Effects of Non-insulin Anti-hyperglycemic Agents on Gut Microbiota: A Systematic Review on Human and Animal Studies

Background: As growing evidence links gut microbiota with the therapeutic efficacy and side effects of anti-hyperglycemic drugs, this article aims to provide a systematic review of the reciprocal interactions between anti-hyperglycemic drugs and gut microbiota taxa, which underlie the effect of the gut microbiome on diabetic control via bug-host interactions. Method: We followed the PRISMA requirements to perform a systematic review on human vs. animal gut microbiota data in PubMed, SCOPUS, and EMBASE databases, and used Cochrane, ROBIN-I, and SYRCLE tools to assess potential bias risks. The outcomes of assessment were trends on gut microbiota taxa, diversity, and associations with metabolic control (e.g., glucose, lipid) following anti-hyperglycemic treatment. Results: Of 2,804 citations, 64 studies (17/humans; 47/mice) were included. In human studies, seven were randomized trials using metformin or acarbose in obese, pre-diabetes, and type 2 diabetes (T2D) patients. Treatment of pre-diabetes and newly diagnosed T2D patients with metformin or acarbose was associated with decreases in genus of Bacteroides, accompanied by increases in both Bifidobacterium and Lactobacillus. Additionally, T2D patients receiving metformin showed increases in various taxa of the order Enterobacteriales and the species Akkermansia muciniphila. Of seven studies with significant differences in beta-diversity, the incremental specific taxa were associated with the improvement of glucose and lipid profiles. In mice, the effects of metformin on A. muciniphila were similar, but an inverse association with Bacteroides was reported. Animal studies on other anti-hyperglycemic drugs, however, showed substantial variations in results. Conclusions: The changes in specific taxa and β-diversity of gut microbiota were associated with metformin and acarbose in humans while pertinent information for other anti-hyperglycemic drugs could only be obtained in rodent studies. Further human studies on anti-hyperglycemic drugs other than metformin and acarbose are needed to explore gut microbiota's role in their therapeutic efficacies and side effects.


INTRODUCTION
Gut microbiota plays a pivotal role in the pathogenesis of diabetes as significant alterations were found in the gut microbiome composition in type 2 diabetes (T2D) patients relative to healthy individuals (1). A metagenome-wide association study reported a moderate degree of dysbiosis associated with depletion in butyrate-producing bacteria, accompanied by increases in opportunistic pathogens among diabetic patients (2). These changes were echoed by a recent systematic review, which shows an inverse association of T2D with the genera Bifidobacterium, Akkermansia and butyrate-producing bacteria (e.g., Roseburia, Faecalibacterium), in conjunction with a positive association with Ruminococcus, Fusobacterium, and Blautia (1).
From a clinical perspective, these findings provide a rationale for targeting gut microbiota imbalance as a potential strategy for T2D treatment by restoring a healthy gut microbiome, including fecal microbiota transplant and probiotic supplements (3,4). However, the efficiency and effectiveness of these treatments remain uncertain due to concerns over the invasive nature of fecal microbiota transplant and the dosage, species, and duration required for an effective probiotic treatment. Emerging evidence indicates that the therapeutic efficacy of antihyperglycemic drugs might, in part, be attributable to their ability to modulate the compositions of gut microbiota (1,3,(5)(6)(7)(8)(9). This compositional change might lead to enrichments in bacterial species exhibiting beneficial effects to intestinal health via the production of health-promoting metabolites, such as short-chain fatty acids (SCFAs) and bile acids (8). Nevertheless, certain anti-hyperglycemic drugs were reported to cause increases in the abundance of Escherichia and Candidatus Arthromitus, which contribute to gastrointestinal side effects and weight gain, respectively (9)(10)(11).
Among various anti-hyperglycemic drugs in clinical use, metformin, acarbose, sitagliptin, and vildagliptin (5,6) have been investigated for their reciprocal interplay with gut microbiota by assessing their effects on human and animal gut microbiota, and vice versa (1,8,11). From a translational perspective, animal models might help to explore the causality of complex host-microbiota interactions and possible mechanisms of action in a controlled experimental setting. However, it should be noted that differences in dietary habits, host metabolism, inflammatory states, and body anatomy contribute to great variations in gut microbiota compositions between humans and animals, and subsequently, the respective drug effects in disease control (12). A meta-analysis of published 16S rDNA sequencing data from mouse and human fecal microbiota showed that there were significant increases in Lactobacillus and Turicibacter genera in mouse gut microbiota while the genera of Streptococcus, Ruminococcus, Lachnospira, Faecalibacterium, Dialister, and Oscillospira were elevated in human gut microbiota (12). Moreover, age, mouse strains/populations, microbiota pools in laboratories, and other practical factors might have varied to a great extent among different studies (12). Previous reviews have suggested the effects of anti-hyperglycemic drugs on gut microbiota (8,11,13), however, the differences in results between human and animal studies have not been differentiated.
Reciprocal interplays between individual anti-hyperglycemic drugs and gut microbiota remain unexplored with respect to the contribution of specific bacterial taxa to drug's therapeutic efficacy in disease control (i.e., the clinical question). Thus, we conducted this systematic review aiming to shed light on the associations among anti-hyperglycemic agents, changes in specific taxonomic groups of gut microbiota, and host glucose control or metabolic profiles mainly in humans, as compared to those reported in animal studies.
after receiving anti-hyperglycemic drugs; (ii) gut microbiota data were analyzed from feces or colonic content specimens; (iii) must be written in English or Chinese. Studies were excluded if they did not provide data of individual bacterial taxa or were only available as conference abstracts or proceedings.

Selection of Studies
Initially, the abstracts and titles of potential articles were screened, followed by the evaluation of the full-text articles for eligibility. Two authors were responsible for screening and evaluating these papers independently. Disagreements were resolved by consensus between these two authors and, if necessary, discussed by additional two authors.

Data Extraction
A standardized form in a Microsoft Excel file (e.g., characteristics of studies, participants, treatments and comparisons, methods to analyze the microbiome, and measures of outcomes) was used for data extraction. Data were extracted by one author and reviewed by a second one. All disagreements were resolved by consensus and a third or fourth author when necessary.

Quality Assessment
We used the Cochrane risk-of-bias tool to assess the risk of bias in selected randomized trials (16). For quasi-experimental and observational studies, we used the Risk of Bias in Nonrandomized studies-of Interventions (ROBIN-I) to assess the risk of bias (17). Further, the SYRCLE's risk of bias tool for animal studies (18) was used to assess the risk of bias. The risks of bias data were extracted by four different authors and all disagreements were resolved by consensus made by the remaining authors.

Outcomes of Assessment
Other than describing the characteristics of the evaluated human or animal studies, the primary outcome was the difference in relative abundance or change patterns of individual intestinal bacterial taxa, categorized based on six common taxonomic categories [Phylum (P)c, Class (C), Order (O), Family (F), Genus (G), Species (S)], in associations with the use of antihyperglycemic agents, among those available human or animal studies, respectively. Secondary outcomes were differences in microbial diversity, changes in intestinal or serum levels of SCFAs and/or bile acids in human or animal hosts, respectively, after taking/using individual drugs, associations between specific taxa and host metabolic parameters, e.g., glucose, body weight, and lipid profile.

Data Synthesis
We classified the primary and secondary outcomes into the following categories: significant increase, significant decrease, and no significant difference between comparison groups. Changes of each taxon were synthesized from at least 2 studies for human or animal studies, respectively. Specifically, the effects of different anti-hyperglycemic drugs on specific taxon among the evaluated human or animal studies were compared. Further, the corresponding effects of each individual drug on specific taxon were compared to explore its consistency, in terms of having the same trend of alteration on the specific taxon caused by the same specific anti-hyperglycemic drug, or not. These findings also were categorized by the target research populations (e.g., obese, prediabetic, newly T2D, prevalent T2D), individual treated drugs or different animal models (mice or rat models with various diets or genetic knockout). For gut microbial diversity, each study might use one or more measures to assess α-(richness and evenness) or β-diversity. We considered α-diversity as "Increase" if at least one measure showed an increase and no measure showed a decrease; "Decrease" if at least one measure showed a decrease and no measure showed an increase; "No difference" if all measures showed no difference. β-diversity was assessed as "Difference" if at least one measure showed a difference; "No difference" if all measures showed no difference. In terms of associations between specific taxa and host metabolic parameters, we collected data from specific taxa that increased or decreased significantly in participants receiving non-insulin anti-hyperglycemic drugs. Only data with statistical significance were extracted for analyses.

RESULTS
The following presented results were mainly focused on human studies, which are compared to those of animal studies.

The Risk of Bias
Among human studies, three randomized studies were at the high risk of bias in performance, detection, and attrition, while four studies were unclear risks in most domains (Supplementary Figure 1).
Among quasi-experimental studies, three studies were at low risk of bias in all domains, whereas two studies were at serious risk in confounding, selection of participants, and classification of interventions (Supplementary Figure 2). All 5 cross-sectional studies were at serious risk in several domains, e.g., confounding, selection of participants, classification of intervention (Supplementary Figure 3). Almost all mouse studies were unclear risk across domains, even if some were at low risk of bias in selective outcome reporting (Supplementary Figure 4).

Bacterial Taxa
Importantly, the synthesized results from animal studies reported all common six taxonomic categories (P, C, O, F, G, S) of bacterial taxa but there were only three common taxonomic categories (F, G, S) of gut microbiota taxa were reported in those from human studies based on the available data (Tables 4-7). Glipizide and  liraglutide were assessed in a single human study. No differences were found in patients treated with glipizide (32), while Wang et al. (34) found the association between liraglutide treatment and the increased abundance of genus Akkermansia in T2D patients. The assessments of the effects of metformin and acarbose on the human gut microbiota composition represented the foci of 14 studies (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)34) and three studies (7,32,33), respectively ( Tables 4, 5).
Of the phylum Bacteroidetes, the genus Bacteroides decreased in two studies treated with metformin among newly diagnosed T2D patients (20,24), and in two studies treated with acarbose among pre-diabetic and newly diagnosed T2D patients (7,32). Additionally, one study treated with metformin (24) and one study treated with acarbose (32) in newly diagnosed T2D patients reported similar results of decreases in seven species (e.g., Bacteroides dorei, Bacteroides finegoldii). For the phylum Firmicutes, the genus Lactobacillus increased in two studies in pre-diabetic (7) and newly diagnosed T2D patients (32) receiving acarbose, and the species L. gasseri increased in one study treated with metformin (21), and with acarbose (32), respectively, among newly diagnosed T2D patients. Meanwhile, the genus Clostridium decreased in one study among healthy participants receiving metformin (22) which was also reported in newly diagnosed T2D patients treated with acarbose (32) ( Table 4). Two species, i.e., C. bartlettii and C. botulinum, consistently decreased among T2D patients receiving metformin in two separate studies (21,31). Three out of four studies showed a decrease in the genus Intestinibacter (21, 22, 30) among healthy participants and T2D patients treated with metformin ( Table 5). With respect to the phylum Actinobacteria, the genus Bifidobacterium with the species B. adolescentis increased in one study with metformin (21) and another study with acarbose (32) among newly diagnosed T2D patients ( Table 4), and B. longum consistently increased among T2D patients treated with acarbose in two studies (32, 33) ( Table 5).
Concerning other phyla, two studies evaluated the genus Fusobacterium (phylum Fusobacteria) (20,27) and the species Akkermansia muciniphila (phylum Verrucomicrobia) (21,29) among T2D patients treated with metformin. Both showed increases in the abundance of these two taxa. In the phylum Proteobacteria, conflicting results were reported in two studies with respect to the family Enterobacteriaceae in healthy participants vs. T2D patients treated with metformin (25,27). Overall, metformin might increase different taxa from the family Enterobacteriaceae and other families in the order of Enterobacteriales. Of six studies that evaluated the genus Escherichia (19-22, 25, 30) and four studies evaluating the genus Shigella (19,20,22,25), the treatment of healthy participants, obese individuals, and T2D patients with metformin all led to increased abundance of these two genera. Another two studies on metformin in newly diagnosed and prevalent T2D patients (21,31) reported consistently increases in eight species of the order Enterobacteriales, including Citrobacter koseri, Escherichia coli, Klebsiella pneumonia (family Enterobacteriaceae), Erwinia amylovora (family Erviniaceae), Pectobacterium wasabiae, and Dickeya dadantii (family Pectobacteriaceae) ( Table 5).
The effects of 13 anti-hyperglycemic drugs on the compositions of gut microbiota were conducted in different rodent models. The results of pioglitazone was inconclusive (78), while the other 12 drugs were presented in Tables 6, 7. The Firmicutes/Bacteroidetes ratios were decreased in two studies treated with metformin in high fat diet (HDF)-fed mice (37,48).     This decrease was also noted in another two studies, in which liraglutide was given to in rat models induced by diets and gene knockout (66,69).
Responses of the phylum Bacteroidetes to different antihyperglycemic agents were investigated in 15 rodent studies. No difference was noted in studies treated with acarbose (5,58). Five out of the seven studies on the genus Bacteroides after using metformin (37,42,48,54,56) revealed increased abundance of this genus in mice and rats.
With respect to other phyla, there was a trend of decrease in the phylum Proteobacteria in mice treated with metformin and liraglutide, while Verrucomicrobia and Tenericutes increased after treated with metformin and liraglutide, respectively. However, results for the phyla Actinobacteria, Cyanobacteria, Elusimicrobia, and Fusobacteria were conflicting. The genus Akkermanisa (phylum Verrucomicrobia) increased in eight studies treated with metformin using dietary or genetic models (35,37,38,41,44,48,54,56). Three of the four studies with metformin (41,53,55) reported an increase in the species A. muciniphila, and another two studies reported a similar increase in this species after treating with liraglutide (65,68).

Short-Chain Fatty Acids (SCFAs)
In human studies, changes in the levels of three main SCFAs (acetate, propionate, and butyrate) in feces and sera were reported in three studies treated with metformin (19, 21, 23) (Supplementary Table 3). Wu et al. (21) found that the levels of fecal butyrate and propionate increased in T2D male patients. However, no difference in fecal levels of these two SCFAs was noted among obese women in Ejtahed's study (19). In contrast, fecal acetate levels decreased in obese women (19) did not change among T2D patients in Wu's study (21). Huang et al. (23) reported that the serum levels of all three SCFAs remained unchanged after treating with metformin in T2D patients. In mouse studies, fecal levels of SCFAs after metformin interventions were assessed in db/db mice (35) and OLETF rats (50) (Supplementary Table 3). It was found that levels of acetate and butyrate increased, but propionate levels remained unchanged. The effects of acarbose on these SCFAs levels were also assessed in dietary models (59,61,62). These studies showed consistent results of increased levels of butyrate in feces and ceca, but the levels of acetate and propionate varied in a dietdependent manner.

Bile Acids
In human studies, three clinical trials treated with metformin (21,24,26) and one randomized trial with acarbose and glipizide (32) were carried out in T2D patients to assess the respective effects on the fecal and serum levels of bile acids (Supplementary Table 4). Regarding metformin, one study (24) showed increases in the fecal level of conjugated secondary bile acids, while no difference was reported in the other two studies (21,26). Two of these three studies (21,24)

reported increases in the blood level of
Frontiers in Endocrinology | www.frontiersin.org  secondary bile acids, while the other one (26) revealed an inverse trend. Concerning changes in total and primary bile acids, their levels in feces were unchanged among these three trials, but results in blood levels were conflicting (21,26). The random trial assessing the effects of acarbose and glipizide on bile acid levels in newly diagnosed T2D patients (32) showed that acarbose might increase the plasma and fecal levels of primary bile acids, accompanied by decreases in secondary bile acids. In contrast, no significant changes in bile acid levels were found in patients treated with glipizide.
As for rodent studies, one study in rats (51) revealed that the fecal level of total bile acids increased while the levels in liver tissues were decreased after metformin intervention. One study in mice (63) found that voglibose treatment was associated with increases in serum levels of primary bile acids, accompanied by decreases in serum levels of secondary bile acids (Supplementary Table 4).

Associations With Host Metabolic Parameters
Among pre-diabetic and T2D patients treated with metformin (20,21) or acarbose (7,32,33), alterations in certain specific taxa in human gut microbiota were associated with improvement in HbA1C and fasting blood glucose values, body weights, and lipid profiles ( Table 10). For instance, increments in the genera Escherichia, Shigella, Subdoligranulum, and Dialister, and the species Bifidobacterium adolescentis, Bifidobacterium longum, and Lactobacillus gasseri were inversely associated with HbA1C after treating with metformin or acarbose (7,20,21,32). In addition, there were inverse associations between increases in the genus Blautia and fasting blood glucose after treating with metformin (20).
Mouse studies treated with metformin (44,50,55,57), liraglutide (65,66,70), and saxagliptin (70) also explored the relationship between changes in the compositions of gut microbiota and improvement in various metabolic parameters ( Table 11). It was found that some related specific taxa after treating with metformin in mice (i.e., Bacteroides spp., Blautia) were different from that in humans.

DISCUSSION
Our study provides a comprehensive review to report human and animal data separately about reciprocal interactions between anti-hyperglycemic drugs and specific taxonomic groups of gut microbiota. While other reviews suggest the effects of antihyperglycemic drugs on gut microbiota without discerning findings from either human or animal studies (8,11,13), this systematic review attempts to fill the gap of these reviews to try to explore the associations among anti-hyperglycemic agents, specific taxonomic patterns of gut microbiota, and glucose control or metabolic profiles mainly in humans, as compared to those reported in mouse studies. Further, the fact that threequarters of included studies were published in and after 2017 implies a growing interest in this clinical question for an up-todate systematic review.
Of the 17 human studies selected, the majority of these studies focus on either newly diagnosed or prevalent T2D patients, and directed toward investigating the interplay of metformin, and to a lesser extent, acarbose, with gut microbiota. Our results suggest that these two drugs mediate their glucose-lowering effect, in part, by stimulating beneficial gut bacteria that could produce metabolites to promote intestinal homeostasis (3,9). We rationalize that alterations in gut microbiota compositions might also underlie the gastrointestinal side effects known to metformin, i.e., diarrhea and fecal incontinence (10,25,80). In contrast, results from other anti-hyperglycemic drugs analyzed in this study showed inconsistency with respect to their effects on the compositions of gut microbiota, which might be attributable to small numbers of studies and, equally important, differences in animal models and experimental conditions used among these studies.
Further, treatment durations of anti-hyperglycemic drugs in available studies, regardless of human or animal, varied to a great extent (i.e., few days to few months). Thus, the reported drug effects on the gut microbiota structure were diverse. Indeed, the anti-hyperglycemic drugs, i.e., metformin, could affect the intestinal bacterial compositions after 1 or several days of treatment (24,25,40,43), or after prolonged periods of treatments (21). For instance, Wu et al. found that gut microbiota compositions after a 2-and 4-month treatment of metformin in newly T2D patients were not identical (21). In contrast, Wang et al. did not find significant changes in the gut microbiota compositions among T2D patients after different periods of metformin or liraglutide treatment, given their baseline gut microbiota compositions were unknown (34). Thus, there are no consistent findings on gut microbiota after various treatment durations of anti-hyperglycemic drugs, and further studies are warranted to explore the treatment duration of anti-hyperglycemics required for emergence of beneficial gut bacteria. Evidence indicates that the use of metformin or acarbose in T2D patients was associated with increases in the abundance of beneficial bacteria, including the genera Bifidobacterium (phylum Actinobacteria) and Lactobacillus (phylum Firmicutes), and the species A. muciniphila (phylum Verrucomicrobia). The increase in the genus Bifidobacterium was positively associated with diabetes control, which is consistent with that reported in the review by Gurung et al. (1). In addition, two included studies showed an increase in two specific species of the genus Bifidobacterium (B. adolescentis and B. longum), which was inversely associated with HbA1C levels or body weights, and positively associated with HDL cholesterol levels among newly diagnosed T2D patients (21,32).
A number of human studies have reported positive associations between the abundance of the genus Lactobacillus (phylum Firmicutes) and improved T2D control (81,82). For example, T2D patients treated with acarbose showed increased L. gasseri levels, accompanied by lower HbA1C and body weights (32). In addition, as several species in the genus Lactobacillus have been used as probiotics, administration of these Lactobacillus strains showed beneficial effects on glycemic control and lipid profiles in T2D patients (4). Moreover, almost all animal studies that tested the efficacy of several species from the genus as probiotics for T2D reported improvements of glucose parameters (1).
A previous report found decreased abundance in the mucindegrading bacterium A. municiphila in patients with metabolic disorders, including obesity, impaired glucose tolerance, and diabetes, which were associated with insulin resistance, dyslipidemia, and overweight (83). Two other studies showed increased amounts of A. municiphila in newly diagnosed and prevalent T2D patients treated with metformin, which, however, did not provide pertinent information on metabolic parameters. The potential role of this mucin-degrading bacterium in ameliorating metabolic disorders was further confirmed by a series of animal experiments. For example, mice treated with metformin and liraglutide showed increased levels of A. municiphila in association with improved control of blood glucose and body weight (55,65). More importantly, HFD-fed mice treated with A. municiphila exhibited similar improvements in glucose tolerance and goblet cell production and inflammatory regulations as compared to the metformin treatment group (56).
The effect of metformin and acarbose on the abundance of different species of the genus Bacteroides (phylum Bacteroidetes) is interesting. The genus Bacteroides seem to play a beneficial role in glucose metabolism where B. intestinalis and B. vulgatus were decreased in T2D patients, and B. stercoris was enriched in patients with diabetes remission (1). The same phenomenon was also noted in experimental animals (1). However, decreased abundance of some Bacteroides species, including B. pleibeius, B. dorei, B. vulgatus, after using acarbose in newly diagnosed T2D patients was reported to be positively associated with body weight in one study (32). As for rodent studies, colonization of B. fragilis was associated with more severe glucose intolerance in HFD-fed mice (24). A recent study, which compared fecal microbiota compositions between T2D patients and non-diabetic individuals, showed that Bacteroides was an independent risk factor of the disease by diminishing insulin sensitivity (84).
The effects of metformin on A. muciniphila were similar in both human and rodent studies. However, there was an inverse association of Bacteroides and metformin use in human and mouse studies in this review. Alterations of many other taxa in humans treated with metformin or acarbose were not the same as in mouse studies, and vice versa. The diverse dietary habits, metabolism or inflammatory statuses of host, body sizes and organs in these human and mouse studies might contribute to inconsistent findings of gut microbiota compositions (12,85). Although the gut microbiota of human and mice are dominated by two major phyla, i.e., Bacteroidetes and Firmicutes, approximately 85% of the representative gut microbiota sequences in mice were not found in humans (86). Further, the genera Escherichia and Shigella, belonging to the order of Enterobacterales in phylum Proteobacteria, were found to increase consistently after metformin treatment in T2D patients. Certain bacteria belonging to the phylum Proteobacteria, including the order Enterobacteriales, was found to be overly present in patients with metabolic disorders and T2D, and were positively related to intestinal permeability and endotoxemia in the pathophysiology of these metabolic diseases (87,88). Enrichment in the order Enterobacterales, especially Escherichia coli, was demonstrated to play an important role in gut inflammation in patients with inflammatory bowel disease and also in various mouse model of colitis (88). Elbere et al. (25) observed an association between the severity of gastrointestinal side effects and increased abundance of the genera Escherichia and Shigella. Thus, the enrichment of the order Enterobacteriales might contribute to gastrointestinal side effects of metformin.
In this review, the results of β-diversity indicate significant changes in gut microbiome structure related to metformin or acarbose treatment. The findings on α-diversity among those treated with metformin were inconsistent while the richness and evenness were decreased after treating with acarbose. For healthy human subjects, the reference microbiome list and abundance profile showed various ratios of Bacteroidetes and Firmicutes, as well as the other phyla, e.g., Acinobacteria, Proteobacteria (89). This might reflect sufficient α-and β-diversities in healthy individuals due to significant regional heterogeneity at the species level and consistency at the higher taxonomic level (89). With regard to T2D patients, the associations between the disease and the diversities of microbiota were inconclusive (1). In the other words, there is no consensus or simple way to make a conclusion on the relationship between diversity and gut microbiota compositions among T2D patients treated with metformin or acarbose.
While metformin and acarbose have been shown to stimulate the growth of SCFA-producing bacteria, e.g., Lactobacillus and Bifidobacterium, information on the effects of these drugs on the fecal levels of various SCFAs in humans is lacking in the literature. Thus, this review entailed data from mouse studies, which are more informative. These mouse studies showed an increase trend in fecal and cecal levels of acetate, propionate, and butyrate in response to treatments of metformin, acarbose, voglibose, dapagliflozin or canagliflozin, of which the impacts on other physiological functions, other than that in gastrointestinal track, warrant further evaluations (90). With respect to bile acids, information on the effects of anti-hyperglycemic agents on their levels is limited and often conflicting in human vs. rodent studies, which merits further investigations to understand the role of other confounding factors, such as diets, antibiotic therapy, and disease states (91).
Again, the most critical limitation is lack of consistency among human and rodent studies. In humans, differences in the health status of participants, disease type or staging, ethnicity, drug dosage, and duration of treatment might directly impact gut microbiota compositions. Furthermore, it becomes difficult to come to a conclusion due to the small number of participants as well as differences in study design in each study. The risk of bias of studies needs to be taken into considerations, in which overall bias of randomized trials was high risk and unclear risk, and two out of five quasi-experimental studies and all of observational studies were at serious risk. Also, there was a high degree of heterogeneity in rodent studies due to differences in species used and environmental factors, as alluded above. Differences in microbiota analysis methods could also be a cause of deviation.
Another major limitation is lack of human studies on antihyperglycemic drugs beyond metformin and acarbose. Thus, no conclusion could be reached regarding the associations between human gut microbiota and these drugs. Because results from rodent models might not always be translatable to humans, conclusions should be made with cautions.
Although findings from rodent studies included in this review suggest potential positive effects of other antihyperglycemic drugs besides metformin and acarbose on human gut microbiota, additional human studies on these drugs are needed to clarify the role of gut microbiota in their therapeutic efficacies.
In light of the enormous amounts of published data, this systematic review aims to provide readers a comprehensive view of this emerging area by taking an integrated approach through an all-inclusive literature search in conjunction with vigorous data extraction and validation, and assessment of risk bias. Moreover, this systematic review has tried to differentiate various aspects of the anti-hyperglycemic drug-gut microbiomehost axis, thereby filling the gap of merging all available data from human or animal studies relevant to the interdependence between anti-hyperglycemic drugs and the specific taxon of gut microbiota. Nevertheless, more investigations are warranted to support the positive contribution of metformin and acarbose to the health of gut microbiome (e.g., A. muciniphila, Lactobacillus, Bifidobacterium longum). In addition, given the limited information available in the literature, more studies are needed to shed light onto the roles of other anti-hyperglycemic drugs (e.g., miglitol, voglibose) in modulating human host taxa of gut microbiome.

CONCLUSION
This review highlights that changes in specific taxa and βdiversity of gut microbiota were associated with metformin and acarbose in humans while pertinent information for other antihyperglycemic drugs could be only obtained in rodent studies. These results support the possible action mechanisms of these drugs, which may have translational potential to foster new approaches for the treatment of diseases related to gut dysbiosis in the future. Mouse studies on the other anti-hyperglycemics suggested the links between these drugs and gut microbiota were inconclusive. Therefore, additional human studies are needed to explore the role of gut microbiota in their therapeutic efficacies or side effects.

DATA AVAILABILITY STATEMENT
The original data presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.