean
age 63.1±10.7 years).
40
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(53) US 333 breast cancer In five neurocognitive domains, there were no statistically significant intervention effects
survivors (mean age for metformin or weight loss.
N
62.6±6.9 years).
(45) China 527 138 middle-aged In non-diabetics, genetically proxied metformin use was linked to a 4% decreased risk of
A
Europeans (24 087 subjects Alzheimer's disease (OR=0.96, 95% CI 0.95-0.98, p=1.06×10−4 ). Mitochondrial complex
with Alzheimer’s disease, 47 1 demonstrated a significant impact on Alzheimer's disease that was independent of
M
793 subjects with AMPK (OR=0.88, p=4.73×10−4 ). A lower risk of Alzheimer's disease was linked to
Alzheimer’s disease-by- reduced Mitochondrial complex 1-related gene (NDUFA2) expression (OR=0.95,
proxy, and 383 378 p=4.64×10−4 ).
(43) D
US
controls).
559 106 subjects with T2D The incidence rate of dementia was lower in the group of patients treated with metformin
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(mean age 65.7±SD 8.7) (6.2 cases per 1000 person-years) compared to the groups treated with sulfonylureas and
thiazolidinedione (13.4 cases per 1000 person-years). Sulfonylurea monotherapy was
associated with a 12% higher risk of all causes of dementia compared to the metformin-
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treated group (HR=1.12, 95% CI 1.09 -1.15). Combination therapy with metformin and
thiazolidinedione reduced the risk of all-cause dementia. After two years of treatment,
these results did not change, although the combination of metformin and sulfonylureas
became protective against all causes of dementia (HR=0.91, 95% CI 0.88-0.95).
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Table 2 Human studies focused on metformin, bacterial taxonomy, and functionality.
U
Reference and Country Study design Findings related to bacterial taxonomy Bacterial
Clinical functionality
N
Trials.gov
identifier
A
(78) Denmark 27 healthy men (mean Genera: Intestinibacter a, Clostridium , Terrisporobacter , NRc
age 26 ± 3.4) immediately after metformin and all the intervention periods.
M
Senegalimassilia , and Lachnospiraceae , temporarily.
(NCT0254605 Escherichia / Shigella b , immediately after metformin and all the
0) intervention periods. Bilophila , Lachnoclostridium,
D Caproiciproducens , Tyzzerella, and Prevotella,at

different time periods.
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(22) Spain 18 recently diagnosed In metformin-treated group compared with the placebo-treated Fecal propionate,
T2D subjects with group: butyrate, lactate,
placebo (mean age 54.9 Genera: Escherichia , Bifidobacterium , Intestinibacter . succinate , Plasma
Akkermansia muciniphila , Bifidobacterium bile acids , in the
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±1.9) Species:
22 recently diagnosed adolescentes , Escherichia coli . metformin group.
T2D subjects with
metformin (mean age
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52.6 ±2.0)
(79) China 100 subjects with T2D In metformin-treated group compared with the herbal formula- NR
(NCT0147127 and metformin (mean age treated group:
alpha-diversity (Simpson’s diversity index): .
A
5) 58.55±9.17).
100 subjects with T2D Genera: Erysipelotrichaceae incertae sedis , Escherichia
and herbal formula (mean /Shigella , Fusobacterium , Flavonifractor , Lachnospiraceae
age 59.00±9.46). , Lachnospiracea incertae sedis , and Clostridium XVIII and IV
, Blautia , Anaerostipes , Bacteroides , Parabacteroides ,
Oscillibacter , Alistipes , Ruminococcaceae .
42
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(75) Latvia 18 healthy subjects Alpha-diversity (Shannon index) . NR
treated with metformin Order: Enterobacteriales 
N
(mean age 25.5±7.5) Family: Peptostreptococcaceae , Clostridiaceae_1 ,
Enterobacteriaceae .
A
Genera: Peptostreptococcaceae_unclassified ,
Clostridiaceae_1_unclassified, Asaccharospora ,
M
Romboutsia , Escherichia-Shigella .
(80) US 121 subjects (mean age In metformin-treated group: Increased serum
60 years) Genus: Roseburia . acetate, butyrate, and
D Species: Escherichia coli , Ruminococcus torques ,

I. bartlettii , Roseburia intestinalis , Roseburia faecis .
valerate at 6 months, in
the metformin-treated
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group.
(81) South 20 healthy adult male Alpha-diversity (Kruskal-Wallis test) , beta diversity . Urine and plasma:
NCT03809260 Korea subjects (between 19–33 Genera: Intestinibacter , Clostridium , Romboutsia , Palmitoleic acid ,
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years old) Escherichia . most

carbohydrates,
amino acids  post
metformin period.
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(109) China 22 subjects with T2D Alpha-diversity (Shannon index) . Serum and stool:
(mean age 55 years old) Genus: Bacteroides . Tauroursodeoxycholic
Species: Bacteroides finegoldii , B. fragilis . acid , conjugated
A
secondary bile acids

,glycoursodeoxychol
ic acid .
(20) Denmark 277 non-diabetic T2D without metformin-treatment:  butyrate and
induvial, 75 T2D Genera: Roseburia spp , Subdoligranulum sp , Clostridiales spp propionate production.
subjects, and 31 Type 1 , Lactobacillus spp .
Diabetes subjects from a T2D with metformin-treatment:
Danish cohort. Genera: Escherichia spp , Intestinibacter spp .
43
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53 T2D female subjects,
and 92 nondiabetic
N
individuals from
Swedish.
A
71 T2D subjects, and 185
M
from China.
(124) Latvia 35 healthy nondiabetic Alpha-diversity  in T2D patients. In healthy subjects:
individuals (mean age Metformin induced changes: folate biosynthesis ,
D 31,5 ± 10,2).
50 newly diagnosed T2D
Genus: Oscillibacter .
Species: Clostridium bartlettii , Barnesiella intestinihominis ,
thiamine and vitamin
B6 biosynthesis .
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patients (mean age 58,6 Parabacteroides distasonis .
±12,5).
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(125) Colombia 28 T2D patients, 14 with T2D patients with metformin compare with nondiabetes: NR
metformin (mean age 50 Class: Mollicutes (OTU284 y OTU067)).
± 10) and 14 without Genera: Butyrivibrio (OTU062), Prevotella (OTU028),
metformin (mean age 44 Megasphaera (OTU069), Bulleidia p-1630-c5  (OTU077).
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± 9) Species: Bifidobacterium bifidum , Akkermansia muciniphila .

84 nondiabetic Enrichment of Prevotella and Megasphaera OTUs was observed.
individuals (mean age 47
A
± 9)
(76) Sweden 30 T2D patients (mean In T2D patients with metformin (n=23): SCFA concentrations
(NCT0240580 age 57.5±8.2) Family: Enterobacteriaceae . reported no related to
6) metformin.
(110) China 51 T2D patients with In T2D patients with metformin: Metformin promoted
metformin treatment alpha-diversity (Shannon index): . lipids biosynthesis.
(mean age 58.1±9.4), 36 Phylum: Actinobacteria . Also, contributed to
T2D patients with insulin Class: Erysipelotrichi , Alphaproteobacteria
44
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treatment (mean age Genus: Fusobacterium , Spirochaete , Turicibacter , the hypotaurine and
59.75±13.0), 17 T2D Ruminococcus . taurine metabolism.
N
patients with α-
glucosidase inhibitor
A
treatment (mean age
62.3±10.4), 26 T2D non
M
treated patients (mean age
56.4±10.6), 50
(77) D
Japan
(mean age 58.5±3.0).
50 T2D patients (mean In T2D patients with metformin: NR
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age 62.5±10.8), 50 Family: Enterobacteriaceae .
nondiabetic individuals Genus: Staphylococcus .
(mean age 60.2±12.9).
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(82) Sweden 53 women with T2D In women with T2D taking metformin treatment. NR
(mean age 70.5±0.1), 49 Family: Enterobacteriaceae .
women with impaired Genera: Escherichia , Shigella , Klebsiella ,Salmonella ,
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glucose tolerance (mean Clostridium , Eubacterium .

age 70.5±0.1), 43 women
with normal glucose
A
tolerance (mean age

70.3±0.1).
(21) United 12 T2D subjects (mean On visits with metformin: Serum bile acids .
(NCT0135787 Kingdom age 56±5.4) Genus: SMB53 , Adlercreutzia , Eubacterium . Fecal bile acids .
6) Glucagon like peptide
1 (GLP-1) .
45
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(115) Netherlan 24 T2D patients, 15 In T2D patients with metformin: SCFA levels 
ds metformin users, 9 Species: Escherichia coli . (specially propionate).
N
metformin users.
a
 (decreased)
A
b
 (increased)
c
Non reported
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D
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A
46
4 Figure Legends
6 Figure 1. Impact of Metformin on cognition. Each chart shows the cognitive domain
7 where metformin has been reported to have effects and the brain region involved in each
8 cognitive function. Clinical and preclinical studies show that metformin has a protective
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9 effect against dementia and decreases the incidence of neurodegenerative diseases such
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10 as Alzheimer's disease. Although the mechanisms by which metformin has
11 neuroprotective effects are not fully elucidated, metformin primarily exerts its effects on
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12 energy homeostasis within neurons by targeting AMPK. Furthermore, the effects of
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13 metformin on cognition could be facilitated through the gut microbiota. Amyloid-beta,
14
N
(Aβ); Adenosine monophosphate-activated protein kinase, (AMPK).
15
A
16 Figure 2. Impact of Metformin on gut microbiota. The figure shows the main effects of
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17 metformin on the gut microbiota that have been reported in clinical studies. GUDCA,
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18 Glycoursodeoxycholic acid; BSH, bile salt hydrolase.

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19
20
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47
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48
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N
135x122 mm ( x DPI)
A
Figure 1
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D
TE
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A
21
22
23
24
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49
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N
135x94 mm ( x DPI)
A
Figure 2
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D
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26
27
28
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Graphical Abstract
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29
30
31
32 Essential points
33
34
35 • Metformin seems to exert neuroprotective, anti-inflammatory and antioxidant
36 effects, including prevention of dementia and neurodegenerative diseases in
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37 humans.
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38 • Clinical studies have also reported an association between metformin and
39 improved memory and executive function.
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40 • Metformin use changes bacterial taxonomy, increasing the relative abundance of
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41 species such as Akkermansia muciniphila and Escherichia coli.
42 •
N
Likewise, bacterial functionality changes after metformin, increasing different
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43 short-chain fatty acids and bile acids.
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44 • Although research on the gut microbiota-gut-brain-metformin axis has reported
45 interesting and consistent associations in some human studies, most studies are
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46 purely associative.
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47 • Understanding the complex metformin-gut-brain axis interactions could allow a
48 personalized medicine, targeting the beneficial actions of metformin as well as

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49 diminish its adverse effects.

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1 Metformin, cognitive function, and changes in the gut microbiome

5 Marisel Rosell-Díaz1,2,3 , José Manuel Fernández-Real1,2,3,4*

12 Keywords: metformin, type 2 diabetes, gut microbiota, cognition.

15 * José-Manuel Fernández-Real, M.D., PhD. e-mail: jmfreal@idibgi.org. ORCID ID: 0000-0002-7442-9323

18 CIBERobn Fisiopatología de la Obesidad y Nutrición, Girona, Spain.

19 Department of Medical Sciences, School of Medicine, University of Girona, Girona, Spain.

29 No conflicts of interest, financial or otherwise, are declared by the authors.

18 changes in taxonomy, function, and circulating metabolomics), the changes in cognitive

function and their possible bidirectional implications.

14 Microbiome-gut-brain axis is a bidirectional communication system. This complex system

18 microbiota on the development of the gut-brain axis is crucial at birth. A microbiome

22 by future generations(6). Therefore, phenotypes in response to a dysbiosis of the gut microbiota

25 like behavioral changes in the offspring (7).

14 microbiota is involved in the development and progression of various psychiatric and

15 neurological conditions, such as depression, autism, stroke, Parkinson's disease, and

16 Alzheimer's disease (10).

23 monophosphate-activated protein kinase (AMPK). Once activated, AMPK activates the

8 concentrations, which appears to be another pathway by which it impacts glucose homeostasis

14 L. plantarum, L. paracasei, L. delbrueckii, and L. brevis) increased plasma GLP-1

15 concentrations, leading to a reduction in brain amyloid load and resulting in modulation of

17 Comparatively to T2D patients on a low-fat diet, T2D patients on a low-carbohydrate almond-

22 and characterized in humans as in animal models.

22 induced by amyloid-beta in hNSCs through downregulation of caspase 3/9 activities and

23 cytosolic cytochrome c. Furthermore, metformin co-treatment was critical in restoring the

24 fragmented mitochondria in amyloid-beta-affected hNSCs, approaching them to normal

25 morphology(33). Besides, metformin has been described to alleviate Alzheimer's disease-

24 estimate for cognition.

5 2.1 Positive effects of metformin on cognition

17 accelerated specifically in executive function (p=0,006). Hence, an 81% reduction in incident

19 0,85; p = 0,030) (42).

2 causes of dementia (HR=0.91, 95% CI 0.88-0.95) (43).

15 <75 years old patients (HR=0,89, 95% CI 0.79–0.99, p<0,033) (27).

22 reduction in the incidence of neurodegenerative disease (dementia, Parkinson’s disease,

4 mechanisms of metformin on Alzheimer's disease. Mitochondrial complex 1 demonstrated a

5 significant impact on Alzheimer's disease (OR=0.88, p=4.73×10−4 ). A lower risk of

14 CI 0.01-0.12, p=0.0202) and delayed memory (β=0.089, 95% CI 0.032-0.146, p=0.0024)

15 performance across time (48).

19 0,38, 95 % CI -0,02-0,79; p=0,115), executive functions (d = 0.51, 95% CI −0.06-1.08; p =

4 A 16-week placebo-controlled crossover study was conducted on 20 non-diabetic subjects with

17 baseline Disease Assessment Scale-cognitive subscale, the metformin group showed a

20 diseases, as well as improved cognition, primarily in subjects with T2D.

22 2.2 No effects or negative effects of metformin on cognition

5 cognitive impairment (subhazard ratio= 2.75; 95% CI 1.64-4.63, p<0.001) (54).

18 were significant results.

21 had only 200 metformin-treated participants. Additionally, metformin-treated subjects had

2 parameter to monitor in patients with T2D treated with metformin.

15 effects (61–63). Metformin reaches 30-300-fold higher concentrations in jejunal tissue

16 compared to plasma concentrations (64–66). Caenorhabditis elegans engages in a beneficial

21 the gut microbiota and dietary intake.

23 3.1 Changes induced by metformin at the level of Bacterial Taxonomy.

12 increased abundance of bacteria belonging to the Enterobacteriaceae family (75–77),

18 decrease in the abundance of genera such as Romboutsia (75,81), Clostridium (20,78,81,82),

23 could provide a competitive advantage to Escherichia spp. This enrichment of Escherichia

8 Escherichia/Shigella was positively correlated with the levels of these proinflammatory

12 have beneficial effects. A detailed mechanistic investigation suggests agmatine as an E. coli-

T2D(73). However, it is not possible to determine if the rise in agmatine in metformin-treated

23 observed with metformin use.

5 human health are uncertain (20).

21 may have a detrimental impact on neurogenesis(97,98). Overproduction of IL-6 impairs

23 prefrontal cortex (99,100).

16 characterized species, including Roseburia intestinalis, all of which produce SCFA.

20 have been seen.

2 approach is more accurate in functional characterization of the microbial community, according