The Role of Gut Microbiota in High-Fat-Diet-Induced Diabetes: Lessons from Animal Models and Humans

The number of diabetes mellitus patients is increasing rapidly worldwide. Diet and nutrition are strongly believed to play a significant role in the development of diabetes mellitus. However, the specific dietary factors and detailed mechanisms of its development have not been clearly elucidated. Increasing evidence indicates the intestinal microbiota is becoming abundantly apparent in the progression and prevention of insulin resistance in diabetes. Differences in gut microbiota composition, particularly butyrate-producing bacteria, have been observed in preclinical animal models as well as human patients compared to healthy controls. Gut microbiota dysbiosis may disrupt intestinal barrier functions and alter host metabolic pathways, directly or indirectly relating to insulin resistance. In this article, we focus on dietary fat, diabetes, and gut microbiome characterization. The promising probiotic and prebiotic approaches to diabetes, by favorably modifying the composition of the gut microbial community, warrant further investigation through well-designed human clinical studies.


Introduction
Diabetes, the epidemic of the 21st century, has become one of the major threats to human health and has greatly increased the global burden of the disease [1,2]. The development of diabetes is associated with a number of factors, including excessive dietary intake, genetics, and a sedentary lifestyle [3]. Dietary composition is an important factor influencing the risk of developing diabetes [4], and the quantity and/or quality of dietary fat in diabetes have attracted considerable interest. Dietary fat, especially saturated fatty acid, has been considered to be an unhealthy dietary component due to its high energy density [5,6]. The excessive intake of dietary fat is thought to be associated with obesity and metabolic disorders [7], and the relationship between high-fat diets (HFDs) and diabetes has received extensive attention in past studies. Studies have confirmed that 59% of safflower oil can lead to insulin resistance in rats [8]. With HFD supplementation, beta cell senescence leads to a reduction in insulin release [9].
Gut microbes refer to microbiota present in the gastrointestinal tract and are associated with energy harvesting and storage and the metabolism of many metabolic functions, such as amino acids and carbohydrates [10,11]. Gut microbes are affected by diet, and when mice were shifted to a high-fat, high-sugar diet, the structure of the microbiota was altered within one day [12]. An HFD of 60% lard and soybean oil resulted in a decrease in Bacteroidetes and an increase in Firmicutes and Proteobacteria in mice [13]. Imbalances in gut microbes are associated with metabolic diseases such as obesity and diabetes through mechanisms such as increasing the amount of energy obtained from the diet, affecting fatty acid metabolism in the liver and adipose tissue, and increasing serum concentrations of branched-chain amino acids causing insulin resistance [14][15][16].
grain intake was negatively associated with type 2 diabetes [53]. With a long-term HFD, a lack of exercise, genetics, and aging, the body becomes metabolically disturbed, and the balance of blood glucose in the body is disturbed, causing an increase in blood glucose and leading to the development of type 2 diabetes. An HFD is currently one of the main methods of inducing diabetes in rodent models, and HFD-induced diabetes in rodents is associated with weight gain, hyperglycemia, insulin resistance, hyperinsulinemia, and accumulation of lipids [50]. An HFD can lead to hyperglycemia, insulin resistance, and damage to pancreatic beta cells by affecting glucose and lipid metabolism in the metabolic organs [51].

HFD-Induced Diabetes in Animal Models and Humans
An HFD leads to fat accumulation and increased blood sugar, causing insulin resistance and beta cell damage, causing diabetes in humans [30]. The ethical aspects of human research have necessitated the development of animal models of diabetes. Animal models of diabetes can better explore the pathogenesis of diabetes and help to reveal the pathogenesis of diabetes. Currently, common models of diabetes include rodents, non-human primate models, large animals, and non-mammalian models [54]. Within these models, HFD induction is a common approach, and common symptoms of HFD-induced diabetes in animal models include weight gain, hyperinsulinemia, and disruption of glucose homeostasis [55].

HFD-Induced Diabetes in Human Intervention Studies
The strongest evidence about the relationship between diet and the progression of disease comes from RCTs. Most of the current studies on the relationship between an HFD and diabetes are cohort studies. An HFD can lead to high type 2 diabetes by affecting glucose and lipid metabolism, which in turn can impair the function of major metabolic organs [56], including adipose tissue, pancreas, and liver. Table 1 summarizes the relationship between dietary fat and diabetes in human studies.
Adipose tissue is a loose connective tissue consisting of cells filled with lipids [57]. As an important organ involved in energy homeostasis, adipose tissue produces various bioactive substances, such as adipocytokines and fatty acids, which play a key role in the development of diabetes [58]. An HFD also has an effect on gene expression in adipose tissue; an RCT of patients with metabolic syndrome found that a high saturated fat diet increased the expression of lipolytic genes, which may be associated with impaired insulin sensitivity [59].
The pancreatic beta cells can maintain blood glucose stability by secreting insulin to promote glucose uptake by peripheral tissues [60]. Type 2 diabetes eventually develops when pancreatic beta cells do not secrete enough insulin to meet the demands of insulin resistance. The decrease in beta cell mass in type 2 diabetics is due to beta cell apoptosis [61]. In pre-diabetes, blood glucose can still be maintained at normal levels due to the compensatory response of the beta cells [62]. As oxidative stress and inflammatory responses proceed in later stages, the compensatory mechanisms of the beta cells are continuously compromised, eventually leading to the development of type 2 diabetes [63].
Under normal physiological conditions, hepatic glucose production is regulated by a combination of insulin and glucagon, with glucagon inducing hepatic glucose production and insulin inhibiting it [64]. As there is insulin resistance in diabetes, the inability of insulin to suppress liver glucose production leads to hyperglycemia [65]. An HFD can lead to fat accumulation in the liver, causing insulin resistance and thus disrupting blood glucose homeostasis. An HFD has been shown to significantly increase liver fat levels in 56% of obese women [66]. Margarine consumption is positively associated with diabetes risk [73] Fat intake (SFA, MUFA, PUFA, TFA, animal fats, vegetable fats, marine omega-3 fatty acids, non-marine omega-3 fatty acids, and omega-6 linoleic acid (18:2n-6)) The people who were free of diabetes but were at high cardiovascular risk were aged  (N = 3349)

years
SAT and animal fats (cheese and butter) are associated with a higher risk of diabetes [74] Fat intake (SFA, MUFA, and PUFA) Healthy women aged 45-50 (N = 8370) 6 years Intake of MUFA, total n-3 PUFA, α-linolenic acid, and n-6 PUFA were positively associated with the incidence of diabetes [75] Total fat, SFA, MUFA, PUFA, and TFA Healthy women aged 45-50 (N = 84,204) 14 years TFA intake was positively associated with the risk of diabetes, while PUFA intake was negatively associated with the direction of diabetes [76]

HFD-Induced Diabetes in Animal Models
Intervention human studies have necessitated the development of animal models of diabetes, including mice, rats, Drosophila, zebrafish, and so on, to better explore the pathogenesis of diabetes and help to reveal the pathogenesis of diabetes. An HFD affects the major insulin-sensitive tissues of the animal model, including adipose tissue, the pancreas, and the liver. Rodent models are the most widely used animal models of diabetes and have been well-studied. Large animal models have physiological conditions more similar to those of humans, including physiological and pathological features. Primate models are very similar to humans but are more expensive and have a longer life cycle. Nonmammalian models, such as fruit flies and zebrafish, have a variety of advantages, such as short growth cycles, simple husbandry, low cost, and high reproductive capacity [54,79]. Table 2 summaries the animal models induced by an HFD, including fat type and amount, duration, animal species, and symptoms Adipose tissue has an important role in the development of diabetes, and adipose tissue is a major site for storing gluconeogenic substrates and energy [80]. An HFD can alter the expression of genes in adipose tissue, down-regulating genes encoding lipid metabolizing enzymes or markers of lipid differentiation, and increasing genes encoding markers of inflammation [81]. The B-cell activating factor (BAFF) is a tumor necrosis factor (TNF) ligand family protein that is a key factor in the development of poor glucose tolerance [82]. Mice fed an HFD had significantly increased BAFF in visceral adipose tissue and serum [83]. The pro-inflammatory cytokine TNF-α is associated with insulin resistance [84], and an HFD of both lard and soybean oil can increase TNF-α expression levels in adipose tissue [85].
The pancreas is a key site for regulating the secretion of insulin and glucagon, and an HFD can have an impact on the pancreas, leading to the development of diabetes. Several studies have pointed out the mechanism by which an HFD can enhance the compensation of pancreatic β-cells. For example, Jonatan Ahrén et al. found an increase in β-cell volume and β-cell numbers after feeding mice with a 60% lard diet for three months [86]. Kanno et al. found that the compensatory mechanism of islet cells in those on an HFD resulted mainly from increased levels of insulin translation [87]. Ribeiro et al. found that an HFD induced islet hypertrophy and a compensatory morpho-functional shift in pancreatic β-cells [88]. However, it has also been suggested that an HFD can directly lead to the degeneration of islet cell function. The levels of glucose transporters (GLUT)2 and glucokinase mRNA in rat pancreas were significantly reduced after 10 weeks of HFD feeding, and an HFD can reduce insulin secretion by impairing signal transduction in pancreatic β-cells [89]. In ZDF rats fed an HFD for a long period of time, the pancreas developed fat accumulation, which may have led to pancreatic fibrosis, acinar cell damage, and pancreatic stellate cell activation [90,91].
The liver is the main site of carbohydrate and fat utilization and plays an important role in controlling glucose intake, fat metabolism, and energy balance [92]. Liver fat accumulation is associated with insulin resistance, and excess fat intake leads to increased levels of free fatty acids and increased triglyceride deposition in the liver [93].

Gut Microbiota Dysbiosis in HFD-Induced Diabetes
From a physiological point of view, one of the most important links between an HFD and diabetes is the gut microbiota-host axis, as well as the factors released from intestinal metabolites, mediating bidirectional communication between the intestines and the host. Specific intestinal flora community profiles have been suggested to promote type 2 diabetes. Type 2 diabetic patients have dysbiosis of gut microbes with a reduced abundance of butyrate-producing bacteria (including Eubacterium rectale, Roseburia intestinalis, and Roseburia inulinivorans) and an increased abundance of pathogens (such as Clostridium ramosum, Clostridium symbiosum, Eggerthella lenta, and Escherichia coli) [102]. Emerging evidence demonstrates that changes in the ratios between gut microbiota, such as the ratios of Bacteroides and Firmicutes, are associated with the development of type 2 diabetes [103].
Recent studies have also found that type 2 diabetes caused by an HFD may be associated with gut microbes. Diet is a major factor influencing the composition and function of gut microbes. Animal experiments have shown that an HFD affects gut microbes. Compared to the normal diet, mice fed a high-fat diet were more susceptible to diabetes, which may be associated with a reduction in Bifidobacteria [104]. In mice on a 45% HFD, there was a decrease in Bacteroidetes and an increase in Firmicutes and Proteobacteria [13]. The HFD reduced the mice's Akkermansia, Coprococcus, and Ruminococcus, increased Odoribacter and Parabacteroides, and led to a reduction in short-chain fatty acids (SCFAs) [105]. SCFAs can alleviate diet-induced insulin resistance, and a reduction in SCFAs may lead to type 2 diabetes [106]. An HFD can affect the production of immunoglobulin A, a key regulator of glucose homeostasis, an immune-derived molecule in the gut [107]. Table 3 below summaries the gut microbiota of animals with high-fat-diet-induced diabetes.

Measurements to Treat Diabetes
With aging and urbanization, the number of people with diabetes is increasing, and the prevalence of diabetes continues to rise [136]. Therefore, the prevention and treatment of diabetes is now an important issue for people. Current treatments for diabetes include medication, dietary interventions, and physical activity [137]. In recent years, with intensive research into gut microbes and diabetes, probiotics may be a new way to treat diabetes.

Therapeutic Interventions for Diabetes
When dietary interventions are not feasible, medication may be considered as a strategy to prevent the development of type 2 diabetics. The chemical drugs used can be divided into biguanides, sulfonylureas, thiazolidinediones, glucosidase inhibitors, etc., according to the mechanism of action and chemical structure [138,139]. Metformin, a biguanide, is the most widely used oral hypoglycemic drug, with the advantages of safety and effectiveness, cardiovascular protection, and low cost, and is recommended as a first-line drug by the American Diabetes Association and the European Diabetes Association [140]. Metformin acts primarily on the liver and can improve hyperglycemia by inhibiting hepatic glucose production [141]. Metformin can act by inhibiting mitochondrial respiratory chain complex I, increasing the AMP/ATP ratio and activating AMPK-activated protein kinase [142,143]. The therapeutic effect of metformin may be related to its effect on gut microbiota, confirmed in animal models and clinical studies. Metformin decreases Bacteroides fragilis in type 2 diabetics and also increases the abundance of the mucin-degrading bacterium Akkermansia in HFD mice [144,145]. Sulphonylureas are a class of drugs that promote insulin secretion and can act by binding to the SUR subunit of the ATP-sensitive potassium channel in pancreatic cells [146]. However, the effects of sulfonylureas on gut microbiota have still not been well studied. Thiazolidinediones improve insulin sensitivity, and rosiglitazone and pioglitazone are representatives of these drugs. Thiazolidinediones are agonists of peroxisome proliferator receptor gamma (PPARγ), which can enhance insulin target tissues (muscle, fat, liver) and accelerate glucose utilization by activating PPARγ to promote the expression of genes related to glucose transport and lipid metabolism [147,148]. The relative abundance of Proteobacteria decreased after the treatment of HFD mice with pioglitazone [149]. Thiazolidinediones may cause a variety of side effects, such as heart failure, cardiovascular death, edema, and fractures [150]. Oral αglucosidase inhibitors can improve hyperglycemia by delaying the breakdown of carbohydrates into glucose. Currently, the three clinically approved glucosidase inhibitors include acarbose, voglibose, and miglitol [151]. A double-blind RCT of acarbose altered gut microbiota in prediabetic patients, decreasing Ruminococcaceae and Lachnospiraceae and increasing Lactobacillaceae, Ruminococcaceae, and Veillonellaceae [152].

Dietary Interventions to Alleviate Diabetes
Most health organizations point to dietary interventions as a powerful treatment for diabetes, with controlled diets improving insulin sensitivity and reducing the risk of diabetes and its complications [153]. The American College of Lifestyle Medicine believes that diabetes can be treated with dietary interventions that use whole food, plant-based eating patterns, and increase the intake of unrefined plant foods in the daily diet while eliminating or minimizing the intake of animal foods and refined foods, and with moderate exercise in life [154]. The impact of dietary interventions on diabetes includes effects through indirect weight loss and direct consumption of a variety of nutrients with health benefits [155]. Being overweight is considered to be one of the important factors associated with the risk of diabetes [156,157]. Dietary interventions can reduce weight and improve diabetes by reducing the intake of fat.
As carbohydrate catabolism causes blood glucose to rise, reducing carbohydrate intake in the daily diet can be a good treatment for type 2 diabetes [158]. A low-carbohydrate diet, as defined by the American Diabetes Association, is 130 g/day or less than 26% of total daily energy intake [159]. The traditional Mediterranean diet of minimally processed whole grains has also been shown to have significant benefits for diabetes [160].
Specific types of dietary fat may affect diabetes. The KANWU study found that replacing a diet with monounsaturated fatty acids (23%E for MUFA, 8%E for SFA, 6%E for PUFA) over saturated fatty acids (17%E for SFA, 14%E for MUFA, 6%E for PUFA) improved insulin sensitivity at a total fat intake below the median (37E%) [68]. Another study also confirmed that a diet rich in MUFA could improve central fat distribution and insulin resistance [161]. The Iowa Women's Study found a reduced risk of diabetes when saturated fatty acids were replaced with unsaturated fatty acids [71], and a study by Summers et al. also noted that replacing saturated fatty acids with unsaturated fatty acids improved insulin sensitivity and abdominal fat accumulation [162]. n-3 PUFA improves high-fat-diet-induced insulin resistance. n-3 PUFA in fish oil improves insulinemia, lipid metabolism, and glucose metabolism in insulin-resistant rats [163]. Different dietary fat types may influence the affinity of insulin receptors by affecting the fatty acid composition of cell membranes [164]. It has also been suggested that dietary fat can modulate the expression of genes involved in lipid metabolisms that affect diabetes, such as fatty acid transport proteins and fatty acid synthases [165]. Increased inflammation may lead to insulin resistance, and PUFA acid intake may improve inflammation, e.g., n-3 PUFA may inhibit Toll-like receptors on the cell surface and reduce the production of inflammatory cytokines [166,167].
Other than the chemical drugs currently used to treat diabetes, natural plant foods such as fruits and vegetables, which are rich in nutrients such as antioxidants and polyphenols, can improve adipokines and oxidative stress, significantly improving beta cell function and insulin sensitivity [168,169]. Table 4 below summaries the potential mechanisms of natural products in food for the treatment of diabetes. Table 4. Potential mechanisms of natural products in foods in animal models of diabetes.

Potential Probiotics Help with Diabetes
Probiotics refer to the beneficial microbiota which inhabit the gut and have a variety of health functions [181]. The development of diabetes is closely linked to gut microbes, and therefore the regulation of gut microbes through probiotics could be a new approach to treating diabetes. The hypoglycemic effect of probiotics has been confirmed in both in vitro and in vivo experiments. Zhu et al. found that most of the Lactobacillus species inhibited dipeptidyl peptidase IV and α-glucosidase by cell-free excretory supernatants and cell-free extracts prepared from 21 Lactobacillus species [182]. Several studies have shown that probiotics can lower blood sugar to varying degrees in diabetic animals, such as Lactobacillus plantarum [183], Lactobacillus casei [184], Lactobacillus rhamnosus [185], and Clostridium butyricum [186]. A meta-analysis indicated that probiotics significantly lowered hemoglobin A1c, fasting blood glucose, fasting insulin, triglycerides, and total cholesterol, and improved the symptoms of diabetes [187]. High endotoxemia was demonstrated in high-fat-fed mice. Supplementation with oligofructose to increase the number of gut Bifidobacteria revealed that endotoxemia was negatively correlated with Bifidobacteria and also improved glucose tolerance and insulin secretion with increased Bifidobacteria [188].
The mechanisms by which probiotics improve diabetes include direct effects on the gut microbiota, anti-inflammatory and immunomodulatory effects, reduction in oxidative stress, and involvement in glucose homeostasis [181,189,190]. Gut microbial dysbiosis in diabetic patients leads to increased gut permeability and increased concentrations of bacterial endotoxins such as lipopolysaccharides, inducing inflammation and, ultimately, systemic insulin resistance [191]. Probiotics such as Lactobacillus paracasei can restore the expression of the tight junction protein in the colon, thereby reducing serum lipopolysaccharide and inflammatory cytokine levels [192]. Diabetes leads to increased systemic oxidative stress, and the intake of beneficial bacteria can significantly improve fasting blood glucose and the antioxidant status in diabetics [193,194]. Bifidobacterium lactis improves glucose uptake and GLU4 translocation through the insulin signaling pathway AKT and insulin receptor substrate-1, increases the expression of GLUT4 and insulin-sensitivity-related genes, and regulates glucose metabolism [195].

Conclusions
A long-term HFD, especially the excessive intake of saturated fats, could have a variety of adverse effects on human body health and even lead to chronic diseases, including diabetes. Animal models of diabetes can better explore the pathogenesis of diabetes and help to reveal the pathogenesis of diabetes. Daily diet can have a direct impact on the composition and function of host gut microbiota. Excessive fat intake can lead to the imbalances of gut microbiota, including changes in the ratio of Bacteroidetes and Firmicutes, a decrease in butyrate-producing bacteria, and an increase in the abundance of pathogens. A gut microbiota imbalance may further disturb host metabolism, such as decreased amounts of SCFA and immunoglobulin A, ultimately leading to diabetes. Within animal models, HFD-induced diabetes is accompanied by weight gain, hyperinsulinemia, and the disruption of glucose homeostasis. At present, the treatment of diabetes includes dietary interventions and medication. The causal relationship between gut microbiota and diabetes and its underlying mechanisms are still not fully elucidated, and further research is needed. In the near future, as research into the mechanisms of diabetes and gut microbes intensifies, probiotics may become a new method of treatment for diabetes.

Conflicts of Interest:
The authors declare no conflict of interest.