Research progress on adaptive modifications of the gut microflora and regulation of host glucose and lipid metabolism by cold stimulation

2.1 The gut microflora affects the metabolism of glucose

The gut microflora chemically transforms exogenous substances^[23], and affects their half-life and bioavailability by altering their chemical structures. Having been ingested, part of carbohydrates are absorbed by small intestine, and the remaining part are absorbed in large intestines after modification by enzymes secreted by gut microbes. Numerous enzymes associated with this process, such as hydrolases, lyases, oxidoreductases, and transferases, are widely present in the gut microflora^[24,25,26]. The gut microflora uses these metabolic enzymes to hydrolyze or reduce heterologous substances and then enters the glucose metabolism^[23]. Glucose tolerance reflects body's regulatory capacity of glucose. When body is in abnormal conditions, the metabolic capacity of the gut microflora is weakened, which is manifested by impaired glucose tolerance. For example, one study investigated the gut microflora of patients with impaired glucose tolerance versus those with normal glucose tolerance and found that those with impaired glucose tolerance had higher levels of Erysipelotrichaceae and Lachnospiraceae that were positively associated with metabolic disorders^[27]. Type 2 diabetes mellitus (T2DM) is a highly prevalent metabolic disease with glucose metabolism disorder caused by insulin dysfunction as the most obvious clinical manifestation is. A research group in China performed high-throughput 16S rDNA sequencing of the intestinal flora of T2DM patients and found that the gut microflora was significantly correlated with T2DM, with the microflora altering with the progression of the disease^[28]. In this study, the subjects were divided into three groups according to their glucose tolerance status: normal glucose tolerance (NGT), early T2DM group and T2DM group. From the NGT to the early T2DM group, the relative abundance of Verrucomicrobia decreased while the relative abundance of Betaproteobacteria increased, and the abundance of Betaproteobacteria further increased in the T2DM group. The relative abundance of Streptococcus continuously decreased from NGT to the early T2DM group and to the T2DM group. The relative abundance of Clostridium in the T2DM group was higher than that in the NGT and early T2DM groups. The abundance of Bacteroides and Clostridium experienced significant fluctuations along with the progression of T2DM. As a result, the gut microflora might be a potential biomarker for diabetes diagnosis^[28]. Metformin is one of the most widely used drugs for treating T2DM nowadays, and it works primarily by transiently blocking the mitochondrial respiratory-chain complex I, thereby stimulating glucose consumption and decreasing hepatic glucose output^[29–30]. Interestingly, a study has reported that gut microbes work together with metformin to regulate blood glucose. When obese mice with metabolic disorders were treated with metformin, eighteen signaling pathways in the gut microbiome were found activated, most of which were associated with glucose metabolic processes^[31]. The above studies suggest that some specific microflora in intestines, such as β-streptococcus and Clostridium, may adjust their relative abundance to adapt to the change of the body or compensate for the impaired glucose tolerance.

The influence of microflora on glucose metabolism is not only limited to intestine but is also reflected on the interaction of the brain-intestine axis. Gut microbes initiate the signal transduction and communications between the gut and the central nervous system by activating the vagus nerve^[32], regulating the homeostasis of microglia^[33], and influencing tryptophan metabolism^[34]. Glucose homeostasis is also an important factor in maintaining cell viability. When digestion process is affected and glucose metabolism is disturbed, the intestinal endocrine cells send signals to the central nervous system through the endocrine pathways, which directly act on the hypothalamus and the pituitary region or affect the central nervous system through the vagus nerve, thereby regulating the brain-intestinal axis to ensure normal digestion, absorption, and energy production and to maintain glucose homeostasis^[35].

2.2 The gut microflora affects the metabolism of lipid

The prevalence of obesity and the associated metabolic diseases is now a public health problem in many countries. Obesity is associated with lipid metabolic disorder, which then leads to abnormal blood lipid levels, ectopic lipid deposition, and other related symptoms^[36–37]. Lipid metabolism includes the biosynthesis and degradation of lipids including fatty acids, triglycerides, and cholesterol, and the gut microflora regulates these metabolic processes^[38]. Lipid dysregulation is found associated with the undesirable alterations of the composition of the gut microflora. Researchers tracked the gut microflora of obese preschool children and found that the composition of gut microflora in obese children changed significantly with increasing proportion of harmful bacteria such as Proteus and decreasing proportion of beneficial bacteria such as Bifidobacterium^[39]. Lipidomic analysis of regular dietary germ-free and commonly fed mice showed that the gut microflora was able to influence the lipid components in host tissue and serum^[40]. Metabolomics analysis of obese people suggested that total serum cholesterol and triglyceride levels were associated with the total genomic gene abundance in gut microbiome of obese people^[41]. It is also found that individuals with fewer gut microbial genes had higher triglyceride levels and lower high-density lipoprotein levels^[42]. In obese mice, the diversity of gut microbes decreased, while the overall number of bacteria increased. After transplantation of gut microbes of obese and lean mice (mice with a leaner body size and less fat content) into normal germ-free mice, mice transplanted with the gut microbes from the obese produced 2% more energy after consuming the same amount of food than those transplanted with the gut microbes from the lean, suggesting that the obesity-associated gut microflora confers richer energy from the diet to the host^[43].

The gut microflora affects not only the host energy intake by changing its composition, but also the synthesis of short-chain fatty acids (SCFA), an important substrate involved in lipid metabolism including lipogenesis, gluconeogenesis and cholesterol synthesis^[44]. SCFA including acetic acid, propionic acid, and butyric acid are produced when dietary fiber is decomposed and fermented. Dietary fiber is positively correlated with the intestinal IgA level^[45], implying that dietary fiber or its metabolites has the potential to support the B lymphocyte response in the gut. As the main microbial metabolite of dietary fiber, SCFA play a role through a variety of mechanisms, such as activating cell surface receptors, inhibiting histone deacetylase, and the like. Besides, SCFA can enhance the function of acetyl-CoA in B lymphocytes, contributing to oxidative phosphorylation, glycolysis, and fat synthesis^[46]. SCFA can bind to specific G protein-coupled receptors (GPCRs) which are involved in the regulation of lipid and glucose metabolisms^[47]. Free fatty acid receptors Ffar2 and Ffar3 are two common receptors for SCFA binding, and SCFA-Ffar regulates the balance among oxidation, synthesis, and breakdown of fatty acids in vivo^[48]. In addition to the SCFA-Ffar signaling pathway, the AMP-dependent protein kinase (AMPK) signaling pathway also plays an important regulatory role in lipid and glucose metabolisms^[49]. The above studies suggest that the gut microflora participates in host lipid metabolism by promoting the production of SCFA from dietary fiber, and then regulates the balance among body fat synthesis and fatty acid oxidation, synthesis, and decomposition.

Gastrointestinal hormones, receptors of the central nervous system, and neuropeptides regulate lipoprotein metabolism through the sympathetic and parasympathetic signaling pathways^[50]. The gut microflora acts on the gut-central nervous system through the production of neurotransmitters to maintain the metabolic balance. One study showed that transplantation of multiple gut microbiomes in germ-free mice promoted the maturation of microglia. And some bacterial strains including Bacteroides distasonis, Lactobacillus salivarius, and Clostridium cluster XIV were able to alter the synthesis of neurotransmitters such as γ-aminobutyric acid, serotonin, dopamine, and norepinephrine, which may affect the activation of microglia in the brain and some brain functions^[51], and those neurotransmitters are involved in lipid metabolism regulation through the endocrine system of the intestine epithelium and the brain-intestinal axis^[52].

2.3 Gut microflora affects mitochondria

Mitochondria are involved in almost all processes of the life cycle, and as energy providers, mitochondria's role in glucose and lipid metabolism cannot be ignored. The gut microflora affects mitochondrial metabolism in certain ways. As a by-product of mitochondrial oxidative phosphorylation, reactive oxygen species (ROS) may be key signaling factors for interactions between mitochondria and gut microbes^[53]. Gut microbes produce short-chain fatty acids including acetic acid and butyric acid, of which acetate can enhance mitochondrial respiration and improve the defense ability of the body's intestinal epithelial cells^[54], while butyric acid can stimulate mitochondrial biogenesis, improve cell respiration capacity, and activate antioxidant enzymes^[55–56]. Enhanced mitochondria biogenesis has a positive regulatory effect on glycolipid metabolism^[57]. Therefore, the influence of the gut microflora on mitochondria is directly related to life activities such as glucose and lipid metabolism.

3.1 Cold exposure alters the composition of the gut microflora

Researchers found that long-term cold exposure alters the composition of the gut microflora. Cold-exposed mouse fecal microbes showed significant changes in phylum abundance compared to room temperature controls. The Verrucomicrobia phylum was almost eradicated (from 12.5% to 0.003%), and the Bacteroides and Firmicutes phyla became two predominantly flora in the gut microflora^[59], within which the proportion of Firmicutes increased from 18.6% to 60.5% while that of Bacteroides decreased from 72.6% to 35.2%, and the ratio Firmicutes/Bacteroidetes increased significantly^[11]. These findings are supported by another study based on 16S RNA sequencing of the microflora of rat exposed to 4°C^[60].

3.2 Cold exposure affects the function of the gut microflora

4.1 Cold exposure regulates glucose and lipid metabolism in the liver

As the main organ for adaptive regulation of metabolism, the liver maintains a stable state of glucose and lipid metabolism. Hepatic glycogen consumption increases during acute cold exposure, and expression of the key enzymes required for hepatic glycogen synthesis increases at the same time^[72]. Liver glycogen of rats was depleted in four hours under the −8°C cold exposure condition, and liver edema and other pathological manifestations quickly developed after cold exposure at lower temperatures such as −20°C^[73–74]. Liu et al.^[75] found that after 0 hour, 2 hours, 4 hours, and 6 hours cold exposure at 4°C, the blood glucose level of C57BL/J mice significantly decreased, while the expression of cold inducible RNA binding protein (CIRP) increased, and the CIRP expression level increased with prolonged duration of cold exposure. The AKT pathway is one of the important regulatory pathways for liver injury protection mechanism^[75]. The CIRP regulates liver glucose metabolism in mice through the AKT signaling pathway to maintain hepatocyte energy balance^[76]. Glucose metabolism and lipid metabolism in the liver are closely related. Wei et al. studied the effect of cold exposure duration at 4°C on the relevant indicators of liver lipid metabolism and found that cold exposure reduced the content of liver triglycerides and cholesterol, downregulated the expression of liver lipogenesis genes such as fatty acid synthase (FASN), and up-regulated the expression of liver gluconeogenic genes such as Pgc-1α and G6p^[77]. However, reports of elevated liver triglyceride levels due to cold exposure are at odds with other results of different cold exposure lengths (4 hours, 24 hours, and 10 days, etc.)^[78–79], which may be due to differences in cold exposure conditions or fasting conditions of mice. Taken together, cold exposure leads to accelerated liver glucose metabolism and decelerated lipid metabolism, which may be a protective mechanism against external environmental stimuli.

4.2 Cold exposure regulates glucose and lipid metabolism in adipose tissue

Mammals fight against the loss of heat in cold environments by increasing thermogenesis, and as one of the important tissues for storage and conversion of energy, adipose tissues play an important role in the process of energy metabolism. Adipose tissues typically include white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is an important substance regulating homeostasis of energy by storing energy in the form of triglycerides (TGS), whereas BAT is the main non-shivering thermogenic tissue by breaking down lipids to produce heat, which is mediated by tissue-specific UCP1 in BAT mitochondria^[80]. Cold stimulation activates BAT, up-regulates UCP1 expression, increases thermogenesis, and regulates energy expenditure^[81]. BAT activation is significantly associated with triglyceride-free fatty acid (FFA) cycling, FFA oxidation, and insulin sensitivity in adipose tissue^[82]. At the same time, cold exposure can induce the browning of white adipocyte to beige adipocyte which has similar characteristics and functions to BAT, and the browning of white adipocyte can significantly increase the body energy consumption^[83–84]. Activating BAT and promoting WAT browning may be new strategies for the treatment of metabolic diseases. Orava et al. explored the role of BAT in human lipid mobilization and clearance by means of isotopic tracing^[85]. The results showed that in BAT compared with in WAT the gene expression associated with lipid metabolism in cold induction was up-regulated, and fat mobilization as well as lipid oxidation was accelerated^[85]. In cold stress, the function of thermogenic tissues including BAT are influenced by sympathetic and thyroid hormones, therefore the glucose and lipid metabolisms of BAT are regulated in synergy with the hypothalamic-pituitary-thyroid axis (HPT axis)^[86].

4.3 Cold exposure regulates glucose and lipid metabolisms in skeletal muscles

Skeletal muscles are an important organ where in active glucose metabolism occurs through decomposing glycogen. Specifically and mechanistically, skeletal muscles regulate glucose metabolism during cold exposure by activating peroxisome proliferator-activator receptor γ coactivator-1α (PGC-1α) which in turn modulates muscle glucose uptake and insulin sensitivity. This process is mediated by the insulin receptors (IR)/serine/threonine kinases (AKT) and AMPK-dependent oxidative phosphorylation signaling pathways^[87]. But there are still many controversies as to the overall regulatory pathways involved. Muscle proteins are the most important component of skeletal muscle, and the rate of muscle protein synthesis in rats exposed to cold was significantly reduced, which may explain the decrease in protein quality following long-term cold exposure^[88]. Manfredi et al. observed a decrease in the rate of protein synthesis and an increase in the rate of decomposition of the cataracts muscle in mice after 24 hours cold exposure at 4°C. Meanwhile, the sympathetic nervous system was activated, and a sharp increase in plasma glucose, cortisol, and thyroid hormone levels as along with a decrease in plasma insulin levels were also observed^[89]. Notably, short-term cold exposure may alter the fatty acid composition of fatty-infiltrating skeletal muscles (intramuscular fat infiltration) by directly affecting the lipid metabolism pathways, including the phosphatidylinositol kinase (PI3K-AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. For example, saturated fatty acids of mice exposed to 4°C cold for 3 days were reduced by 6.4% compared with counterparts raised at room temperature. Additionally, the expression level of genes associated with fat and energy metabolism including Leptin (Lep), stearyl-CoA desaturase genes 1 (Scd1), uncoupling protein factors 2, 3 (Ucp2, Ucp3) and cytochrome c oxidase 5A (Cox5a) were increased in skeletal muscles. Especially, the myogenic differentiation factor 1 (Myod1), a muscle differentiation marker, was also significantly increased after cold exposure^[90]. However, the results from another study showed a 28% reduction of the protein content in gastrocnemius and flounder muscles following 20 days of cold exposure compared to the control group, and there were no significant differences in protein synthesis and degradation index of skeletal muscles in cold-exposed rats, including cold stress (5 days of cold exposure) and cold acclimatization (20 days of cold exposure), compared to control ones kept in normal conditions^[91]. The specific causes for the observed alterations in this stress model remain unclear, and the alterations of muscle protein metabolism following cold stress also remain controversial. The signaling pathways regulating the glucose and lipid metabolism processes of skeletal muscles during cold exposure merit further studies.