Circulating microbiome and its clinical implications in diabetes mellitus: Mechanistic insights and therapeutic perspectives

Abstract

The circulating microbiome, comprised of microbial DNA, metabolites, and cell-derived fragments, has emerged as a potential contributor to the pathophysiology of diabetes mellitus. This review summarizes current evidence on how microbial translocation and blood-borne microbial components influence metabolic and immune dysfunction in type 1 and type 2 diabetes. Quantitative studies report elevated circulating lipopolysaccharide levels (often > 2-fold higher in diabetic cohorts), increased trimethylamine-N-oxide, and reduced short-chain fatty acids, all of which correlate with systemic inflammation, insulin resistance, and glycemic variability. Mechanistic data indicate that microbial products activate Toll-like receptors (TLRs), particularly TLR4 and TLR2, amplifying cytokine release and impairing β-cell function. Distinct microbial DNA signatures identified through metagenomics further link the circulating microbiome to complications, such as nephropathy, cardiovascular dysfunction, and impaired wound healing. Emerging therapeutic approaches, including microbiome-directed diets, prebiotics, probiotics, and strategies that reduce microbial translocation, show promise in modulating these systemic effects. Despite rapid advances, major gaps remain in establishing causality, standardizing detection methods, and defining clinically actionable microbial biomarkers. Addressing these challenges will be essential for translating circulating microbiome research into diagnostic tools and personalized interventions for diabetes management.

Key Words: Diabetes mellitus; Circulating microbiome; Blood microbiome; Microbial components; Toll-like receptors; Trimethylamine N-oxide; Short chain fatty acids

Core Tip: The circulating microbiome, encompassing microbial DNA, products, and microbial components detected in blood, links gut barrier dysfunction to chronic, low-grade inflammation in diabetes. Quantifying blood microbial signatures (16S rDNA/cell free DNA, endotoxin activity, metabolites) may enable early risk stratification for insulin resistance and prediction of micro- and macrovascular complications. Integrating longitudinal cell-free microbiome profiling with glycemic and inflammatory indices could refine diagnosis, personalize therapy, and monitor responses to interventions (dietary fiber, pre/probiotics, postbiotics, barrier-restoring strategies, and microbiome-informed pharmacotherapy, such as metformin). Standardized sampling, contamination control, and causal studies are critical to translate these insights into routine diabetes care.

INTRODUCTION

The human microbiome profoundly influences host immunity, metabolism, and chronic disease. Although most investigations have centered on the gut microbiome, recent evidence demonstrates the presence of microbial DNA and extracellular vesicles in systemic circulation, collectively termed the circulating or blood microbiome, challenging the historic assumption of blood sterility[1]. This emerging concept is particularly relevant to diabetes mellitus, where chronic inflammation, endothelial dysfunction, and impaired mucosal immunity increase the likelihood of microbial translocation into the bloodstream.

Foundational studies have shown that gut barrier dysfunction in early type 2 diabetes facilitates the passage of commensal bacterial products into circulation, promoting systemic inflammation and metabolic deterioration[2]. Metabolic endotoxemia, first described by Cani et al[3], established lipopolysaccharide (LPS) as a driver of insulin resistance. Subsequent work confirmed that diabetic individuals exhibit altered microbial signatures and detectable bacterial DNA in blood[4-7]. More recently, Li et al[7] emphasized the mechanistic relevance of gut-blood microbial interactions in metabolic disease progression.

Modern sequencing studies demonstrate that circulating microbial profiles are not simple reflections of gut dysbiosis. Giacconi et al[8] showed in 2025 that older adults with type 2 diabetes harbor distinct blood-microbiome DNA patterns enriched in Proteobacteria and Actinobacteria, linking these shifts with inflammatory markers[8]. Complementing this, Yuan et al[9] identified unique microbial DNA signatures in children with new-onset type 1 diabetes, suggesting that systemic microbial disturbances may precede long-term metabolic decline[9]. Zhai et al[10] further demonstrated that circulating microbial DNA carries functional metabolic pathways relevant to immune activation and glucose dysregulation.

Beyond microbial DNA, circulating metabolites provide mechanistic support for this disease axis. Trimethylamine-N-oxide (TMAO) has been associated with insulin resistance and cardiometabolic risk in recent meta-analyses[11], while LPS-induced Toll-like receptor (TLR) 4 (TLR4) activation remains a central pathway linking microbial components to impaired insulin signaling[5].

Many recent studies show that gut microbial dysbiosis increases the translocation of microbial products into circulation, shaping the blood or circulating microbiome. Foundational methodological reviews emphasize that most microbial signals detected in blood arise from gut or oral barrier leakage rather than a true endogenous bloodstream microbiome, highlighting the importance of contamination control and low-biomass rigorous workflows[12]. Clinical and metagenomic studies show that blood microbial profiles vary with health status, including family-based and age-dependent shifts dominated by Proteobacteria, where elderly individuals exhibit greater Gammaproteobacteria abundance, consistent with metabolic endotoxemia and inflammaging. Additional sequencing analyses corroborate that unmapped reads from whole-genome sequencing consistently contain bacterial signatures in healthy individuals, reinforcing that microbial fragments circulate even without overt infection[13]. Cardiovascular epidemiology further demonstrates that specific blood bacterial genera (Kocuria, Enhydrobacter, Paracoccus) associate with mortality risk, supporting a mechanistic link between gut barrier dysfunction, microbial translocation, and vascular inflammation[13]. Virome profiling in acute myocardial infarction, many of whom are diabetes patients, reveals distinct viral signatures between patients and controls, yet systemic inflammation, not viral abundance, appears to be the key driver, consistent with a model in which microbial products potentiate acute and chronic inflammatory injury[14]. Finally, an integrative cardiometabolic review supports that gut dysbiosis-driven leakage of endotoxins, microbial metabolites, and bacterial DNA into blood fuels chronic low-grade inflammation central to metabolic, renal, and cardiovascular disease progression[15].

Despite growing interest, significant challenges remain. Blood is a low-biomass environment prone to contamination, requiring strict methodological controls[16]. The origin, viability, and functional relevance of circulating microbial products are still debated, with potential contributions from gut, oral, skin, or respiratory sources. Moreover, heterogeneity in sequencing methods, sample handling, and analytical pipelines limits cross-study comparability. Given these gaps, studying the circulating microbiome, distinct from the gut microbiome, is essential for understanding systemic immune-metabolic interactions in diabetes. This review synthesizes current evidence, highlights mechanistic pathways (LPS, TMAO, TLR signaling), addresses methodological controversies, and outlines future directions to integrate circulating microbiome profiling into precision diabetes care.

THE CIRCULATING MICROBIOME IN DIABETES

Mechanisms of microbial translocation

Bloodstream microbial translocation primarily arises from gut dysbiosis and barrier disruption: The human gastrointestinal tract harbors trillions of microorganisms, collectively termed the gut microbiota, which play critical roles in nutrient metabolism, immune regulation, and maintenance of gut homeostasis[17,18]. A healthy, balanced microbiome is typically dominated by beneficial commensals that promote epithelial health and immune tolerance. However, perturbations in microbial composition and function, referred to as gut dysbiosis, profoundly impacts intestinal barrier function and facilitate microbial or microbial product translocation into systemic circulation, a process implicated in chronic inflammation and diverse systemic diseases[19,20] (Figures 1 and 2).

Figure 1 Factors influencing bacterial translocation.

Figure 2 Factors with regulatory action on the mucosal intestinal barrier through paracellular permeability. Ca²⁺: Calcium ion.

Gut dysbiosis-a shift towards pathobionts: Gut dysbiosis is commonly characterized by reduced microbial diversity, depletion of beneficial commensals such as Faecalibacterium prausnitzii and Bifidobacterium spp., and enrichment of opportunistic organisms including Enterobacteriaceae, Proteobacteria, and Clostridioides difficile[21,22]. Dysbiosis may arise due to antibiotics, Western-type diets, infections, stress, or host genetics[23]. These alterations disrupt host-microbiome symbiosis, leading to metabolic imbalance and immune dysregulation. Importantly, dysbiotic microbiota produce excess microbial toxins such as LPS, hydrogen sulfide, and ethanol, which compromise epithelial integrity[24], while reducing beneficial metabolites such as short-chain fatty acids (SCFAs) like butyrate, which normally support mucosal immunity and epithelial tight junctions[25]. This imbalance creates a permissive environment for barrier damage.

Microbial translocation-from lumen to bloodstream: Under normal physiological conditions, microbial passage into systemic circulation is minimal. However, barrier disruption allows microbial components, including LPS, peptidoglycan, and bacterial DNA, to translocate, fueling systemic inflammation. Once across the epithelium, microbes can reach mesenteric lymph nodes and ultimately the bloodstream, particularly if immune surveillance is impaired[26]. This systemic microbial translocation is increasingly recognized as a key driver of chronic inflammation in metabolic, autoimmune, and infectious diseases[19].

Factors that contribute to increased gut permeability in diabetes patients: Increased gut permeability, commonly referred to as “leaky gut”, is a pathological condition characterized by the disruption of tight junctions between intestinal epithelial cells, allowing translocation of microbial products such as LPS into systemic circulation. This phenomenon plays a crucial role in metabolic endotoxemia and systemic inflammation, both of which are implicated in diabetes mellitus pathogenesis and progression. Several common diabetes-associated factors, including hyperglycemia, a pro-inflammatory diet, and the use of non-steroidal anti-inflammatory drugs (NSAIDs), contribute to this altered gut barrier integrity. Hyperglycemia, a defining feature of diabetes, can directly impair intestinal barrier function. Chronic high glucose levels lead to oxidative stress and inflammatory signaling in the intestinal epithelium, disrupting tight junction proteins such as zonula occludens-1, occludin, and claudins. These molecular disruptions weaken the gut epithelial barrier, increasing paracellular permeability.

In vitro studies have demonstrated that elevated glucose concentrations reduce tight junction integrity in intestinal epithelial cell lines (e.g., Caco-2 cells), associated with increased production of reactive oxygen species (ROS) and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6[27,28]. In vivo, diabetic animal models exhibit higher intestinal permeability and endotoxemia, which correlate with systemic inflammation and insulin resistance[3]. This increased LPS translocation due to hyperglycemia-induced gut barrier dysfunction activates TLR4 pathways, aggravating inflammation in diabetes patients. Diets rich in saturated fats, refined sugars, and low in fiber, hallmarks of a Western or pro-inflammatory diet, significantly contribute to increased gut permeability. Such diets promote dysbiosis, characterized by reduced microbial diversity and an imbalance in commensal and pathogenic bacteria. Dysbiosis leads to the overproduction of microbial metabolites and endotoxins that damage the epithelial lining.

Moreover, saturated fats can directly increase intestinal permeability by modulating tight junction protein expression and promoting inflammation via activation of TLRs. In diabetic individuals, this results in a vicious cycle where dietary-induced inflammation further exacerbates metabolic dysfunction. Conversely, diets rich in polyphenols, omega-3 fatty acids, and dietary fiber promote the growth of beneficial bacteria like Akkermansia muciniphila and Faecalibacterium prausnitzii, which help maintain tight junction integrity and mucosal barrier function[29]. The production of SCFAs like butyrate from fiber fermentation is especially critical in preserving gut barrier function and modulating inflammation. NSAIDs are commonly used in diabetes patients to manage pain and inflammation, particularly those with comorbid conditions like neuropathy or arthritis. However, chronic use of NSAIDs is a well-established contributor to increased intestinal permeability. NSAIDs disrupt mitochondrial function and inhibit prostaglandin synthesis in intestinal epithelial cells, leading to enterocyte apoptosis and decreased mucus production. This creates breaches in the epithelial barrier, allowing luminal antigens and bacteria to penetrate the mucosa. NSAIDs like ibuprofen and diclofenac have been shown to reduce the tight junction protein expression and increase intestinal permeability in both animal models and human studies[30]. In diabetic patients, who are already predisposed to gut barrier dysfunction, NSAID use may amplify intestinal inflammation and systemic immune activation, thereby worsening metabolic control.

Translocated microbial components interact with TLRs on immune cells: The human immune system constantly surveys for microbial threats using pattern recognition receptors (PRRs), which detect pathogen-associated molecular patterns (PAMPs) and are conserved structures found in bacteria, viruses, fungi, and other microbes. Among PRRs, TLRs represent a highly conserved family of receptors predominantly expressed on innate immune cells such as macrophages, dendritic cells, and monocytes. TLRs play a central role in initiating immune responses and orchestrating inflammatory signaling cascades. Under physiological conditions, microbial components such as LPS, flagellin, and unmethylated microbial DNA are confined to mucosal surfaces. However, during mucosal barrier dysfunction or disease states like gut dysbiosis, infections, or tissue injury, these microbial products translocate into the bloodstream and gain access to systemic immune cells[3,31].

LPS and TLR4 activation: LPS is a component of the outer membrane of gram-negative bacteria and is a potent immune activator. Once in systemic circulation, LPS forms a complex with LPS-binding protein (LBP) and cluster of differentiation (CD) 14, which facilitates its presentation to TLR4 and the co-receptor myeloid differentiation factor 2 (MD2) on immune cells. Upon recognition, TLR4 initiates a dual signaling cascade via both myeloid differentiation primary response protein 88 (MyD88)-dependent and Toll/IL-1 receptor domain-containing adaptor-dependent TLR signaling-dependent pathways. These pathways lead to activation of transcription factors nuclear factor kappa-B (NF-κB) and interferon regulatory factor 3, promoting the production of inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and type I interferons[32]. Sustained LPS exposure, known as metabolic endotoxemia, is a feature of many chronic inflammatory diseases, including type 2 diabetes, atherosclerosis, and non-alcoholic fatty liver disease (NAFLD)[33,34].

Flagellin and TLR5 signaling: Flagellin, the principal protein of bacterial flagella, is another important PAMP detected by TLR5, primarily located on mucosal surfaces and immune cells. Once flagellin binds to TLR5, MyD88-dependent signaling is initiated, leading to NF-κB activation and cytokine secretion, including IL-8 and IL-6, which contribute to neutrophil recruitment and amplification of inflammatory responses[35]. Flagellin also plays a role in intestinal epithelial barrier dysfunction and mucosal immune activation, and its presence in circulation is associated with inflammatory disorders like inflammatory bowel disease and metabolic syndrome. Chronic TLR5 activation by translocated flagellin may contribute to systemic inflammation, particularly when gut permeability is compromised[36].

Microbial DNA and TLR9 activation: Bacterial and viral DNA, particularly unmethylated CpG motifs, are sensed by TLR9, which is localized to endosomal compartments of plasmacytoid dendritic cells, B cells, and macrophages. Upon endocytosis of DNA-containing particles or apoptotic material harboring microbial DNA, TLR9 is activated and signals through MyD88, inducing type I interferons, pro-inflammatory cytokines, and B cell activation[37]. In conditions of gut barrier dysfunction or bacterial translocation, microbial DNA can enter the bloodstream and activate TLR9, fueling systemic inflammation. TLR9 activation has been implicated in autoimmune diseases, such as systemic lupus erythematosus and atherosclerosis, where inappropriate immune activation leads to chronic inflammation and tissue damage[38,39].

Systemic implications and chronic inflammation: Chronic exposure to microbial components in the bloodstream promotes a state of low-grade systemic inflammation, which is a hallmark of numerous chronic diseases, including diabetes, cardiovascular disease, obesity, and neurodegenerative disorders[40]. Activation of TLRs by microbial ligands does not remain confined to immune cells; endothelial cells, adipocytes, neurons, and hepatocytes also express TLRs and participate in the inflammatory cascade. For instance, TLR4 and TLR2 activation on adipocytes contributes to insulin resistance, while endothelial TLR activation promotes vascular inflammation and atherosclerotic plaque formation[41,42]. Moreover, persistent TLR signaling can induce innate immune memory (trained immunity), resulting in prolonged pro-inflammatory responses to secondary stimuli, even in the absence of ongoing microbial exposure. This amplifies the chronic inflammatory loop and contributes to the progression of inflammatory diseases[43]. A network-pharmacology study used gut microbiota metabolite data to map how microbial metabolites may influence diabetes via IL-17, phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), hypoxia inducible factor-1 and vascular endothelial growth factor (VEGF) signaling, with key host targets including IL6, AKT1, and PPARG[44]. A recent editorial emphasized that circulating microbial products, including cell-free microbial DNA, LPS, extracellular vesicles, metabolites (SCFAs, TMAO, bile acids) likely contribute to immune-metabolic dysregulation in diabetes via translocation from the gut barrier and activation of pattern-recognition receptors [TLRs, nucleotide-binding oligomerization domains (NODs)][45].

Systemic inflammatory responses

Chronic exposure to circulating microbial components results in low-grade systemic inflammation (metaflammation): The human body exists in a state of dynamic equilibrium with the microbial communities that colonize mucosal barriers, especially the gastrointestinal tract. Under healthy conditions, these microbes or their products are restricted to the gut lumen. However, in the context of compromised mucosal integrity, caused by factors like dysbiosis, stress, chronic disease, or poor diet, microbial components such as LPS, flagellin, bacterial DNA, and peptidoglycan may translocate into systemic circulation[46,47]. Chronic exposure to these components triggers the activation of innate immune system PRRs, particularly TLRs and NOD-like receptors (NLRs), resulting in sustained, low-grade systemic inflammation, a phenomenon referred to as metaflammation[48]. The gastrointestinal tract is a major interface between the host and the microbial world. Several metabolic and environmental stressors can lead to increased intestinal permeability, enabling microbial components to enter the circulation[46] (Table 1 and Figure 3).

Figure 3 A schematic representation of the concept of metaflammation.

Table 1 The relationship between key microbial components/metabolites (lipopolysaccharide, short-chain fatty acids, trimethylamine-N-oxide) and their associated clinical outcomes in diabetes[200-202].

No.	Microbial component/metabolite	Source/mechanism	Key biological effects	Clinical outcomes in diabetes	Ref.
1	Lipopolysaccharide	Outer membrane of gram-negative bacteria; translocates into circulation during gut barrier dysfunction	Activates TLR4; drives systemic inflammation, insulin resistance, and β-cell stress	Chronic low-grade inflammation (“metabolic endotoxemia”)	Kim and Sears[200]
	Worsened insulin resistance
	Increased risk of type 2 diabetes
	Endothelial dysfunction and cardiovascular complications
2	Short-chain fatty acids (acetate, propionate, butyrate)	Fermentation of dietary fibre by beneficial gut bacteria	Regulate gut barrier integrity, GLP-1 secretion, energy homeostasis, and immune tolerance	Improved insulin sensitivity	Nogal et al[201]
	Enhanced glycemic control
	Reduced inflammation
	Butyrate deficiency linked to dysbiosis and impaired metabolic regulation
3	TMAO	Produced by gut microbial metabolism of choline/carnitine converted to TMAO in liver	Promotes inflammation, oxidative stress, and vascular dysfunction	Increased risk of type 2 diabetes	Brunt et al[202]
	Higher incidence of atherosclerotic cardiovascular disease
	Elevated diabetic nephropathy progression risk

TMAO: Trimethylamine-N-oxide; TLR: Toll-like receptor; GLP-1: Glucagon-like peptide-1.

Metaflammation and disease progression: Metaflammation has been implicated in the pathophysiology of several chronic diseases. In obesity and type 2 diabetes, LPS binding to TLR4 on adipocytes and macrophages triggers cytokine release, disrupts insulin signaling, and contributes to insulin resistance[41]. Similarly, activation of TLR9 by microbial DNA exacerbates hepatic inflammation in NAFLD[49]. Activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome by microbial-derived peptidoglycans leads to IL-1β secretion, linking innate immunity with adipose tissue dysfunction and metabolic dysregulation[50]. Moreover, microbial PAMP-induced metaflammation has been linked to atherosclerosis, neurodegeneration, and autoimmune conditions. In atherosclerosis, circulating LPS and other bacterial products activate vascular endothelial cells and monocytes, leading to plaque formation and instability[42]. In the central nervous system, chronic peripheral immune activation due to microbial translocation can prime microglia, increasing susceptibility to neuroinflammation and cognitive impairment[51].

Elevated circulating LPS levels correlate with insulin resistance, adipose tissue inflammation, and endothelial dysfunction in diabetic patients: In the setting of metabolic dysfunction, increased gut permeability facilitates LPS translocation into circulation, a phenomenon termed metabolic endotoxemia[3]. Chronic low-grade endotoxemia has been observed in individuals with type 2 diabetes mellitus (T2DM), and accumulating evidence links elevated circulating LPS levels with insulin resistance, adipose tissue inflammation, and endothelial dysfunction, which are hallmarks of the diabetic phenotype.

LPS and metabolic endotoxemia in diabetes: The intestinal microbiota exerts a pivotal influence on host metabolic processes and immune homeostasis. Dysbiosis in T2DM patients is characterized by a shift toward gram-negative bacterial populations, which leads to increased LPS production and accumulation in the gut[4]. Concurrently, hyperglycemia, high-fat diets, and inflammation compromise the intestinal barrier, allowing LPS to enter the bloodstream[52]. Cani et al[3] first demonstrated that mice fed a high-fat diet exhibited a two- to three-fold increase in plasma LPS levels, correlating with the onset of insulin resistance, glucose intolerance, and weight gain. Similar observations have been reported in human studies, where diabetic and obese individuals exhibit significantly higher serum LPS and LBP levels compared to healthy controls[2,53].

LPS-induced insulin resistance: LPS contributes to insulin resistance by activating the TLR4 signaling pathway on insulin-sensitive tissues such as liver, muscle, and adipose tissue. Binding of LPS to TLR4, in association with the co-receptors CD14 and MD2, triggers downstream signaling cascades involving MyD88, NF-κB, and C-Jun N-terminal kinase, ultimately leading to the expression of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β[38]. These cytokines interfere with insulin signaling by promoting serine phosphorylation of insulin receptor substrate-1 (IRS-1), thereby impairing downstream activation of the PI3K-AKT pathway critical for glucose uptake[41]. Experimental models support the direct role of LPS in insulin resistance. Mice injected with low-dose LPS for 4 weeks develop metabolic abnormalities that mimic high-fat diet-induced insulin resistance[3]. In vitro studies using cultured adipocytes and hepatocytes show that LPS treatment reduces insulin-stimulated glucose uptake and glycogen synthesis[54]. These mechanistic insights are consistent with clinical studies, where circulating LPS levels positively correlate with insulin resistance indices, such as homeostatic model assessment of insulin resistance in T2DM patients[55].

Adipose tissue inflammation: Beyond its role in nutrient storage, adipose tissue functions as an active endocrine organ that regulates systemic metabolism. In T2DM, adipose tissue becomes infiltrated by immune cells, primarily macrophages, which shift toward a pro-inflammatory M1 phenotype under the influence of LPS[56]. These macrophages produce large amounts of inflammatory mediators that exacerbate local and systemic insulin resistance. LPS acts as a chemoattractant and immune modulator in adipose tissue. TLR4 is highly expressed in both adipocytes and resident macrophages. Upon LPS stimulation, adipocytes release monocyte chemoattractant protein-1 (MCP-1), which recruits additional monocytes, further amplifying inflammation[57]. Notably, adipocytes also directly respond to LPS by upregulating cytokine production, suggesting they are not passive bystanders but active participants in inflammation[58]. Furthermore, LPS impairs the normal function of adipose tissue by inhibiting adiponectin secretion and promoting lipolysis, resulting in elevated free fatty acids (FFAs) that further disrupt insulin signaling[59]. These changes contribute to adipose tissue dysfunction, systemic insulin resistance, and increased cardiovascular risk in diabetic individuals.

Endothelial dysfunction and LPS: The onset of diabetic vascular complications is strongly driven by endothelial dysfunction as a key pathogenic mechanism. Chronic exposure to LPS impairs endothelial nitric oxide synthase activity, reduces nitric oxide bioavailability, and promotes vascular inflammation and oxidative stress[60]. LPS-mediated TLR4 activation on endothelial cells leads to increased expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), facilitating leukocyte recruitment and promoting atherogenesis[61]. In diabetic patients, elevated LPS levels are associated with impaired flow-mediated dilation, a measure of endothelial function[53]. Moreover, high circulating levels of LPS and sCD14 (a marker of LPS-TLR4 interaction) correlate with increased levels of C-reactive protein (CRP), endothelial microparticles, and vascular stiffness, all of which are indicators of endothelial damage and cardiovascular risk[48]. Mice deficient in TLR4 exhibit protection from LPS-induced vascular inflammation and endothelial impairment, underscoring the role of innate immune signaling in metabolic vascular injury[42].

Upregulated inflammatory cytokines exacerbate metabolic dysregulation: Metabolic dysregulation, particularly in conditions such as obesity, T2DM, and metabolic syndrome, is increasingly recognized as a consequence of chronic low-grade inflammation. Among the most influential mediators of this inflammatory state are the pro-inflammatory cytokines IL-6 and TNF-α. These cytokines are persistently elevated in individuals with metabolic disorders and are key contributors to insulin resistance, lipid abnormalities, adipose tissue dysfunction, and systemic inflammation[62,63]. Rather than being passive byproducts, IL-6 and TNF-α actively drive metabolic disturbances by interfering with insulin signaling and altering energy homeostasis at multiple levels.

TNF-α, a master regulator of inflammatory metabolic disruption: Elevated levels of TNF-α were observed in the adipose tissue of obese rodents, and neutralization of TNF-α led to improved insulin sensitivity[62]. This cytokine is predominantly produced by adipose tissue macrophages (ATMs), and to a lesser extent, by adipocytes, particularly under metabolic stress or nutrient excess[64]. At the cellular level, TNF-α impairs insulin signaling by promoting serine phosphorylation of IRS-1, thereby preventing effective downstream activation of the PI3K-AKT pathway[65]. This disruption inhibits glucose uptake in muscle and adipose tissue and suppresses glycogen synthesis in the liver. Moreover, TNF-α increases the release of FFAs by stimulating lipolysis, which further exacerbates insulin resistance through lipotoxicity and mitochondrial stress[66]. In addition to its metabolic effects, TNF-α induces endothelial dysfunction, promotes oxidative stress, and upregulates adhesion molecules, which contribute to the vascular complications commonly seen in metabolic syndrome[67]. TNF-α also amplifies inflammatory cascades by inducing the expression of other cytokines, such as IL-1β, and chemokines like MCP-1, establishing a positive feedback loop of inflammation.

IL-6, a dual-edged sword in metabolism: IL-6 is another key cytokine that is elevated in obesity and insulin resistance. Unlike TNF-α, IL-6 exhibits both pro- and anti-inflammatory properties, depending on the context and signaling pathway involved[68]. Chronic elevations of IL-6, particularly from non-immune tissues such as visceral adipose tissue, are associated with negative metabolic consequences. In obesity, adipocytes and ATMs release IL-6 into circulation, and its levels strongly correlate with body mass index and waist circumference[69]. IL-6 exerts detrimental effects on insulin sensitivity by activating suppressor of cytokine signaling 3 (SOCS3), which interferes with insulin receptor signaling through inhibition of IRS proteins[70]. SOCS3 expression is upregulated in response to both IL-6 and leptin and is considered a molecular bridge between inflammation and insulin resistance. Additionally, IL-6 has been shown to impair lipid metabolism by decreasing lipoprotein lipase activity in adipose tissue, leading to elevated plasma triglyceride levels[71]. It is important to note, however, that acute elevations of IL-6, such as during exercise, have beneficial effects and may improve insulin sensitivity by promoting adenosine 5’-monophosphate-activated protein kinase (AMPK) activity[72]. Thus, the timing, source, and duration of IL-6 exposure are key in determining its impact on metabolism.

Cytokine crosstalk and immune-metabolic feedback: The metabolic impact of IL-6 and TNF-α is further amplified through their interactions with other immune mediators. For example, both cytokines stimulate CRP production in the liver[73]. TNF-α also induces IL-6 expression, and both acts synergistically to disrupt metabolic homeostasis[74]. In adipose tissue, cytokines modulate immune cell composition, shifting macrophage populations from an anti-inflammatory M2 phenotype to a pro-inflammatory M1 state. This phenotypic switch results in increased TNF-α and IL-6 production, creating a vicious cycle of inflammation and insulin resistance[56]. The gut microbiota is also implicated in this inflammatory network. Increased intestinal permeability leads to microbial translocation and systemic exposure to LPS, which activates TLR4 signaling and further enhances IL-6 and TNF-α production[3]. This mechanism reinforces the idea that chronic immune activation is not isolated to adipose tissue but is systemic and multifactorial.

Effects on insulin resistance and beta-cell function

Circulating microbial metabolites have diverse effects on metabolic pathways: The human gut microbiota metabolizes dietary substrates to produce a range of bioactive compounds, some of which can enter systemic circulation and influence host metabolism. Among these, TMAO and SCFAs have garnered significant attention for their contrasting roles in metabolic health and disease. These microbial metabolites not only reflect dietary patterns and microbiota composition but also modulate host physiology through interactions with metabolic, inflammatory, and cardiovascular pathways (Figure 4).

Figure 4 Schematic representation of the link between diet, gut microbiota, trimethylamine-N-oxide and the clinical manifestations of type II diabetes mellitus. TMAO: Trimethylamine-N-oxide; TMA: Trimethylamine.

TMAO, a pro-atherogenic and metabolically disruptive metabolite: TMAO is generated from the microbial metabolism of dietary nutrients such as choline, L-carnitine, and phosphatidylcholine, commonly found in red meat, eggs, and dairy. The gut microbiota converts these precursors into trimethylamine (TMA), which is then oxidized in the liver by flavin-containing monooxygenases (primarily FMO3) into TMAO[75]. Elevated plasma TMAO levels have been associated with an increased risk of cardiovascular events, type 2 diabetes, and chronic kidney disease. Mechanistically, TMAO promotes atherosclerosis by enhancing foam cell formation, impairing reverse cholesterol transport, and increasing platelet hyperreactivity[76]. Moreover, TMAO can interfere with bile acid signaling and cholesterol metabolism, thereby contributing to metabolic dysregulation[77]. In diabetic individuals, elevated TMAO levels correlate with markers of insulin resistance, systemic inflammation, and endothelial dysfunction[78]. TMAO can also activate pro-inflammatory pathways through the NLRP3 inflammasome and NF-κB signaling, further exacerbating metabolic imbalance[79]. Interestingly, hepatic FMO3 activity, which determines TMAO production, is itself regulated by insulin and metabolic hormones, indicating a bidirectional link between host metabolism and TMAO biosynthesis[80]. These findings highlight TMAO as a microbial-derived mediator that bridges dietary intake, gut microbiota function, and host metabolic health.

SCFAs, beneficial regulators of metabolic homeostasis: In contrast to TMAO, SCFAs, primarily acetate, propionate, and butyrate, are produced through the microbial fermentation of non-digestible carbohydrates. These metabolites play crucial roles in maintaining gut barrier integrity, modulating immune responses, and regulating metabolic processes[25]. SCFAs exert their effects through G-protein coupled receptors (GPR) such as GPR41 (FFAR3), GPR43 (FFAR2), and GPR109A, and function as histone deacetylase (HDAC) inhibitors, thereby influencing gene expression[81]. Butyrate in particular, serves as an energy source for colonic epithelial cells and reinforces intestinal barrier function, thereby preventing microbial translocation and systemic inflammation. Propionate and acetate enter the portal circulation and affect hepatic gluconeogenesis, lipogenesis, and cholesterol synthesis[82]. Through GPR41 activation, SCFAs can influence sympathetic nervous system activity and energy expenditure[83]. Additionally, SCFAs stimulate the release of gut hormones such as glucagon-like peptide-1 and peptide YY, which enhance insulin sensitivity and reduce appetite[84]. In metabolic disorders such as obesity and type 2 diabetes, the abundance of SCFA-producing bacteria is often reduced and circulating SCFA levels are altered[85]. Experimental supplementation of SCFAs or prebiotics that promote their production has been shown to ameliorate insulin resistance, reduce adipose tissue inflammation, and improve lipid metabolism[86].

TMAO is associated with insulin resistance and cardiovascular risks: TMAO is a gut microbiota-derived metabolite that has gained significant attention due to its strong association with metabolic disorders and cardiovascular diseases. TMAO is produced through a two-step process: Dietary nutrients such as choline, phosphatidylcholine, and L-carnitine, which are abundant in red meat, eggs, and dairy products, are metabolized by gut microbes into TMA. TMA is then absorbed into the portal circulation and oxidized to TMAO in the liver by FMO3[76]. Studies have shown that elevated TMAO levels correlate with impaired glucose metabolism and insulin signaling. For instance, Gao et al[87] demonstrated that TMAO-treated mice fed with a high-fat diet exhibited greater fasting glucose levels, glucose intolerance, and insulin resistance compared to controls. Mechanistically, TMAO has been implicated in promoting endoplasmic reticulum (ER) stress and inflammation in insulin-responsive tissues, such as liver and adipose tissue, which interferes with insulin receptor signaling[88]. Additionally, TMAO induces inflammatory gene expression via NF-κB activation, contributing to systemic insulin resistance[89]. Beyond its influence on glucose metabolism, TMAO has a well-established link to cardiovascular risks. Initial studies by Wang et al[76] reported a strong association between plasma TMAO concentrations and the risk of major adverse cardiovascular events, including myocardial infarction, stroke, and death. This relationship was independent of traditional risk factors such as age, LDL cholesterol, or blood pressure. Mechanistically, TMAO has been shown to promote foam cell formation by enhancing cholesterol uptake in macrophages, impairing reverse cholesterol transport[77]. Furthermore, TMAO increases platelet reactivity and thrombosis potential, contributing to atherothrombotic events[90].

Another pivotal aspect of TMAO’s impact on cardiovascular health is its contribution to endothelial dysfunction. Endothelial cells exposed to TMAO display increased oxidative stress and reduced nitric oxide bioavailability, which impairs vasodilation and promotes vascular inflammation[91]. These changes set the stage for early atherogenesis and arterial stiffness. Moreover, TMAO promotes the expression of adhesion molecules (e.g., VCAM-1, ICAM-1) in endothelial cells, facilitating the recruitment of monocytes and enhancing vascular inflammation[79]. Interestingly, elevated TMAO levels have also been reported in patients with T2DM, and the metabolite is being considered a potential biomarker for metabolic-cardiovascular risk stratification. A study by Shan et al[92] showed that T2DM patients had significantly higher plasma TMAO concentrations compared to normoglycemic individuals, and these levels positively correlated with markers of insulin resistance and subclinical atherosclerosis. Moreover, intervention studies suggest that modulating gut microbiota composition through antibiotics or dietary interventions can reduce TMAO production and improve metabolic profiles[93].

SCFAs support gut barrier integrity but are often reduced in diabetic patients: SCFAs, especially butyrate, are important microbial metabolites that play a crucial role in maintaining intestinal health and regulating host metabolism. SCFAs, including acetate, propionate, and butyrate, are primarily produced through the fermentation of indigestible dietary fibers by commensal gut bacteria, particularly by taxa belonging to the Firmicutes phylum such as Faecalibacterium prausnitzii, Roseburia, and Eubacterium species[94]. Among these, butyrate is of special interest due to its significant immunomodulatory and barrier-supportive properties, which are often compromised in metabolic diseases like T2DM. One of the key beneficial effects of butyrate lies in its ability to maintain and enhance gut barrier integrity. Butyrate serves as the primary energy source for colonocytes and promotes the expression of tight junction proteins, such as claudins and occludins, thereby preserving epithelial barrier function[95]. This helps to prevent the translocation of harmful microbial components like LPS into systemic circulation, which can trigger low-grade inflammation and contribute to insulin resistance. In diabetic individuals, reduced levels of butyrate-producing bacteria correlate with increased gut permeability[96].

Butyrate also exhibits potent anti-inflammatory effects by inhibiting HDACs, leading to epigenetic modulation of gene expression. This inhibition dampens the activation of NF-κB, a major transcription factor involved in pro-inflammatory cytokine production[97]. In macrophages and intestinal epithelial cells, butyrate has been shown to suppress the production of IL-6, TNF-α, and other inflammatory mediators, thereby contributing to the maintenance of immune tolerance and metabolic homeostasis[98]. The impact of butyrate extends to peripheral metabolic tissues, as well. Studies have indicated that butyrate can enhance insulin sensitivity by modulating mitochondrial function and reducing oxidative stress. For example, Gao et al[86] utilized mouse models to demonstrate that butyrate supplementation improved insulin sensitivity and increased energy expenditure, partly through AMPK activation in skeletal muscle and liver.

However, in individuals with T2DM, fecal SCFA levels, particularly butyrate, are often significantly reduced. A study by Qin et al[99] showed that diabetic patients had a lower abundance of butyrate-producing bacteria compared to healthy controls, suggesting a dysbiosis that may impair SCFA synthesis. This microbial imbalance is thought to be driven by a Western-style diet low in fiber and high in fat and simple sugars, which reduces the availability of fermentable substrates necessary for SCFA production[100]. Furthermore, clinical studies have confirmed the link between reduced butyrate levels and metabolic dysfunction in humans. In a cohort study by Karlsson et al[101], individuals with T2DM showed not only reduced microbial diversity but also significant depletion of genes involved in butyrate synthesis pathways. This deficiency contributes to chronic intestinal inflammation, compromised barrier function, and systemic metabolic abnormalities characteristic of diabetes. Therapeutically, interventions aimed at restoring butyrate levels, such as high-fiber diets, prebiotics (e.g., inulin, resistant starch), and probiotic supplementation with butyrate-producing strains, have shown promise in improving metabolic parameters. For instance, Ghanbari-Gohari et al[102] reported that a Mediterranean diet enriched in fiber enhanced butyrate production and improved glycemic control in prediabetic individuals.

Microbial translocation-induced chronic inflammation impairs pancreatic beta-cell function, reducing insulin secretion: Microbial translocation acts as a chronic inflammatory trigger, activating PRRs such as TLRs on immune cells and various tissues, including the pancreas. A growing body of evidence suggests that chronic exposure to these microbial components promotes systemic low-grade inflammation and oxidative stress, which contribute significantly to pancreatic beta-cell dysfunction and impaired insulin secretion[3,103]. LPS is the most widely studied translocated microbial product linked to metabolic endotoxemia. Chronic exposure to LPS stimulates TLR4 signaling pathways in immune cells and metabolic tissues, triggering the release of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β[104]. In turn, these cytokines induce insulin resistance in peripheral tissues and have direct cytotoxic effects on pancreatic beta cells. Specifically, IL-1β impairs insulin gene expression and promotes beta-cell apoptosis via NF-κB signaling and ER stress[105].

The pancreas is not immune to the inflammatory effects of systemic endotoxemia. Beta cells express TLR4 and are susceptible to LPS-mediated damage. Exposure of islets to LPS in vitro reduces glucose-stimulated insulin secretion and increases pro-apoptotic signaling[41]. In vivo models also support this. Mice chronically exposed to low-dose LPS develop glucose intolerance and exhibit beta-cell dysfunction[106]. Chronic inflammation within pancreatic islets not only reduces insulin production but also alters beta-cell identity and promotes dedifferentiation, contributing to the progressive loss of beta-cell mass in T2DM[107]. Oxidative stress is a key mediator of beta-cell impairment in the context of chronic inflammation. Beta cells are particularly vulnerable to oxidative damage due to their relatively low expression of antioxidant enzymes like catalase and glutathione peroxidase[108]. When microbial translocation activates immune cells, ROS are generated as part of the inflammatory response. If not neutralized, these ROS induce mitochondrial dysfunction, DNA damage, and lipid peroxidation in beta cells, exacerbating functional decline and promoting apoptosis[109]. The unfolded protein response, activated under ER stress conditions, is further aggravated by ROS, leading to beta-cell exhaustion and impaired insulin synthesis[110]. Persistent exposure to LPS and systemic inflammation has been shown to upregulate ER stress markers such as CCAAT/enhancer binding protein homologous protein and binding immunoglobulin protein in pancreatic tissue, indicating a convergence of inflammatory and oxidative pathways in beta-cell damage[111].

Clinical studies reinforce the link between microbial translocation, inflammation, and beta-cell dysfunction. In individuals with T2DM or prediabetes, circulating levels of LPS and bacterial DNA are elevated and correlate with markers of inflammation and insulin resistance[33]. Furthermore, diabetic patients often exhibit reduced gut microbial diversity and a higher abundance of LPS-producing bacteria, suggesting that gut dysbiosis contributes to increased endotoxemia[99]. Therapeutic strategies aimed at reducing microbial translocation and systemic inflammation have shown potential in preserving beta-cell function. Prebiotic and probiotic supplementation to restore gut barrier integrity, as well as pharmacological interventions targeting TLR4 or oxidative stress pathways, have demonstrated benefits in experimental models[85]. For example, supplementation with Akkermansia muciniphila, a mucin-degrading bacterium that strengthens the gut barrier, has been associated with reduced endotoxemia and improved glucose homeostasis[112].

Link to diabetic complications

Circulating microbial components contribute to the development of diabetic nephropathy: Diabetic nephropathy (DN), a leading cause of end-stage renal disease, is a serious microvascular complication of diabetes. While hyperglycemia and hypertension are well-established drivers of DN, recent research has illuminated the pivotal role of circulating microbial components, particularly LPS, bacterial DNA, and other PAMPs, in exacerbating renal injury through innate immune activation, glomerular inflammation, and fibrosis. In patients with T2DM, increased intestinal permeability facilitates the translocation of microbial components into the systemic circulation[3]. Among these, LPS, derived from the outer membrane of gram-negative bacteria, is a potent activator of TLR4 on immune and renal cells. Binding of LPS to TLR4 triggers downstream NF-κB signaling and the release of proinflammatory cytokines, including IL-6, TNF-α, and MCP-1, which contribute to leukocyte recruitment and glomerular inflammation[113] (Figure 5).

Figure 5 Schematic overview of vasculopathy in diabetes mellitus. Diabetic vasculopathy involves the cerebral, ophthalmic, cardiac, renal, and peripheral systems.

The kidneys, particularly the glomeruli, are highly susceptible to inflammatory damage due to their rich capillary networks and immune cell infiltration in response to TLR activation. Studies in murine models have shown that LPS administration exacerbates albuminuria and mesangial expansion, mimicking features of DN[114]. Moreover, renal TLR4 expression is upregulated in diabetic states, amplifying sensitivity to circulating microbial stimuli[115]. Human studies corroborate these findings. Elevated circulating LPS levels have been correlated with increased urinary albumin excretion and deteriorating glomerular filtration rates in diabetic individuals[58]. Chronic activation of the TLR4-NF-κB axis stimulates the release of transforming growth factor-β1, a key fibrogenic cytokine, which induces extracellular matrix deposition by renal fibroblasts and podocytes[116]. Additionally, bacterial DNA recognized by TLR9 has been shown to activate inflammasomes and induce mitochondrial oxidative stress, further driving fibrogenic responses in the kidney[117]. Emerging research on the gut-kidney axis also highlights the systemic impact of microbial dysbiosis in DN pathogenesis. Dysbiosis-induced translocation of LPS and other PAMPs exacerbates metaflammation, which in turn affects renal endothelial and epithelial integrity[118]. This systemic burden contributes to endothelial dysfunction, altered glomerular permeability, and progressive renal injury. Moreover, circulating LBP, which complexes with LPS and facilitates its recognition by immune cells, is elevated in diabetic patients with nephropathy compared to those without renal complications[119]. LBP has thus emerged as a potential biomarker of microbial translocation and renal inflammation severity in diabetes.

Microbial metabolites and endotoxins increase the risk of cardiovascular events in diabetic patients: Cardiovascular disease remains the leading cause of mortality in individuals with diabetes mellitus. Beyond classical risk factors, such as hyperglycemia, dyslipidemia, and hypertension, accumulating evidence points to the significant contribution of gut microbiota-derived metabolites and endotoxins in exacerbating vascular inflammation and promoting atherosclerosis. These microbial products not only influence systemic immune responses but also directly affect endothelial function and plaque stability, thereby increasing the risk of cardiovascular events in diabetic patients. One of the most extensively studied microbial metabolites associated with cardiovascular risk is TMAO. Formed in the liver from TMA, a product of microbial metabolism of dietary choline and carnitine, TMAO has been shown to enhance foam cell formation, impair reverse cholesterol transport, and promote vascular inflammation[76]. Elevated circulating TMAO levels are strongly associated with increased atherosclerotic burden and incident major adverse cardiovascular events[75]. In diabetic populations, plasma TMAO concentrations are often elevated and correlate positively with markers of vascular dysfunction, including arterial stiffness and endothelial injury[88].

TMAO exerts its pro-atherogenic effects through multiple mechanisms. It enhances the expression of scavenger receptors such as CD36 and scavenger receptor class A1 on macrophages, facilitating the uptake of oxidized low-density lipoprotein and foam cell formation[77]. It also promotes vascular inflammation by upregulating inflammatory cytokines (e.g., IL-6, TNF-α) and activating the NLRP3 inflammasome in endothelial and immune cells[79]. These processes contribute to endothelial dysfunction, an early hallmark of atherosclerosis, and are particularly relevant in diabetes, where chronic low-grade inflammation already primes the vasculature for injury. LPS, a gram-negative bacterial endotoxin, is another key microbial component implicated in atherogenesis. Diabetic patients frequently exhibit elevated serum LPS levels due to increased gut permeability, often referred to as metabolic endotoxemia[3]. LPS binds to TLR4 on vascular endothelial and immune cells, triggering MyD88-dependent signaling pathways that culminate in NF-κB activation and production of proinflammatory cytokines[120]. Chronic LPS exposure fosters monocyte adhesion to the endothelium, oxidative stress, and vascular smooth muscle cell proliferation[41].

Furthermore, LPS-induced endothelial activation results in increased expression of adhesion molecules (ICAM-1, VCAM-1), facilitating leukocyte infiltration into the vascular intima and plaque development[121]. In diabetic individuals, this process is amplified by hyperglycemia-induced advanced glycation end-products and oxidative stress, compounding vascular injury[122]. Murine studies have shown that antibiotic depletion of gut microbiota or the use of LPS-neutralizing agents can significantly attenuate atherosclerotic lesion development, underscoring the causal role of microbial endotoxins in vascular pathology[123]. While SCFAs such as butyrate and propionate are generally considered protective, maintaining gut barrier integrity and modulating inflammation, their reduced production in diabetes contributes to gut dysbiosis and increased endotoxemia[124]. The consequent rise in LPS and other microbial-derived proinflammatory mediators sets off a cascade of immune activation that perpetuates vascular inflammation. Importantly, microbial metabolites not only affect atherosclerosis progression, but also influence plaque stability and thrombosis. TMAO has been reported to enhance platelet hyperreactivity and thrombotic potential by modulating calcium signaling and promoting platelet aggregation[89]. This has direct implications for cardiovascular events, such as myocardial infarction and stroke in diabetic patients, who are already at heightened risk. A comprehensive recent review highlights the central role of the gut microbiota in T2DM pathophysiology and management, detailing how microbial metabolites (SCFAs, TMAO, bile acids, LPS) regulate host metabolism, inflammation, and therapeutic responses[125]. In a 2025 study on diabetic cardiomyopathy, gut-microbiota derived metabolites (SCFAs, TMAO, bile acids, LPS, tryptophan-catabolites, sulfuretted hydrogen) were mechanistically linked to cardiac metabolic reprogramming, mitochondrial dysfunction, inflammation, and fibrosis, demonstrating how microbiota can directly affect diabetic heart disease[126].

Altered microbial signatures have been linked to diabetic retinopathy: Emerging evidence suggests that altered gut microbial signatures contribute to the progression of diabetic retinopathy (DR), a microvascular complication of diabetes mellitus. The dysbiotic microbiome in diabetic patients has been associated with systemic inflammation and oxidative stress, both of which are critical in DR pathogenesis. The gut-retina axis has become a focal point in understanding how microbial metabolites and immune-modulating components affect retinal health. In individuals with T2DM, gut microbiota composition is significantly altered, typically marked by a reduction in butyrate-producing bacteria and an increase in pro-inflammatory taxa. This dysbiosis leads to impaired intestinal barrier integrity and increased systemic translocation of microbial products like LPS. LPS activates TLRs, particularly TLR4, stimulating a cascade of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6[2].

Inflammation and oxidative stress in retinal tissues: Once microbial components enter systemic circulation, they can reach distant organs, including the retina. Retinal endothelial cells and microglia express TLRs, and upon stimulation by microbial products, these cells release pro-inflammatory mediators that lead to increased vascular permeability and leukostasis, hallmarks of DR[127]. Moreover, the chronic inflammatory environment induces ROS generation through triphosphopyridine nucleotide oxidase activation, mitochondrial dysfunction, and xanthine oxidase pathways[128]. Elevated ROS in retinal tissues leads to DNA damage, apoptosis of pericytes, and breakdown of the blood-retinal barrier, accelerating DR progression.

Microbial metabolites modulating retinal health: Beyond LPS, microbial metabolites such as TMAO and SCFAs also influence retinal outcomes. TMAO, elevated in diabetic individuals, has been linked to vascular inflammation and endothelial dysfunction[90]. In contrast, SCFAs like butyrate exhibit anti-inflammatory properties and help maintain epithelial barrier function[129]. Reduced SCFA levels in diabetic patients contribute to enhanced systemic inflammation and compromise mucosal immunity, indirectly worsening retinal inflammation.

Evidence from clinical and experimental studies: Several clinical studies support the association between microbial dysbiosis and DR. For instance, Bai et al[130] found that patients with proliferative DR had distinct gut microbial compositions compared to diabetics without DR, with an enrichment of pro-inflammatory genera such as Ruminococcus and depletion of beneficial Faecalibacterium. Similarly, intravitreal cytokine levels such as IL-6 and VEGF were significantly elevated in patient data repository patients, correlating with microbial dysbiosis indices[131]. Germ-free mice or those treated with antibiotics showed altered susceptibility to retinal inflammation in diabetic settings[132]. Fecal microbiota transplantation (FMT) from DR patients into diabetic mice exacerbated retinal vascular leakage and increased expression of inflammatory markers, further demonstrating a causal link between microbiota and DR[133]. A 2025 integrated analysis comparing gut microbiota, fecal and serum metabolites in T2DM patients with peripheral neuropathy identified distinct metabolomic signatures linked to disease complications, underlining the mechanistic relevance of gut metabolites beyond glycemic control[134].

CLINICAL IMPLICATIONS

Biomarkers for diagnosis and prognosis

Specific microbial DNA sequences and metabolites in circulation can serve as biomarkers: Microbial translocation leads to systemic exposure to PAMPs, particularly LPS and bacterial DNA. These molecules activate PRRs such as TLRs and NLRs on immune cells, leading to downstream activation of NF-κB and pro-inflammatory cascades (Figure 6).

Figure 6 Classification of various biomarkers implicated in diabetes. mRNA: Messenger RNA.

High-throughput sequencing technologies and metabolomic profiling are crucial for identifying these microbial signatures: Advances in high-throughput sequencing (HTS) and metabolomic profiling technologies have revolutionized our understanding of the human microbiome and its association with health and disease. These tools are critical for identifying disease-specific microbial signatures, including taxonomic, functional, and metabolic alterations. In the context of chronic diseases such as diabetes, cardiovascular disorders, and neurodegeneration, these platforms enable a comprehensive analysis of the microbiome-host interactions, facilitating biomarker discovery and the development of personalized interventions.

HTS in microbiome profiling: HTS, also known as next-generation sequencing, allows for the deep and accurate characterization of microbial communities. The two principal approaches in microbiome research include 16S rRNA gene sequencing and whole metagenome shotgun (WMS) sequencing. While 16S rRNA sequencing targets conserved regions of the bacterial ribosomal RNA gene to infer taxonomic identity, WMS sequencing captures the entire genetic repertoire of microbial communities, offering insights into microbial function, strain-level variation, and resistance genes[135,136]. In studies of diabetic patients, 16S rRNA sequencing has revealed reduced diversity and an enrichment of pro-inflammatory taxa such as Bacteroides and Ruminococcus, which correlate with elevated glycemic markers[4]. More recently, WMS sequencing has provided high-resolution insights into functional dysbiosis in T2DM, identifying genes linked to LPS biosynthesis, oxidative stress resistance, and SCFA metabolism[99]. These findings highlight the importance of sequencing depth and coverage provided by WMS in identifying clinically relevant microbial traits. Emerging platforms such as Oxford Nanopore and PacBio long-read sequencing are further enhancing microbiome research by enabling more complete genome assemblies, improving the detection of complex genomic features, such as clustered regularly interspaced short palindromic repeats elements, plasmids, and phage genomes[137]. These long-read technologies complement short-read sequencing by resolving ambiguities in highly similar genomic regions and capturing strain-level variation that may be masked in conventional HTS datasets.

Metabolomics-functional fingerprinting of the microbiome: While sequencing technologies provide insights into microbial composition and functional potential, metabolomics measures the biochemical output of the microbiota, capturing real-time interactions between the microbiome and host physiology. Metabolomics relies on mass spectrometry and nuclear magnetic resonance spectroscopy to detect a broad range of metabolites such as SCFAs, bile acids, amino acids, and microbial toxins in biological samples including feces, serum, urine, and tissues[138]. In diabetic cohorts, metabolomic profiling has revealed distinct metabolic fingerprints, including elevated levels of branched-chain amino acids and TMAO, and decreased levels of butyrate and propionate, which are key SCFAs with anti-inflammatory roles[7,76]. These metabolic shifts reflect not only microbial dysbiosis but also altered host-microbiome crosstalk in glucose metabolism, lipid regulation, and immune modulation. Importantly, integrating metagenomics with metabolomics provides a systems-level understanding of how microbial genes translate into functional metabolites that impact host physiology. For example, a study by Wang et al[76] demonstrated that gut microbial metabolism of dietary choline and L-carnitine to TMAO is mediated by microbial enzymes, and this pathway is significantly enriched in patients with metabolic syndrome. These integrative analyses uncover functional pathways that are not evident through genomics alone, emphasizing the complementary value of metabolomic data.

Integrative multi-omics approaches and future prospects: The integration of HTS and metabolomics, commonly referred to as multi-omics, enables a comprehensive understanding of microbial signatures across different layers viz., genetic, transcriptomic, proteomic, and metabolic. Advanced bioinformatics pipelines now allow for the correlation of microbial taxa with metabolite profiles, facilitating causal inference and biomarker discovery[139]. For example, Franzosa et al[139] applied multi-omics profiling in the human microbiome project (HMP) phase II and identified unique microbial and metabolomic shifts associated with inflammatory bowel disease. Similar approaches are being applied in diabetes research to identify microbial signatures that precede complications such as nephropathy and retinopathy, potentially enabling early intervention. Furthermore, machine learning models built on multi-omics datasets are being used to predict disease risk and treatment outcomes. These predictive models rely heavily on large-scale, high-quality sequencing and metabolomics data, underscoring the critical role of these technologies in clinical microbiome science. As technologies continue to advance, real-time sequencing, point-of-care metabolomics, and cloud-based data analysis will become integral in personalized medicine. The continued evolution and democratization of these platforms promise to expand microbiome research beyond research laboratories to clinical and even community health settings.

Therapeutic strategies

Modulating the gut microbiome may reduce microbial translocation and systemic inflammation: Given the central role of gut dysbiosis and barrier dysfunction in microbial translocation, therapeutic approaches aim to restore microbial balance and reinforce epithelial integrity (Table 2 and Figure 7).

Figure 7 Using gut microbiota modulation as a precision strategy against type II diabetes mellitus. FMT: Fecal microbiota transplantation; TLR: Toll-like receptor; TMAO: Trimethylamine-N-oxide.

Table 2 Potential therapeutic interventions and their reported efficacy[203-211].

No.	Intervention	Putative mechanism	Reported efficacy (diabetes/cardiometabolic)	Evidence level	Key notes/source	Ref.
1	FMT	Replaces dysbiotic gut community with a healthier donor community improves barrier function, metabolism	Some small trials and pilot studies report improved insulin sensitivity and metabolic markers; results are heterogeneous and short-term	Early clinical/pilot RCTs; heterogeneous	Promising but inconsistent; safety, donor selection, and durability remain concerns. Reviews in the set recommend FMT as a research tool rather than routine therapy	Wu et al[203]
2	Probiotics/single-strain (e.g., Blautia spp.)	Restore beneficial taxa, increase SCFA production, improve gut barrier and metabolic signalling	Specific strains (preclinical and some human data) linked to improved glucose/weight outcomes; Blautia wexlerae showed benefit in obesity/T2D models and human-associated data cited in your reprints	Preclinical + small clinical/translational evidence	Strain-specific effects; some encouraging translational evidence in the uploaded reprint (Blautia example). Larger RCTs needed	Kocsis et al[204]
3	Prebiotics/dietary fibre/dietary patterns (e.g., Mediterranean diet)	Enrich SCFA-producing bacteria, strengthen barrier, reduce endotoxemia	Mediterranean-style diets linked to favorable microbiome-mediated cardiometabolic risk reduction and improved metabolic markers	Observational + controlled dietary interventions	Diet is low-risk, widely recommended; effects likely mediated by microbiome changes per reviews in your files	Salas-Salvadó et al[205]
4	Synbiotics (prebiotic + probiotic)	Synergistic restoration of beneficial microbes and their substrates	Small trials show modest improvements in insulin sensitivity, lipids, and inflammation in some cohorts	Small RCTs/pilot studies	Effects variable; likely dependent on component selection and baseline microbiota. (Supported by intervention-review discussion)	Zhang et al[206]
5	Antibiotics/targeted antimicrobials	Reduce/pathogen-deplete specific taxa driving dysbiosis; transiently alter metabolite production (e.g., TMA producers)	Short-term metabolic changes reported; long-term benefits unclear and potential harms (resistance, loss of beneficial taxa)	Mostly short-term clinical/observational	Not recommended broadly; may be useful experimentally to probe causal roles. Reviews warn about unintended consequences	Mikkelsen et al[207]
6	TLR antagonists/innate-immune modulators (e.g., TLR4/TLR9 inhibitors)	Block host sensing of microbial PAMPs (LPS, bacterial DNA) to reduce inflammation and insulin resistance	Preclinical models show reduced metabolic inflammation and improved insulin sensitivity; limited human data	Preclinical/early-phase	TLR4/TLR9 implicated in sensing circulating microbial products in your reprints; clinical development is early and safety must be established	Yehualashet[208]
7	TMA/TMAO pathway inhibitors (microbial enzyme inhibitors or host FMO inhibitors)	Reduce production or hepatic conversion of TMA lower plasma TMAO, a pro-atherogenic metabolite	Preclinical and small translational studies show reduced TMAO and atherogenic readouts; clinical outcome data lacking	Preclinical/early translational	Considered a promising target for cardiometabolic risk reduction; reviewed as an emerging therapeutic approach in your files	Jaworska et al[209]
8	SCFA/butyrate supplementation or butyrate-promoting strategies	Restore epithelial energy, tighten barrier, modulate GLP-1 and immune responses	Preclinical and small human studies indicate improved insulin sensitivity and gut integrity; direct supplementation trials limited	Preclinical + small human studies	Benefits likely when produced endogenously by fiber fermentation; direct supplementation faces formulation/tolerability issues	Arora and Tremaroli[210]
9	Microbiome-derived metabolite modulation (e.g., bile acid modulators, indoxyl sulfate lowering)	Alter signalling metabolites that influence host metabolism, inflammation and vascular function	Early-stage; some interventions (bile acid modulators) affect metabolic parameters in trials outside uploaded files; specific microbiome-targeted metabolite therapies are emergent	Early translational/mechanistic trials	Concept supported across reviews; direct clinical evidence in diabetes remains limited within your reprints	Calvo-Barreiro et al[211]

FMT: Fecal microbiota transplantation; TLR: Toll-like receptor; TMAO: Trimethylamine-N-oxide; TMA: Trimethylamine; SCFA: Short-chain fatty acid; FMO: Flavin-containing monooxygenases; GLP-1: Glucagon-like peptide-1; PAMPs: Pathogen-associated molecular patterns; LPS: Lipopolysaccharide; T2D: Type 2 diabetes; RCT: Randomized controlled trial.

FMT restores gut microbial diversity and metabolic homeostasis: FMT involves the transfer of stool from a healthy donor into the gastrointestinal tract of a recipient with the aim of restoring the recipient’s gut microbiota composition and functionality. Initially developed to treat recurrent Clostridioides difficile infections, FMT has recently gained attention for its potential therapeutic role in metabolic disorders, particularly T2D and obesity, which are closely linked to gut microbial dysbiosis and chronic inflammation[140,141]. Studies have demonstrated that individuals with T2D and metabolic syndrome exhibit reduced microbial diversity and altered ratios of key microbial phyla, including a higher abundance of pathobionts and reduced beneficial taxa like Faecalibacterium prausnitzii and Akkermansia muciniphila[99,100]. FMT offers a strategy to reverse this dysbiosis by introducing a healthy and diverse microbial ecosystem that can re-establish balance in the recipient’s gut, potentially improving metabolic outcomes. In a pioneering randomized controlled trial, Vrieze et al[142] transferred intestinal microbiota from lean donors to male recipients with metabolic syndrome. The recipients exhibited increased insulin sensitivity, improved gut microbial diversity, and elevated levels of butyrate-producing bacteria post-FMT. These findings highlighted that microbial alterations via FMT could influence host glucose metabolism independently of changes in diet or body weight.

The beneficial effects of FMT in metabolic diseases are thought to be mediated through several mechanisms. First, the reintroduction of beneficial bacteria can increase the production of SCFAs, particularly butyrate, which can exert anti-inflammatory effects, support gut barrier integrity, and modulate energy metabolism[143]. Second, FMT may reduce circulating endotoxins such as LPS, thereby lowering systemic inflammation and associated insulin resistance[144]. Third, altered bile acid metabolism induced by new microbial populations can influence lipid and glucose homeostasis via signaling pathways involving farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5)[145]. The variability in donor microbiota, delivery methods (e.g., colonoscopy, capsules, nasoenteric tubes), and recipient response contribute to heterogeneous outcomes in pilot studies. For instance, Kootte et al[144] conducted a double-blind, placebo-controlled study where insulin-resistant individuals received FMT from lean donors. While insulin sensitivity improved, the response was more pronounced in individuals with low baseline microbial diversity, suggesting that recipient microbiome composition plays a key role in determining FMT efficacy. Safety is another concern in the broader application of FMT. While generally considered safe in well-screened donors, there have been isolated reports of transmission of pathogenic bacteria, leading to severe infections in immunocompromised patients[146]. Thus, regulatory frameworks and standardized protocols are essential for the clinical translation of FMT beyond infectious diseases. The beneficial impact on insulin sensitivity may be transient unless supported by lifestyle modifications, such as dietary changes that favor the engraftment and persistence of the introduced microbial strains[147]. Hence, combining FMT with prebiotic or dietary interventions could enhance its long-term metabolic benefits.

Targeted pharmacological interventions represent novel therapeutic approaches: Emerging evidence has demonstrated that microbial dysbiosis plays a key role in the pathogenesis of chronic diseases such as T2DM, atherosclerosis, and cardiovascular disease. Central to this pathogenesis are microbial metabolites like TMAO and PAMPs such as LPS, which activate innate immune receptors, particularly TLRs. Consequently, targeted pharmacological interventions, such as TLR inhibitors and TMAO pathway modulators, have garnered significant attention as promising therapeutic strategies to combat inflammation-driven metabolic disorders[148,149].

TLR inhibitors: TLR activation leads to NF-κB signaling, promoting the release of pro-inflammatory cytokines and contributing to insulin resistance, vascular inflammation, and atherosclerotic plaque formation[150,151]. Inhibiting TLRs can thus downregulate chronic inflammation associated with metabolic and cardiovascular diseases. Small molecule TLR4 antagonists such as TAK-242 (resatorvid) have been shown to suppress LPS-induced cytokine production by selectively binding to the intracellular domain of TLR4, thereby preventing downstream signaling[152]. In preclinical studies, administration of TAK-242 improved insulin sensitivity and reduced systemic inflammation in obese mouse models[153]. Similarly, eritoran, a synthetic lipid A analogue, blocks the LPS-TLR4-MD2 interaction and attenuated atherosclerosis in apolipoprotein E (ApoE)-deficient mice by reducing macrophage activation and inflammatory cytokine expression[154]. Despite promising preclinical results, clinical translation has been slow. For example, eritoran failed to show efficacy in reducing mortality in patients with sepsis in a large phase III trial[155], though its role in chronic metabolic inflammation remains under investigation. Further refinement of TLR inhibitors, along with patient stratification based on inflammatory biomarkers, may enhance future therapeutic outcomes.

TMAO pathway modulators: Gut bacteria convert these precursors into TMA, which is absorbed and oxidized to TMAO by hepatic flavin monooxygenases, particularly FMO3[80]. Elevated plasma TMAO levels have been strongly correlated with an increased risk of major adverse cardiovascular events, thrombosis, and mortality in both healthy individuals and patients with diabetes[156,157]. Given its causal link to atherosclerosis and inflammation, several strategies have been proposed to modulate TMAO levels. One approach involves the inhibition of microbial TMA formation using small molecules like 3,3-dimethyl-1-butanol (DMB), a structural analogue of choline. Wang et al[158] demonstrated that DMB significantly reduced TMAO production and atherosclerotic lesion formation in ApoE-/- mice without affecting gut microbial diversity. Alternatively, inhibiting FMO3 in the liver has also shown potential. Knockdown of FMO3 in mice using antisense oligonucleotides led to reduced circulating TMAO levels, improved glucose tolerance, and protection against diet-induced obesity and insulin resistance[159]. However, long-term inhibition of FMO3 may interfere with hepatic detoxification pathways, necessitating careful consideration in human studies. Probiotic and dietary interventions that reduce TMAO-producing microbial species or compete with TMA-producing pathways offer another avenue. For example, resveratrol and grape polyphenols have been shown to reduce TMAO levels in rodents, possibly by altering gut microbial composition[160]. Furthermore, bile acid signaling has emerged as an intersecting pathway between gut microbiota, TMAO, and metabolic health. FXR and TGR5, both regulated by bile acids, influence lipid metabolism, insulin sensitivity, and inflammation. Pharmacological modulation of FXR using agonists like obeticholic acid has shown benefits in non-alcoholic steatohepatitis and may indirectly reduce TMAO levels[161].

Personalized medicine

Individual variability in the gut microbiome necessitates personalized therapeutic strategies: The human gut microbiome is increasingly recognized as a dynamic and highly individualized ecosystem that plays a critical role in host metabolism, immunity, and disease progression. Inter-individual variability in microbiome composition, shaped by genetics, diet, environment, medication use, and early-life exposures, has led to the growing recognition that a “one-size-fits-all” approach to microbiome-based therapies is unlikely to be effective[162]. Instead, there is a compelling need to develop personalized therapeutic strategies that account for the unique microbial signatures and metabolic profiles of each individual, especially in complex metabolic diseases such as T2D and obesity. Studies using 16S rRNA sequencing and shotgun metagenomics have revealed substantial heterogeneity in gut microbial composition across individuals and populations[163]. For instance, the presence or absence of key genera such as Akkermansia, Bacteroides, or Prevotella has been associated with differential glucose metabolism, insulin sensitivity, and inflammation[164,165]. Moreover, microbial functions, including the capacity to produce SCFAs or metabolize dietary compounds into potentially harmful metabolites like TMAO, vary substantially between individuals and can influence disease trajectories[85,76] (Figure 8).

Figure 8 Causes of the individual variability in drug response.

Metabolomic profiling further deepens this personalized landscape by identifying specific microbial metabolites linked to health or disease. For instance, elevated circulating levels of TMAO are associated with increased cardiovascular risk in some individuals but not others, suggesting inter-individual differences in microbial metabolic pathways and host responses[77]. Similarly, SCFA levels vary widely between individuals and are influenced by both microbiota composition and dietary fiber intake, modulating inflammation, gut barrier integrity, and insulin sensitivity[82]. These insights underscore the value of multi-omics approaches, including metagenomics, transcriptomics, proteomics, and metabolomics, to build comprehensive individual profiles that can inform therapeutic decisions. Precision medicine initiatives are beginning to incorporate microbiome data to tailor interventions. For example, personalized dietary interventions based on microbiome composition have been shown to more effectively modulate postprandial glycemic responses than standardized diets[166]. Additionally, predictive models integrating microbiota features with clinical parameters can forecast patient-specific responses to prebiotics, probiotics, or FMT[167]. However, implementing personalized microbiome therapies faces several challenges, including the need for standardized and reproducible microbiome analytics, regulatory hurdles, and ethical concerns related to privacy and data use. Furthermore, longitudinal studies are needed to understand the stability of microbial traits and the durability of personalized interventions over time[168].

Multi-omics approaches will enhance the precision of these interventions: The integration of multi-omics technologies, encompassing genomics, transcriptomics, metabolomics, and often proteomics and epigenomics, has revolutionized our understanding of host-microbiome interactions and their influence on health and disease. These complementary layers of biological information offer a systems-level perspective that can enhance the precision and efficacy of microbiome-targeted interventions, particularly in complex, multifactorial diseases such as metabolic syndrome, diabetes, inflammatory bowel disease, and cardiovascular disorders. Genome-wide association studies have revealed that specific host genotypes can influence gut microbial composition and function[169]. For example, polymorphisms in genes related to immunity (e.g., NOD2, TLR5) are associated with altered microbial diversity and an increased risk of inflammatory conditions[170]. By integrating host genomic data, personalized interventions can be designed to account for genetic predispositions and optimize therapeutic outcomes. Transcriptomics, which profiles RNA expression patterns, offers insights into how both host and microbial cells dynamically respond to environmental stimuli and therapeutic interventions. Shotgun metatranscriptomics enables the simultaneous evaluation of microbial gene expression and host immune responses within the gut environment[171]. For instance, transcriptomic analyses have shown that individuals with obesity and insulin resistance exhibit upregulated pro-inflammatory gene expression in both gut epithelial cells and circulating immune cells, which correlates with specific microbial metabolites[172]. Many key microbial metabolites, including SCFAs, bile acids, indole derivatives, and TMAO, have been implicated in modulating immune responses, metabolic homeostasis, and gut barrier integrity[173,174]. Personalized profiling of the metabolites helps predict therapeutic response and disease risk. For example, individuals with a high urinary excretion of TMAO, often driven by microbiota, may benefit from TMAO-lowering interventions like dietary modifications or inhibitors of microbial TMA formation[158] (Figure 9).

Figure 9 Integrating multi-omics to unravel host-microbiome interactions in type II diabetes mellitus.

When integrated, multi-omics data offer a comprehensive map of the host-microbiome interface, revealing mechanistic pathways and actionable biomarkers that single-omics approaches may overlook. Several proof-of-concept studies have demonstrated the power of this strategy. For example, the integrative HMP applied multi-omics profiling to individuals with inflammatory bowel disease and uncovered host genetic variants, microbial shifts, transcriptomic patterns, and metabolite signatures[175]. These insights are critical for guiding interventions such as FMT, dietary regimens, or pharmacologic agents targeting microbial enzymes or receptors. Furthermore, machine learning and artificial intelligence are increasingly used to interpret the high-dimensional datasets generated by multi-omics studies, enabling the construction of predictive models for therapy selection, response monitoring, and disease prevention[176]. Despite its promise, multi-omics integration presents technical, computational, and interpretive challenges. These include sample heterogeneity, data normalization, and the need for robust computational pipelines. However, the continued evolution of bioinformatics tools and reduction in sequencing and metabolomics costs make it increasingly feasible to apply multi-omics approaches in both research and clinical settings.

CHALLENGES AND FUTURE DIRECTIONS

Establishing causality between circulating microbial components and diabetes pathogenesis requires longitudinal studies and interventional trials

The association between gut microbiome dysbiosis and metabolic diseases, particularly T2DM, has been increasingly documented in recent years. A growing body of evidence links circulating microbial components such as LPS, flagellin, peptidoglycan, and microbial DNA to low-grade systemic inflammation and insulin resistance, which are the central features in T2DM pathogenesis[96,106]. However, despite these advances, establishing a direct causal relationship between these microbial components and the onset or progression of diabetes remains challenging. Rigorous longitudinal cohort studies and interventional trials are essential to delineate temporal relationships, control for confounders, and determine mechanistic pathways that can guide therapeutic interventions (Table 3 and Figure 10).

Figure 10  Summary of various microbiome model types. Model types include in vitro models, in vivo models, organoid and organ-on-a-chip models and human microbiota-associated models.

Table 3 Evaluation of interventions that are experimental vs clinically applicable[203-205,207-210,212].

No.	Category	Intervention	Clinical status	Key rationale (with citations)	Ref.
1	Clinically applicable	Dietary modification (high-fibre, Mediterranean-style diet)	Clinically recommended	Improves glycaemic control, reduces inflammation, increases SCFA-producing taxa; supported by clinical and mechanistic evidence summarized in reviews	Salas-Salvadó et al[205]
2		Lifestyle interventions (exercise, weight loss)	Clinically established	Proven metabolic benefits with beneficial shifts in microbiome composition; supported by human observational and mechanistic summaries	Amerkamp et al[212]
3		Commercial probiotics (adjunctive, strain-specific)	Limited clinical applicability	Human studies show modest, strain-dependent metabolic improvements; discussed in multiple review articles as adjuncts rather than stand-alone therapies	Kocsis et al[204]
4	Experimental/research-stage	Fecal microbiota transplantation	Investigational	Pilot RCTs show transient improvements but inconsistent outcomes; safety and standardization concerns limit clinical use	Wu et al[203]
5		TLR antagonists (TLR4/TLR9 inhibitors)	Preclinical/early translational	Reduce metabolic inflammation in models; minimal human data and ongoing safety concerns	Yehualashet[208]
6		TMA/TMAO pathway inhibitors	Preclinical/early translational	Lower TMAO and inflammatory signaling in animal models; no large human trials	Jaworska et al[209]
7		Next-generation or engineered probiotics	Early clinical/experimental	Promising metabolic effects in early translational studies; require larger clinical trials	Kocsis et al[204]
8		SCFA/butyrate supplementation	Experimental	Mechanistically beneficial but limited robust human evidence; diet-induced endogenous SCFA remains preferred	Arora and Tremaroli[210]
9		Antibiotic-based microbiota depletion	Experimental	Mechanistically informative but clinically unsuitable due to resistance, dysbiosis risk and lack of durable benefits	Mikkelsen et al[207]

TLR: Toll-like receptor; TMAO: Trimethylamine-N-oxide; TMA: Trimethylamine; SCFA: Short-chain fatty acid; RCT: Randomized controlled trial.

Current evidence and the need for causal inference: Numerous cross-sectional studies have shown elevated levels of microbial components, especially endotoxins like LPS, in individuals with insulin resistance and metabolic syndrome[20]. However, cross-sectional and case-control designs cannot establish temporal precedence, whether microbial translocation precedes insulin resistance or is a consequence of it. Additionally, such designs are prone to residual confounding from diet, medication, genetics, and lifestyle. Thus, while associations are strong and biologically plausible, causality remains unproven.

Longitudinal studies-understanding temporal dynamics: Longitudinal cohort studies, which involve repeated measurements of microbial components, glycemic status, and host responses over time, are critical for establishing whether increased microbial translocation precedes and predicts incident T2DM. For example, studies such as the KORA and FINRISK cohorts have demonstrated that higher baseline levels of LBP are predictive of future T2DM development[33,177]. These prospective designs support the hypothesis that chronic exposure to microbial products is not just correlated with, but may contribute to, diabetes pathogenesis. Moreover, microbiome-wide association studies tracking microbiota composition and plasma metabolites over time can help identify microbial strains or genes responsible for producing diabetogenic components. Integration of metagenomic, metabolomic, and immunologic data from such cohorts will enhance mechanistic insight and refine risk stratification[176].

Interventional trials-proving mechanism and therapeutic potential: Trials that reduce gut permeability, alter microbial composition, or neutralize circulating microbial components can provide definitive evidence. For instance, studies using animal models have demonstrated that antibiotic-induced depletion of gram-negative bacteria reduces LPS levels and improves insulin sensitivity[96]. Similarly, prebiotic supplementation in humans reduces systemic inflammation and improves glucose metabolism, potentially via alterations in microbial activity and reduced endotoxemia[178]. Randomized controlled trials in humans that directly target microbial products, such as with TLR antagonists, dietary fibers, or FMT, are still limited but promising. Vrieze et al[179] have extensively conducted studies on FMT. However, these studies often lack long-term follow-up and mechanistic biomarkers, underscoring the need for better-designed trials.

Causal inference tools and systems biology: When genetic instruments are available, causal inference frameworks such as Mendelian randomization can be employed to infer directionality. Moreover, systems biology approaches integrating longitudinal multi-omics data can map causal pathways from microbial exposure to metabolic dysfunction. When combined with machine learning models, these strategies enhance the resolution of complex host-microbiome-disease interactions[180].

Standardizing methodologies is essential for reproducibility and clinical translation

The advancement of microbiome science and its implications in metabolic diseases such as diabetes have ushered in a new era of diagnostic and therapeutic exploration. However, a major roadblock to the clinical translation of microbiome-based findings lies in the lack of standardized methodologies for the detection, quantification, and interpretation of microbial taxa and their metabolites[181]. Variability in sample collection, DNA extraction, sequencing platforms, bioinformatics pipelines, and statistical analyses severely compromises the reproducibility and comparability of studies across laboratories and populations[182].

Challenges in sample handling and DNA extraction: Variations in storage conditions can lead to selective degradation or overgrowth of microbial species, skewing community profiles[183]. Similarly, DNA extraction methods differ in their efficiency to lyse gram-positive vs gram-negative bacteria, resulting in biased microbial representation[184]. The adoption of uniform protocols for fecal, blood, and tissue sample processing is essential to reduce batch effects and enhance cross-study reliability.

Sequencing and bioinformatics variability: HTS platforms such as Illumina, PacBio, and Oxford Nanopore each offer distinct advantages and limitations in terms of read length, accuracy, and throughput. In 16S rRNA gene sequencing, choice of hypervariable regions (e.g., V1-V3 vs V4) significantly influences taxonomic resolution and comparability[185]. In WMS sequencing, bioinformatics pipelines, ranging from reference-based (e.g., Kraken, MetaPhlAn) to de novo assembly-based approaches, yield different microbial abundance profiles, further emphasizing the need for harmonized analytical frameworks[186].

Metabolomic profiling constraints: Metabolomic analyses of microbial metabolites such as SCFAs, TMAO, and bile acids are similarly affected by technical variation. Detection platforms including gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry differ in sensitivity and specificity, and results are often influenced by factors such as sample matrix, derivatization steps, and ionization efficiency[187]. Moreover, databases for microbial metabolite identification are not universally standardized, leading to inconsistent metabolite annotations and quantifications.

Importance of cross-laboratory standardization initiatives: Efforts like the HMP and the microbiome quality control project have highlighted the necessity of benchmarking protocols and inter-laboratory calibration[188]. By distributing standardized samples across multiple centers and comparing outputs, these initiatives revealed substantial technical variability that can overshadow biological signals if not controlled[189].

Toward clinical translation: Without methodological harmonization, the transition of microbiome research from bench to bedside remains fraught with uncertainty. Diagnostic signatures or therapeutic targets identified in one cohort may not be validated in another due to underlying technical artifacts. Therefore, consensus-driven guidelines on sampling, processing, sequencing, and bioinformatics are a prerequisite for reliable biomarker development and therapeutic interventions[181].

Ethical considerations in microbiome-targeted therapies

Microbiome-targeted therapies have garnered significant attention as potential tools for addressing a range of diseases, from Clostridioides difficile infection to metabolic disorders and inflammatory bowel diseases. Among these, FMT has emerged as one of the most promising interventions. While the therapeutic potential of FMT is increasingly recognized, its rapid clinical adoption raises critical ethical, regulatory, and safety concerns that must be carefully addressed to ensure responsible and equitable use[190].

Informed consent and risk communication: One of the foremost ethical challenges in FMT and related microbiome interventions is the issue of informed consent. Patients must be made aware of the potential risks, benefits, and unknowns associated with these therapies, especially given that the long-term consequences of manipulating the gut microbiota are still poorly understood[191]. The gut microbiome influences not only gastrointestinal health but also systemic processes including immunity, neurodevelopment, and metabolism[192]. Thus, altering its composition could theoretically lead to unintended systemic effects. Clear communication regarding these possibilities is essential in obtaining valid informed consent.

Donor screening and safety: Ensuring the safety of microbiome-based interventions depends heavily on rigorous donor screening protocols. FMT involves the transfer of complex microbial communities, and current standards for screening are not globally harmonized[193]. Inadequate screening has resulted in severe adverse outcomes, including the transmission of multi-drug-resistant organisms[146]. Ethical implementation of FMT requires stringent, transparent, and universally accepted screening guidelines to protect recipients from avoidable harm.

Therapeutic misuse and access inequities: With increasing media and commercial interest, some individuals seek unsupervised FMT procedures. These practices pose considerable health risks and underscore the need for ethical oversight and public education[194]. Moreover, disparities in access to regulated microbiome therapies could widen existing health inequities. For example, while FMT is widely available in high-income countries for recurrent Clostridioides difficile infection, patients in low-resource settings may face limited access despite high disease burden. Ensuring ethical equity in the deployment of microbiome therapies requires proactive policy frameworks and cost-effective implementation strategies[195].

Regulatory oversight and classification: How microbiome therapies, particularly FMT, should be classified, whether as biological drugs, tissue transplants, or novel therapeutics, has been debated. In the United States, the Food and Drug Administration currently regulates FMT as an investigational new drug, allowing its use under enforcement discretion for recurrent Clostridioides difficile infection unresponsive to standard therapies[196]. This regulatory grey zone introduces challenges in ensuring product consistency, quality control, and reporting of adverse events. Establishing a unified classification system is crucial for ethical oversight, safety monitoring, and promoting evidence-based use[197].

Long-term monitoring and ethical responsibility: Because microbiome interventions may induce persistent changes in host physiology, long-term monitoring is ethically necessary. Recipients of FMT or other microbiome therapies should be followed systematically to assess efficacy, identify delayed adverse effects, and understand durability of response[198]. Such monitoring requires infrastructure, funding, and ethical commitment to post-treatment surveillance, which are often lacking in current clinical practice.

Ownership and commercialization of microbial data: The commercialization of gut microbiome data and donor material also raises ethical questions. As companies begin to patent specific microbial strains or formulations, issues of intellectual property, benefit sharing, and commodification of human-derived biological material become increasingly salient[199]. Ethical microbiome research and therapy development must ensure transparency, fair compensation for donors, and protection of privacy for individuals whose microbiota are being studied or used therapeutically.

CONCLUSION

The emerging evidence linking gut microbial dysbiosis and circulating microbial components with metabolic dysfunction underscores the need for deeper mechanistic and translational research in diabetes. First, standardization of circulating microbiome detection methods, including sample processing, contamination control, sequencing depth, and analytical pipelines, is essential to ensure reproducibility and comparability across studies. Second, establishing causal relationships between circulating microbial DNA and metabolic endpoints remains a major scientific priority; longitudinal cohorts, multi-omics integration, and interventional models will be critical to determine whether microbial products act as biomarkers, mediators, or both. Finally, advances in microbial profiling, metabolomics, and host-microbiome interaction studies open the door to personalized microbiome-based therapies for diabetes, including precision probiotics, targeted metabolite modulation, and individualized dietary interventions. Together, addressing these knowledge gaps will accelerate the translation of microbiome science into clinically meaningful strategies for the prevention and management of diabetes.