The gut microbiota acts as a real organ, which can generate monosaccharides and SCFAs by hydrolyzing and fermenting dietary polysaccharides from the host, including acetate, propionate, and butyrate. The mechanism involving the production of adipose-tissue-derived satiety hormone leptin under the action of SCFAs is the most studied[24]. Nevertheless, recent studies have illustrated that SCFAs act against obesity by preventing fat accumulation. The production of SCFAs is different in obese and insulin-resistant subjects via regulation of metabolism in several organs and tissues, for instance, adipose tissue, the pancreas, and the brain[25,26]. The increased level of butyrate is linked to improved insulin sensitivity[27,28], highlighting the significance of butyrate-secreting bacteria in modulation of glycemia. By further analyzing stool samples from patients, Psichas et al[29] illustrated that high rates of colonic fermentation were closely related with high SCFA products in obese individuals. Therefore, the effect of SCFAs on energy metabolism is multidimensional, including involving lipid oxidation, appetite modulation, as well as glucose metabolism (Figure 1)[24].

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Figure 1 Mechanisms of action of short-chain fatty acids in type 2 diabetes mellitus. The effect of short-chain fatty acids on energy metabolism is multidimensional, including appetite regulation, energy intake/expenditure, and lipid oxidation, as well as glucose metabolism. SCFA: Short-chain fatty acid; GLP-1: Glucagon-like peptide 1; PYY: Peptide YY; ER: Endoplasmic reticulum; UCP-1: Uncoupling protein-1; mTOR: Mammalian target of rapamycin.

Several studies have shown that SCFAs trigger the secretion of Peptide YY (PYY) along with glucagon-like peptide 1 (GLP-1), by recognizing and stimulating G-protein-coupled receptor (GPR)41/43 in rodent and human cell lines[30-32]. Similarly, the same effect and mechanisms have been documented and confirmed in vivo[33]. Moreover, numerous studies have demonstrated that exogenous GLP-1 and PYY acutely reduce food intake in humans, which has been widely used in the treatment of T2DM[34-38]. Therefore, SCFAs not only contribute 5%-10% of energy to the host, but are also recognized by endogenous ligands of GPR41/43, and act as signaling molecules to participate in adjustment of energy[39-41]. Kimura documented that GPR43-modulated adipose-insulin cascades and sympathetic activity were controlled by GPR41, which detected SCFAs released from gut microbiota[42]. Nevertheless, in GPR41/FFAR3^-/- and GPR43/FFAR2^-/- knockout cells, GLP-1 secretion activated by SCFAs was remarkably attenuated. However, unlike GPR41/ FFAR3^-/- mice, GPR43/FFAR2^-/- showed markedly downregulated GLP-1 in circulation, indicating that GPR43/FFAR2 plays a more important role in these effects. Confusingly, the level of satiety hormones at 24 wk following supplementation of inulin propionate ester at 10 g/d was not different from those in groups treated with 10 g/d inulin alone in overweight adults[43], emphasizing mechanisms of crosstalk between the gut microflora and host. Therefore, SCFAs, as metabolites of the gut microflora, are important signaling molecules in the regulation of host energy metabolism. Relevant research on the metabolic influences of SCFA delivery or production on the host is urgently needed, especially the influences on satiety-inducing hormones.

Besides, in rodent receiving acute and chronic oral supplementation of SCFAs, besides affecting the production of satiety hormones, SCFAs could also advantageously impact body weight via impacting energy expenditure[44]. Another study documented that a single oral administration of 1.5% AcOH with a stomach tube, in comparison to distilled water, elevated energy expenditure along with lipid oxidation[45]. Similar to the above results, injection with acetate (5.2 mg/kg) elevated whole-body oxygen consumption and decreased the body weight at 6 mo post-treatment in rats[46]. Moreover, butyrate (5% w/w) prevented high-fat diet (HFD)-induced obesity via enhanced lipid oxidation[47]. Studies on the mechanism showed that these effects were linked to the UCP-1 (uncoupling protein-1) and elevation of peroxisome proliferator-activated receptor-(PPAR)-γ co-activator 1α (PPARGC1A, coding for PGC1α) in brown adipose tissue[47]. The animal experimental data proved that SCFAs upregulate genes that modulate lipid oxidation and thermogenesis, thereby eliminating adiposity and weight gain[24]. Human studies have indicated that colonic infusion of SCFA mixtures, including acetate, butyrate, and propionate, reduces lipolysis and elevates energy expenditure and PYY, as well as fat oxidation in overweight/obese individuals[48]. In addition, acute oral sodium propionate ingestion elevated resting expenditure of energy along with lipid oxidation in 18 healthy volunteers in contrast with a sodium chloride control, and these effects were independent of insulin and glucose contents and sympathetic nervous system activity[49]. The result from a randomized double-blind crossover trial proved that sodium acetate infusion into the distal colon (180 mmol/L) enhanced lipid oxidation relative to sodium chloride placebo, and the resting energy expenditure between overnight-fasted overweight and obese individuals was similar[44]. Jocken et al[49] showed that SCFAs reduced lipolysis and promoted lipid oxidation in white adipose tissue (WAT), and that antilipolytic effect was orchestrated by FFAR3 and/or FFAR2 levels in WAT[50]. Therefore, these results strongly suggest that SCFAs are beneficial for weight control and influence energy expenditure. Research on the mechanism of action of SCFAs in DM has suggested that SCFAs induce GLP-1 and amylin secretion via FFA2 receptor, hence modulating glucose metabolism and insulin levels[51]. The Akt/mTOR pathway, ER stress, and release of intracellular Ca²⁺ play important roles in this process[51]. There is need to understand the pathways and regulators involved in a variety of cell models, for instance, human-derived adipocytes, hepatocytes, or skeletal muscle.

BCAAs have increasingly been studied as playing a role in diabetes[52]. BCAAs constitute nearly approximately 20% of the amino acids used to form proteins[53]. More studies have documented that plasma content elevations of BCAAs have been linked to obesity, as well as diabetes[54]. Previously, this phenomenon was thought as a consequence and not a cause of IR[55]. Nevertheless, recently, growing research evidence opines that BCAAs elevations contribute to IR: (1) Exogenous BCAAs remarkably diminish the sensitivity to insulin, as illustrated via hyperinsulinemic euglycemic clamps measurements[56,57]; and (2) coadministration of BCAAs with HFD generally worsens the ensuing IR in rodents[58]. The presence of HFD or lipids in all these rodents played a critical role in this effect, and BCAAs alone elicited insignificant or no impact, illustrating that BCAAs crosstalk with fatty acids (FAs) to promote IR[59]. Further studies have proved that FA oxidation disorders elevated BCAAs contents in plasma, and aggregation of the intermediate metabolites of BCAAs, for instance, C3 and C5 acylcarnitines along with acetyl-CoA can inhibit complete FA oxidation[60]. The crosstalk of BCAAs with FAs induces energy metabolism disorder, including ATP production, TCA (tricarboxylic acid cycle) cycle, as well as oxidative phosphorylation, and leads to mitochondrial dysfunction and inflammation, which are critical for the progression of DM[61-63]. Inflammatory factor signaling pathways, including the nuclear factor (NF)-κB pathway and mammalian target of rapamycin complex 1 (mTORC1), might be candidate therapeutic targets in this process[54,64]. Moreover, BCAAs supplementation could repress stimulation of Akt2 via the mTORC1- and mTORC2-dependent cascades and enhanced degradation that is dependent on Akt2 ubiquitin-proteasomes via mTORC2 signaling, indicating that mTORC2 might also play an important role in this process[65]. More importantly, by using metagenomics and metabonomics methods, increased concentrations of BCAAs were linked to a gut microbiome that has an abundant biosynthetic ability for BCAAs. Prevotella copri coupled with Bacteroides vulgatus are recognized as the primary species modulating the relationship of biosynthesis of BCAAs with IR[66], indicating that gut microbes affect host serum metabolome along with insulin sensitivity via regulating the level of BCAAs (Figure 2).

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Figure 2 Increased plasma levels of branched-chain amino acids induced by dysbiosis are closely associated with obesity and diabetes. The accumulation of branched-chain amino acids inhibits complete oxidation of fatty acids, induces energy metabolism disorder, including ATP production, tricarboxylic acid cycle, and oxidative phosphorylation, thereby causing energy metabolism disorder, and induces inflammation by targeting nuclear factor-κB and mammalian target of rapamycin complex 1. TCA: Tricarboxylic acid; NF-κB: Nuclear factor-κB; mTORC1: Mammalian target of rapamycin complex 1; BCAAs: Branched-chain amino acids.

Terrestrial mammal trimethylamine N-oxide (TMAO) is derived from exogenous arsenals, with the TMA serving as the precursor, which is a metabolite of diverse other precursors, primarily choline, as well as carnitine originating from ingested foods[67]. Bacteria metabolize choline and L-carnitine into TMA, and flavin-containing monooxygenase (FMO)-3, a hepatic enzyme, oxidizes TMA into TMAO. There are two key steps for the generation of TMAO, illustrating that the gut microflora is an independent risk factor for DM[68,69]. Further analysis of the microbiota has shown that the primary bacterial phylum that degrades carnitine to TMA is Proteobacteria along with bacteria of the family Prevotellaceae, phylum Bacteroidetes. In contrast, the S24-7 family of Bacteroidetes (the family majorly involved in plant polysaccharides) is related with diminished TMAO contents[70-72].

The initial findings suggested a positive relationship of high plasma TMAO content with an elevated risk for major severe cardiovascular diseases including myocardial infarction, stroke, and atherosclerosis. More studies have documented that TMAO is opined to serve as a biomarker, as well as an independent predisposing factor for many diseases, for example, kidney failure, DM, and cancer[69]. Some research evidence opines that TMAO influences glucose metabolism, and remarkably higher median TMAO contents in plasma occur in individuals with diabetes in contrast with persons without DM[73,74]. A prospective mechanism connecting TMAO with IR is TMAO-dependent elevated concentrations of N-nitroso compounds and upregulated activity of FMO-3[75,76]. Research documents reduced TMAO and choline contents in the hepatic tissues of diabetic mice[77]. Metformin has been shown to decrease glucose and increase plasma TMAO[77]. TMAO measurements have low diagnostic significance in diabetic individuals who are obese due to the high variability of TMAO contents in plasma[78]. More importantly, treatment with TMAO promotes normal protein folding, counteracting ER stress in diabetic rats, illustrating a potentially beneficial impact of TMAO in DM[79]. Therefore, from the results so far, the characteristics of TMAO in DM are still controversial.

Dietary fibers are compounds of natural origin present in plants. Chemically, these compounds are defined as nondigestible carbohydrates (with ≥ 3 monomeric units), for instance, polysaccharides and oligosaccharides[114]. Some prospective cohort studies have shown that individuals with high intake of dietary fiber are inversely linked to the risk of DM compared with low intake[115]. Although most dietary fibers belong to prebiotics, which are not digested and absorbed by the human gut and remain intact while passing through the gastrointestinal tract, they selectively arouse the growth and activity of potential beneficial bacteria in the gut. The human gut microflora encodes several types of carbohydrate-active enzymes, including glycoside hydrolases, polysaccharide lyases, glycosyltransferases, and carbohydrate esterases, which are capable of degrading dietary fiber and then generating small-molecular-weight metabolites (degradation products), which may display antidiabetic effects in T2DM[116]. Studies on anti-T2DM activity of fibers have shown that dietary fibers increase the abundance of some species, for instance, Eubacterium rectale, Roseburia, Prevotella, Ruminococcus bromii, Bacteroides, and Bifidobacterium, and decrease the number of some Gram-negative bacteria, for instance, Desulfovibrio and Enterobacteriaceae (LPS-producing bacteria)[117]. More importantly, these changes induced by dietary fiber intervention enhance the production of SCFAs, which can bind to the GPR and enhance the level of the enteroendocrine hormones PYY and GLP-1 in gut epithelial L-cells, thereby improving IR, appetite regulation, and energy intake/ expenditure, as well as lipid oxidation[118]. The alleviating effect of pumpkin polysaccharide in HFD-fed mice is linked with increased SCFA production and selective enhancement of some bacteria, for instance, Bacteroidetes, Prevotella, and Deltaproteobacteria[119]. Administration of inulin can reduce the fasting blood glucose level, increase GLP-1 level, and alleviate glucose intolerance as well as blood lipid contents in rats with T2DM induced by HFD and streptozotocin[120]. SCFA-producing bacteria have a key role in the process, including Lachnospiraceae, Phascolarctobacterium, and Bacteroides[120]. A double-blind, randomized, controlled clinical trial of 60 patients with T2DM found that supplementation with 10 g/d inulin powder promoted gut health by increasing the proportion of Akkermansia muciniphila[121].

Dietary polyphenols are natural compounds that occur in many plant foods, such as fruits and vegetables. These compounds constitute a large heterogeneous collection of compounds, but with structural units common to all phenolic compounds (hydroxylated aromatic rings or phenol rings)[122]. Numerous studies have proved that the beneficial effects of dietary polyphenols may reduce the risk of T2DM and/or its complications[123]. However, it is proved that most polyphenols are not digested and absorbed by the small intestine, but remain in the colon, and are metabolized by gut microflora including demethylation, dihydroxylation, and decarboxylation[124]. Focusing on the changes of the intestinal microbiota, polyphenols improve intestinal health by promoting the growth of beneficial bacteria and inhibiting the pathogenic bacteria[117]. More importantly, the main antidiabetic actions of dietary polyphenols include: Protection of pancreatic β-cells against stimuli-induced oxidative stress; inhibition of the activities of various enzymes (for instance, α-amylases, α-glucosidases, and pancreatic lipase); promotion of β-cell proliferation and survival; and repression of advanced glycation end products formation[117]. An ethanolic extract of Lessonia nigrescens (rich in phenolics and flavonoids) displays its hypoglycemic effect by increasing the abundance of Bacteroidetes and decreasing Firmicutes in the intestines[118]. A recent study illustrated that the antidiabetic effects of polyphenols were also linked to changes in the markers of gut barrier function, for instance, ZO-1 and occludin[125]. Grape pomace extract (mixture of polyphenols consisting of anthocyanins, flavanols, and flavanol glycosides) improves fat mass gain, adipose tissue inflammation, impaired glucose tolerance, and IR by reducing the concentrations of Clostridium sensu stricto, Lactococcus, Desulfovibrionaceae, and Streptococcaceae, and increasing the abundance of Allobaculum, Prevotellaceae, Roseburia, and Erysipelotrichaceae, and improving gut barrier function[126]. Besides these extracting mixtures, the effects of several polyphenol monomeric compounds in T2DM/obesity are also closely linked to the alternation of gut microflora. Resveratrol attenuates HFD-induced nonalcoholic steatohepatitis and ameliorates the intestinal barrier dysfunction and inflammation in rats[127]. Quercetin reverted gut microflora imbalance and related endotoxemia-mediated TLR4 pathway induction, with subsequent repression of inflammasome response and reticulum stress pathway activation, leading to the blockage of lipid metabolism gene expression deregulation in obese mice[63].

Alkaloids have antimalarial, antihyperglycemic, antiasthma, anticancer, and antibacterial activities[128]. Many of them have been utilized in traditional or modern medicines for drug discovery. Recent studies have illustrated that the pharmacological activity of alkaloids is mainly mediated by the gut microflora[129]. Because most alkaloids usually exhibit low oral bioavailability, their absorption into the bloodstream is difficult. The gut microbiota has a variety of enzymes, consisting of β-glucuronidase, β-glucosidase, β-galactase, nitroreductase, azoreductase, 7α-hydroxylase, and protease, and various carbohydrates, which can metabolize alkaloids into many different metabolites that are closely linked to DM[130]. Capsaicin improves glucose homeostasis and insulin tolerance in obese diabetic ob/ob mice by increasing the ratio of Firmicutes to Bacteroidetes and the number of Roseburia, which could decrease the contents of proinflammatory cytokines, for instance, TNF-α and IL-6[131]. Among the alkaloids, the most widely studied is berberine. As an isoquinoline alkaloid, berberine occurs in various medicinal plants, including Coptis chinensis Franch and Phellodendron chinense Schneid. Numerous experimental models have proved that the antiobesity and anti-hyperlipidemic effects of berberine are closely related to changes in the gut microbiome[128]. Researchers have shown that the blood level of BBR in hyperlipidemic patients was higher than that in healthy individuals owing to the differential microbiota composition[132]. Further studies have shown that the effects of berberine on DM are multidimensional: (1) Modulation of the ratio of Firmicutes to Bacteroidetes, thereby increasing SCFA content in feces[133]; (2) Regulation of BCAA biosynthesis and catabolism in liver and adipose tissue[134]; (3) Reduction of the increased expression of inflammatory mediators by decreasing LPS level in plasma and alleviation of gut permeability[135]; and (4) Modulation of the BA cycle and subsequently the ileal FXR signaling pathway[136].

In summary, numerous natural products, for instance, fruits, vegetables, and medicinal plants, possess prebiotic effects and have been illustrated to ameliorate T2DM by modulating gut microflora composition and abundance, reducing gut permeability, and subsequently increasing production of SCFAs and BCAAs, decreasing the level of LPS, and inhibiting inflammation. Current studies mainly focus on modulating the action of natural products and their bioactive components on the gut microbiota for preventing and managing T2DM. However, because the composition of natural products is so complex that the gut microflora may also influence host metabolism of natural products, further studies should focus on the metabolism of natural products and their bioactive components by the gut microbiota. This is important for the pharmacokinetic parameters, enhancing drug efficacy, and finding a novel lead compound via gut microflora-related mechanisms.