*Review* **Diet Modifications towards Restoration of Insulin Sensitivity and Daily Insulin Fluctuations in Diabetes**

**Ana Magalhães 1,†, Cátia Barra 1,2,\*,†, Ana Borges 1,3 and Lèlita Santos 1,2,4**


**Abstract:** The circadian rhythm is essential in order to maintain metabolic homeostasis and insulin sensitivity. Disruption of circadian mechanisms is associated with the development of metabolic diseases, such as diabetes. Lifestyle changes such as an equilibrated diet and physical activity are known to improve glycaemic control in diabetic patients. One of the mechanisms possibly involved in such an improvement is the restoration of insulin circadian rhythms. There are several available dietary schemes based on circadian rhythms. Some of them are associated with better regulation of daily insulin fluctuations and the improvement of Type 2 Diabetes and metabolic syndrome. In the current review, we aim to explore how the different types of diet can impact glucose metabolism and insulin sensitivity in patients with diabetes, highlighting the interactions with the mechanisms of circadian insulin rhythm and the prevention of hyperinsulinemia.

**Keywords:** daily insulin fluctuation; insulin sensitivity; diets; circadian rhythm

**Citation:** Magalhães, A.; Barra, C.; Borges, A.; Santos, L. Diet Modifications towards Restoration of Insulin Sensitivity and Daily Insulin Fluctuations in Diabetes. *Diabetology* **2022**, *3*, 606–614. https://doi.org/ 10.3390/diabetology3040046

Academic Editor: Peter Clifton

Received: 30 September 2022 Accepted: 18 November 2022 Published: 22 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Insulin was discovered by Frederick Banting and Charles Best in 1921. It is a pancreatic beta-cell anabolic hormone initially produced by as pre-insulin and then as proinsulin after a maturation process [1]. Subsequently, it is translocated from the endoplasmic reticulum to the Golgi apparatus, where it is cleaved into C-peptide and insulin, which are simultaneously released by exocytosis [2]. Its production by pancreatic beta cells is enhanced in response to glucose, and it is responsible for maintaining constant blood levels of glucose and metabolic homeostasis. However, it has been demonstrated that amino acids and fatty acids are also capable of stimulating insulin secretion [3,4]. Its secretion from pancreatic beta cells is biphasic: the first phase occurs with a rapid release, while the second phase is characterized by a more sustained and less elevated release [5]. Insulin levels are increased in the postprandial period when the circulating glucose levels increase, making the first phase very significant. In this phase, insulin inhibits glucagon secretion from the pancreatic alpha cells through paracrine action at the beginning of the meal. On the other hand, the second phase is critical for maintaining this inhibition and increasing glucose storage and utilization [6]. Thirty minutes after the beginning of the meal, insulin suppresses lipolysis in the adipose tissue by inactivating the hormonesensitive lipase, leading to a decrease in non-esterified fatty acids (NEFA) and glycerol in the blood [7,8]. The lower blood NEFA and higher insulin levels induce the suppression of glucose production in the liver and an increase in glucose utilization (glycolysis) by the muscle [3,9,10]. Insulin and glucagon secretion, as well as glucose homeostasis, are also regulated by the incretins, which are secreted by the gut in response to food intake [11,12]. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)

<sup>9</sup>

are the two major incretins derived from gut neuroendocrine cells and are involved in these mechanisms, and their receptors may be found both in alpha and beta cells [11,13].

Insulin secretion dysregulation due to several causes promotes the development of diabetes. Type 1 diabetes (T1D) is characterized by auto-immune destruction of beta cells, while type 2 diabetes (T2D) is related to the development of insulin resistance, which is associated with obesity and metabolic syndrome. In T2D, beta-cell destruction is also observed through their progressive exhaustion and dysfunction in a later phase [14,15]. Consequently, beta-cell hyperplasia and a hyperinsulinemic state are often observed in obese patients in order to compensate for chronic low-grade inflammation and insulin resistance [14,16,17]. Moreover, hyperinsulinemia itself contributes to insulin resistance due to insulin receptor downregulation [11]. Lifestyle changes such as an equilibrated diet and physical exercise may prevent these events and thus contribute to maintaining a normal weight and insulin sensitivity in the peripheral tissues.

#### **2. Circadian Rhythm Influences Insulin Secretion**

The circadian rhythm is essential for maintaining a normal body physiology. It is mediated by specific components that are controlled by the daily light–dark and feeding– fasting cycles. The central nervous system plays an important role in the circadian rhythm through synapse networking, which acts in different brain regions and in endocrine cells, contributing to the circadian regulation of metabolism [18]. These outputs are integrated into the hypothalamus, which is responsible for hunger–satiety, thermoregulation, sleep– arousal, and osmolarity [19].

The metabolic activity of several organs is also mediated by their internal clocks and circadian rhythms. In the intestine, the expression of sodium-glucose transport protein 1 (SGLT-1) has a rhythmic cycle, which is increased when the glucose intake is anticipated. Similar mechanisms regulate insulin secretion. Besides being regulated by glucose levels and incretins, its exocytosis also has a circadian regulation, possibly because incretins also have a circadian rhythm themselves [20]. Some studies in healthy human subjects have shown daily fluctuations in glucose tolerance, suggesting that it is greatest in the morning, while a significant decrease occurs during the night [21,22]. This is not only because of the oscillations in peripheral insulin sensitivity, but also the variations of glucose-stimulated insulin secretion during the daily 24 h period [23].

Uncoupling protein 2 (UCP2) has been described to influence metabolism, as it is expressed by endogenous circadian oscillators in pancreatic islets and acts in beta cells as a negative regulator of glucose-stimulated insulin secretion (GSIS). As mentioned before, insulin regulates the metabolic response to fasting and its suppression is crucial to allow for endogenous glucose production in the liver and lipolysis in the adipose tissue in order to release metabolic fuel. Therefore, Ucp2/UCP2 has been suggested to coordinate insulin secretion according to nutrient ingestion, so an upregulation of Ucp2/UCP2 prevents hypoglycaemia during the fasting period by inhibiting insulin secretion and promoting fuel mobilization [24,25]. This type of regulation influences the activity of several other endocrine pathways such as melatonin, glucocorticoids, and growth hormone (GH) [20]. GH and cortisol secretion are regulated by their own circadian rhythm, and are higher in the nocturnal period, which contributes to insulin resistance in the early morning hours [22].

It has been demonstrated that chronic disruption of these circadian mechanisms is related to the development of metabolic diseases, as established by several studies in shift workers [26,27]. In a retrospective study conducted by Garaulet and her collaborators, eating early (lunch time before 15:00 p.m.) results in enhanced weight loss effectiveness, suggesting a relation between eating intervals and the day–night cycle in the metabolism [28]. Other studies have revealed that time-restricted feeding is capable of improving metabolic diseases due to sustained diurnal rhythms and imposing daily feeding–fasting cycles [29,30]. Similar to what happens with GH and cortisol, melatonin (Figure 1) also promotes a hyperglycaemic state in the evening, given that melatonin binding to its receptor in pancreatic islets inhibits insulin release from the beta cells [31]. Reciprocal interactions between

metabolism and the circadian clock imply that nutrition quality, quantity, and daily eating patterns can affect diurnal rhythms, which in turn determine whole-body physiology.

**Figure 1.** Effects of different diets on insulin and incretins secretion, as well as hyperglycaemic and inflammatory states. KD, characterized by the ingestion of <50 g of carbohydrates/day, promotes a decrease in insulin and increase in glucagon and GIP fasting levels. MD, because of its higher fiber supply and reduced sugar ingestion, is capable of decreasing blood insulin levels and increasing GIP secretion, similarly decreasing pro-inflammatory cytokine activation and reducing the risk of diabetes development. VD, based on reduced calorie ingestion and being rich in magnesium and fibre, promotes a decrease in GI. A lipid diet is associated with diabetes development by increasing glucagon secretion and decreasing GLP-1.

#### **3. The Impact of Different Diets on the Levels of Insulin Secretion**

#### *3.1. The Impact of Diet*

Nowadays, most diets that are called healthy can be included in a few categories: they match the Mediterranean Diet, or they are low in fat, low in carbohydrates (CH), or are vegetarian [32]. Nutrition is considered one of the pillars for the prevention and treatment of several diseases, namely metabolic syndrome [33]. Besides the amount of food ingested, growing attention has been devoted to the quality of macronutrients and the role they play in the regulation of T2D [34,35]. The current discussion about food standards suggests that it is not just a question of quantity, but also of quality and, importantly, time and schedule. For example, eating most calories and carbs at lunchtime or in the early afternoon, avoiding

late dinners, and keeping the number of meals and the timing of meals consistent, all play a role in regulating postprandial blood glucose and insulin sensitivity [36].

This section of the review will focus on postprandial insulin, and the long-term insulin response in relation to the glycaemic index (GI), the area under the curve (AUC) of the glucose response after CH consumption [37].

#### *3.2. Mediterranean Diet*

The Mediterranean Diet emerged in the 1960s as the diet with the most beneficial impact on health and the one recommended by experts. This diet is characterized mainly by the consumption of vegetables, with the regular consumption of protein (meat, fish, or egg), CH, and a limited amount of fats, usually from olive oil. Nonetheless, it is a diet low in fat and rich in vitamins with antioxidant and anti-inflammatory properties [35]. Therefore, as it decreases the activation of the pro-inflammatory cytokines (Figure 1), it also reduces chronic inflammation and the risk of T2D. As it includes a low consumption of sugar/CH, it also has a role in regulating diabetes. So, it is suggested that by resulting in a greater supply of fibre and less sugar, it is effective at decreasing the load of this component on the body, while also decreasing the circulating insulin levels [38].

#### *3.3. Vegetarian Diet*

Adherence to a vegetarian diet is growing worldwide. This is characterized by the exclusion of foods of an animal origin. However, if this diet is not supplemented, macro and micronutrient deficiencies such as omega-3 fatty acids, iron, vitamin D, calcium, zinc, iodine, and vitamin B12 may occur [32]. The reinforced intake of vegetables and cereals reduces the risk of T2D as they are rich in magnesium and fibre (Figure 1). The insulin signalling pathway is magnesium-dependent and fibre helps to lower the GI [39]. The low GI of this diet may explain the lower cardiovascular risk and greater insulin sensitivity compared with omnivores [37].

Within the scope of T2D, a plant-based diet will reduce body weight, as it reduces the ingested calories, and increases postprandial metabolism [34]. Some studies suggest that vegetarians have a lower amount of intramuscular lipids than omnivores, and this proportion also occurs at a hepatic level. By having this metabolic (catabolism) effect, it will decrease HbA1c and postprandial lipids. Visceral fat reduction also decreases the levels of plasma inflammatory adipokines and oxidative stress markers, which may have an effect on beta cell function and insulin regulation. An unexpected effect of this diet is that it increases the thermic effect of food, supporting this theory of catabolism [40]. The question that arises in this diet is its suitability for the entire population as it is a difficult diet to maintain, at least for the Western population, as a high intake of CH has to be monitored by a nutritionist [32].

#### *3.4. Paleolithic Diet*

This diet is based on the consumption of fruits, vegetables, eggs, fish, and meat, with the latter as a source of protein. Thus, it excludes all processed food, including dairy products. Thus, it contains less CH but a greater protein intake [41]. In fact, the distribution of nutrients occurs as follows: 35% lipids, 35% CH, and 30% protein [42]. The Paleolithic diet causes a reduction in insulin concentration after a meal, that is, less insulin is needed with this nutrient distribution than with other diets [43]. Studies have suggested that this approach decreases insulin secretion in the long-term and increases beta cell sensitivity to postprandial glucose load [42]. However, Genoni et al. suggest that this adaptation may not be beneficial in terms of intestinal motility [44]. Biologically, a catabolic state is stimulated and postprandial glucagon suppression is observed. Together with insulin secretion, this may result from improved postprandial secretion of the incretins GLP-1 and GIP into circulation. Their increase also promotes satiety and thus weight loss [41].

#### **4. Diets That Promote Ketosis**

This group of diets is divided into three types: low in CH, normoprotein, and high in lipids, such as the ketogenic diet. It also includes the famous «fasting» and calorie restriction, which increase ketosis due to the lack of calories. Despite a restriction of nutrients, some studies have considered this type of diet more healthy as it helps to preserve muscle mass, increase weight loss, reduce appetite, and above all decrease insulin resistance and circulating insulin levels [45,46].

#### *4.1. Ketogenic Diet*

It consists of a normocaloric diet well known for rapid weight loss. However, in terms of insulin patterns and its effect on the liver, it is still controversial [47]. The proposal is to ingest less than 50 g of CH per day, which will mimic a catabolic state. Insulin secretion will decrease as the main nutrient will be ketone bodies (from lipids) as an alternative substrate [45]. Fasting glucagon and GIP levels increase, while, after ingestion, postprandial glucose and insulin AUC are lower than in a control meal (Figure 1). The increased adiponectin observed after this diet may also promote insulin sensitivity [46].

#### *4.2. Lipid-Modified Diet*

As would be expected, this diet may increase the risk of obesity and insulin resistance [48], but some studies claim it is a matter of quantity. The type of ingested lipids affects the insulin signalling cascade. Contrary to expectations, intervention studies have reported that replacing monounsaturated with saturated lipids alters the plasma fatty acids, but may increase insulin sensitivity [34]. However, some studies report that this diet causes insulin resistance. A 2-week lipid-rich diet leads to insulin resistance in muscle tissue due to metabolic changes, specifically in the mitochondria (incomplete oxidation of molecules), and impaired signalling of the insulin cascade [49]. Given that this type of diet causes obesity, this may have other consequences, such as a decreased secretion of GLP-1 (Figure 1) [41]. Thus, the ratio between insulin and glucagon production in the pancreas is changed, leading to an increased appetite and increased caloric intake, which will cause an overstimulation of insulin and induce resistance [41].

#### *4.3. Low Carb Diet (LCD)*

This diet is often used by nutritionists as it promotes weight loss in a short period of time and, consequently, an improvement in the glycaemic profile. This diet is based on the consumption of vegetables and protein, removing virtually all CH (consuming < 130 g/day, the main foods originating glucose) [50]. In this context, the mathematical formula is simple: reducing the intake of glucose (present in CH) reduces hyperinsulinemia, which can be adapted for patients with diabetes [51]. There is evidence that this diet allows 93% of patients to regress to pre-diabetes, 46% of patients no longer need medication, and 60% of patients regress from diabetes within 1 year [51]. However, it has to be noted that a low glycaemic diet (GD) is not a low GI diet [52]. LCD improves the glycaemic profile because it rapidly increases postprandial insulin levels. However, a high glycaemic peak will cause patients to have less satiety and avoid a further increase in dietary intake [46].

#### *4.4. Diet Rich in Carbohydrates*

Controversial studies report that the ingestion of a diet rich in CH and fibre can lead to normoglycemia in prediabetic individuals [53]. After eating a meal, insulin in fat cells stimulates the entry of glucose into the tissues, decreasing the release of lipids and inhibiting the production of ketone bodies. Thus, this diet will produce hyperinsulinemia that will force the deposition of lipids instead of their oxidation [54]. The mechanisms are not clear; there are studies reporting that a diet rich in CH reduces blood glucose through glucose metabolism at the muscle level, improving the glycaemic profile in the short and long term [55]. This occurs if CH is not refined, with an increased adiponectin concentration, a decrease in body weight, and thus an improvement in the metabolic profile [56]. However, it is not clear what would be the long-term effects on beta cell function, because their overstimulation is expected to increase the risk of exhaustion.

Once again, nutrients are paramount, and diets with a GI < 55 are digested slowly and those with a high GI (>70) are digested quickly [52]. Importantly, high GI foods can alter leptin by interfering with satiety beyond the absorption spectrum [57]. So, foods that contain CH, such as vegetables and fruit, have better GI values, satisfying appetite for a longer time and improving the glycaemic profile [52].

#### *4.5. Caloric Restriction*

This dietary intervention consists of reducing the caloric intake by 25–30% from the baseline, but without restricting the necessary nutrients, that is, decreasing the quantity without decreasing the quality [45]. This diet can exacerbate some metabolic problems as it can increase appetite and stress hormones [54]. In this catabolic context, glucagon is favored and the secretion of GLP-1 is inhibited [41]. As a consequence, the hepatic glucose output is increased. However, pancreatic apoptosis decreases and insulin sensitivity increases, mainly due to the lower insulinemia [45]. It is especially problematic in patients with T2D, possibly causing hypoglycaemia and difficulties in the long-term regulation of glucose metabolism. Caloric restriction is more suitable for patients in the early stages of metabolic dysregulation, where it will favour body weight and fat mass loss, while decreasing lipotoxicity in the muscle, liver, and adipose tissue. This will decrease low-grade inflammation and increase insulin sensitivity. Thus, caloric restriction is suitable to prevent insulin resistance and beta-cell exhaustion in patients with obesity or prediabetes, although it is a dietary scheme that is very hard to follow by patients with T2D (reviewed by Joaquim et al., 2022 [58]). For patients with T2D, other less severe schemes are recommended, such as time-restricting fasting (TRF). In this dietary approach, patients are recommended to eat only during one part of the day, not consuming any calories during the other part, usually for at least 10 h. This approach often does not require caloric restriction, only fasting for half of the day in order to regulate relevant biological mechanisms such as circadian rhythms, stress hormones suppression, and autophagy (reviewed by Joaquim et al., 2022 [58]). As mentioned above, dietary consumption at specific times may coordinate the response to the presence or absence of nutrients, namely their gut metabolization and absorption; insulin secretion and action; and all the necessary changes in the liver, muscle, and adipose tissue metabolism. TRF has been shown to promote overnight mobilization of energy reserves through lipolysis, lipogenesis, and hepatic glucose production, contributing to a higher catabolic activity [59–61]. The rhythmic regulation of SGLT1 in the gut and UCP2 in beta cells, as already discussed, may in fact explain the positive effects of this type of diet on the beta-cell function and insulin sensitivity. It is possible that the overnight suppression of insulin may be a key factor in better diurnal insulin sensitivity and preserved beta-cell function (reviewed by Joaquim et al., 2022 [58]).

#### **5. Conclusions**

In this review, we focus on daily insulin fluctuation and glucose metabolism, as well as the influence of diet on these mechanisms. Metabolic homeostasis is highly coordinated by the circadian rhythm, which regulates insulin and glucagon secretion in order to maintain a normal fuel balance between tissues. However, it is well known that disruption of these mechanisms can lead to metabolic disease development, such as T2D. Current dietary habits are known to cause beta-cell overstimulation due to the high consumption of CH, especially refined ones. This will not only cause long-term beta-cell exhaustion but also insulin resistance, as hyperinsulinemia is one of the major drivers of insulin receptor inactivation. Nowadays, there are several diets known to improve insulin sensitivity and better control glycaemic profile in patients with T2D. Postprandial hyperglycaemia depends mainly on meal composition, but other factors also contribute to its magnitude, such as glucose absorption; secretion; and the action of incretin hormones, insulin, and glucagon. Thus, it is important to evaluate the quality of the nutrients rather than only their quantity, and, most importantly, their glycaemic impact. In T2D, it is important to adjust the diet to control glycemia after meals and the postprandial and fasting insulin levels.

**Author Contributions:** Conceptualization: A.M., C.B., A.B. and L.S. Methodology: A.M. and C.B.; A.B. and L.S. Writing—original draft preparation: A.M. and C.B. Writing-review and editing: A.B. and L.S.; Supervision: A.B. and L.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Foundation for Science and Technology (FCT), Portugal (Pest UID/NEU/04539/2013 and UID/NEU/04539/2019: CNC.IBILI; Pest UIDB/04539/2020 and UIDP/04539/2020: CIBB).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Cátia Barra is a CIBB member, who is supported by the Portuguese Science and Technology Foundation (FCT), Strategic Projects UIDB/04539/2020 and UIDBP/4539/2020.

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

#### **References**


**Tamaeh Monteiro-Alfredo 1,2 and Paulo Matafome 1,2,3,4,5,\***


**Abstract:** Glycotoxins include the group of advanced glycation end-products (AGEs) and their precursors, most of them highly reactive intermediary compounds of sugar metabolism. Glycotoxins and products of the Maillard reaction are present in high concentrations in foods rich in sugars and processed at high temperatures and are often associated with the flavour of the food. Proteins undergoing this type of molecular modification are targets for gut peptidases and may be absorbed into circulation. AGEs are associated with the toxic effects of glucose in diabetic patients, and some studies have shown that they also contribute to metabolically unhealthy obesity and prediabetes development. Restriction of dietary glycotoxins was shown to improve insulin resistance in humans. However, the real contribution of dietary AGEs to such mechanisms is still not understood. This review summarizes the current knowledge about glycotoxin formation from dietary sugars, their digestion throughout the gastrointestinal system, and the mechanisms of their intestinal absorption.

**Keywords:** glycation; glycotoxins; dietary sugars; AGEs digestion; intestinal absorption; gut microbiota

#### **1. Introduction**

Free sugars are often very reactive and are non-enzymatically degraded under physiological conditions, leading to the formation of intermediary compounds that may react with other molecules and cause harmful effects. This pool of intermediary and advanced products is generally called glycotoxins. This is a heterogeneous group of compounds that includes advanced glycation end-products (AGEs) and their precursors, most of them highly reactive intermediary compounds. Methylglyoxal (MG) is a dicarbonyl and a major precursor that originates as a by-product of glucose and fructose metabolism (reviewed by [1]). It modifies arginine and lysine residues of biomolecules, namely, proteins and DNA, forming AGEs [1–4]. The AGEs Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) and argpyrimidine are formed after MG-induced arginine modification, whereas lysine modification leads to methylglyoxal lysine dimer (MOLD) and (carboxyethyl)lysine (CEL) formation [5–8]. Alternatively, NE-(carboxymethyl)-lysine (CML), one of the major AGEs, may be formed directly from the Maillard reaction between lysine residues and reducing sugars, fructose being more harmful than glucose [1]. Such reactions may occur intracellularly (cytoplasmic proteins and transcription factors) or with circulating (haemoglobin, albumin, or lipoproteins) extracellular matrix and food proteins [1–4,9]. The endogenous formation of MG-derived AGEs has been associated with diabetes-like vascular and metabolic complications. Their contribution to nephropathy and retinopathy

**Citation:** Monteiro-Alfredo, T.; Matafome, P. Gut Metabolism of Sugars: Formation of Glycotoxins and Their Intestinal Absorption. *Diabetology* **2022**, *3*, 596–605. https:// doi.org/10.3390/diabetology3040045

Academic Editor: Peter Clifton

Received: 9 October 2022 Accepted: 7 November 2022 Published: 10 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

has been consistently shown, not only due to direct effects on endothelial cells but also on podocytes and pericytes. In the development of the diabetic foot, glycotoxins have been shown to contribute both to vascular dysfunction and neuropathy, leading to hypersensitivity of nerve fibres and neuronal degeneration (reviewed by [1,8]). They have also been shown to cause insulin resistance, mainly in already obese models and beta-cell dysfunction. Although a few reports show that their decrease in diet may reduce insulin resistance in diabetic patients, rodent models of AGEs supplementation fail to show insulin resistance. Otherwise, if AGE supplementation is made to high-fat diet-fed rodent models, they potentiate the diet's effects and trigger insulin resistance (reviewed by [10]). Glycotoxins and AGEs are well-known contributors to a myriad of other metabolic diseaserelated disorders. Their contribution has been shown in cardiovascular and cerebrovascular diseases due to endothelial dysfunction in macrovessels, alterations in cardiac muscle contractility/relaxation, tendon stiffness, and even rheumatoid arthritis. Glycotoxins have also been linked with the molecular mechanisms of central nervous system disorders, namely, mitochondrial dysfunction and oxidative stress in Alzheimer's disease and glycation of alpha-synuclein in Parkinson's disease (reviewed by [1,8,11]).

Modification of intracellular proteins by dicarbonyls like MG changes the cellular redox state (oxidative and nitrosative stress), proteasomal activity, and gene expression modulators, while modification of extracellular and circulating proteins leads to increased stiffness of the matrix and alteration of biological properties of circulating factors, such as hormone loss of function and modification of albumin and haemoglobin [1]. MG may be detoxified into D-lactate through the glyoxalase system (GLO-1 and GLO-2), while AGEs and their intermediary Amadori products, such as fructoselysine, may be cleared by amadoriases. Of those, the fructosyl amine oxidase (FAOX) amadoriase, it is the most well studied and characterized for its catalytic activity, cleaving the adducts between sugars and amino acids. Other enzymes include amadoriase II and other amadoriases occurring in other organisms (reviewed by [1,12]). The GLO system is GSH-dependent and its downregulation is associated with higher endogenous AGE formation and the development of diabetic complications in humans and animal models [8].

Despite their quantitative contribution to the circulating pool is not completely understood, glycotoxins may also originate in the diet, namely, in foods rich in sugars and cooked at high temperatures. Using human serum albumin as a model, the dicarbonyls glyoxal and methylglyoxal have been shown to modify the lysyl and arginyl groups in 9 and 14 sites, respectively [13]. Although this depends on each specific protein and the sugars involved, sugar-rich foods and cooking also lead to the initiation of similar reactions involving food proteins. Even in healthy individuals, circulating AGE levels are correlated with their intake, contributing to the endogenous pool of AGEs and activation of inflammatory signals [14–16]. Intestinal absorption of glycotoxins and AGEs has been shown, as well as their deposition in tissues [17]. In foods, the initial steps of the Maillard reaction originate from such Amadori products as fructoselysine and lactuloselysine and AGEs, which are estimated to be consumed on a scale of daily 500–1200 mg in the diet. About 25–75 mg are estimated to be AGEs. Pasteurized milk and bakery products are the main dietary sources of Amadori products [18,19]. However, the digestion and absorption processes are very different among distinct AGEs, imposing extra difficulties in their study.

Although AGEs may be absorbed from the diet, mainly through the Maillard reaction, in sugar-rich foods, there are also other components that may prevent such reactions. The presence of antioxidants and scavenging molecules may hamper AGE formation, although this is still mostly unknown ground. Nevertheless, pectin oligosaccharides were recently shown to prevent food browning (Maillard reaction) and inhibit AGE formation. This is a complex group of non-digestible oligosaccharides originating from pectin, present in the plant cell wall. These compounds also reduced intestinal absorption in an in vitro system, suggesting their use as a dietary supplement to prevent food glycation [20]. Another way to promote the detoxification of AGEs is through the ingestion of vegetables, fruits, and

natural foods rich in phenolic compounds, which have anti-glycating [21] and antioxidant activity [22,23].

#### **2. Formation of Glycotoxins in the Gut and Their Digestion**

Advanced glycation end-product (AGE) formation during carbohydrate digestion: Carbohydrates can be found in the most varied sources: they can be intrinsic to fruit and milk or they can be added to food (extrinsic sugars) to increase palatability and conservation [24]. In recent times, the new habits of the world population are associated with a greater consumption of sugars and consequently AGEs. In general, AGEs provide greater palatability and help to increase shelf life [25]. Although food is an important exogenous source, they may also be produced during digestion, becoming an endogenous source of AGEs [14] (Figure 1). The energy value offered by sugar is 4 kcal g−1, and the recommended intake of carbohydrates is based on the minimum amount of sugar consumed by the brain, which is 130 g day−<sup>1</sup> for both adults and children [26]. Despite this, the actual consumption is above the recommended daily average, according to the Centers for Disease Control and Prevention [27]. The increased consumption of sugars is directly related to the development of obesity, type 2 diabetes, and associated pathologies [23].

**Figure 1.** Overview of the events involved in glycotoxin consumption from sugar-rich foods, their digestion in the gastrointestinal tract, and their intestinal absorption. The lower part of the image shows the different mechanisms described to be involved in intestinal AGE absorption.

Carbohydrate digestion begins in the mouth with the entire physical and chemical process that occurs with the concomitant action of α-amylase and the subsequent effect of pancreatic amylase, located in the small intestine, which is responsible for the digestion of 60% of carbohydrates. Then, the monosaccharides are absorbed by enterocytes into the bloodstream through intestinal epithelial cell enzymes [28]. Finally, they are transferred to cells to participate in energy production, which can be aerobic or anaerobic [29], and are eventually oxidized [30]. As consequence of the exaggerated intake of sugars, the reactions that occur from glucose with the body's proteins give rise to dicarbonyls and AGEs, which gradually accumulate in the body. One of the sugar-driven modifications is the case of glycated haemoglobin (HbA1c), a biochemical marker of glycaemic control in diabetic patients, which reflects the mean glycaemia of the last 90 to 120 days [31].

In addition to the ingestion of exogenous AGEs, their formation can occur through chemical processes of food degradation. Diet-derived MG interacts with intestinal proteins and culminates in the formation of the derivative of arginine MG-H1, which is absorbed in the intestine by enterocytes [32]. In simulations of digestive conditions, the concentrations of these AGEs were reduced in the presence of pancreatic enzymes. Regarding MG, its stability is greater in the gastric juice, but its degradation occurs in the intestinal juice, which probably is related to an enzymatic action [33]. It is believed that the digestive characteristics of AGEs in general lead to their susceptibility to the metabolism of reversibly bound adducts during digestion, and may later promote some local or systemic effect if they are reabsorbed [34] (Figure 1).

For the digestion of AGEs, gastric and intestinal functions are critical and depend on the function of such enzymes as elastase, peptidases, trypsin, and chymotrypsin. Glycation of arginine and lysine residues may directly block the trypsin site (which occurs by the attraction of negative charges between the trypsin active centre (negatively charged) and the amino acid groups (positively charged), being responsible for the impairment of the protein digestive process. Additionally, this modification may also hinder the effect of other digestive proteases, such as β-lactoglobulin and β-casein [35]. On the other hand, in another study, it was confirmed that alterations in the conformation of lactoferrin, caused by glycation, can lead to greater exposure of cleavage sites, increasing susceptibility to proteolysis [36]. The anti-digestion profile of AGEs ends up functioning as a natural barrier against the absorption of AGEs that are bound to proteins, because they have a lower bioavailability than free fractions [37].

Other studies with mice revealed the formation of specific AGEs from dietary sugars, namely, the detection of AGEs derived from MG in the liver tissue after glucose intake, and the detection of glyoxal (GO) when ingesta were based on fructose [32]. A study carried out by Martínez-Saez [38], which aimed to define the products derived from the Maillard reaction from meals, determined that physiological concentrations of sugars (43 mM) already caused the formation of these products. Fructose at a concentration of 314 mM was able to promote the formation of fructosamine and other AGEs, and fructose at 43 mM with lysine was able to give rise to CML [38]. In another assay, where different products obtained from gastric and intestinal digestion were analysed, namely, CEL, CML, MG-H1, and hydroimidazolone 1 derived from GO (G-H1), the binding of these structures in the free form associated with proteins was observed, showing that the link between both survives after the gastrointestinal digestion process, in addition to having the ability of entering the human gastrointestinal tract and triggering a pro-inflammatory environment [39] (Figure 1).

Regarding conformational changes, dietary glycated proteins may naturally trigger changes that can play an important role in the digestion of these sets of glycated amino acids. Studies show that the non-cross-linked structures display a reduction in the digestibility of the glycated protein, as occurred with CML-casein. When submitted to a proteolysis procedure, its digestibility was considerably lower than the native casein [40]. This condition is probably associated with a reduction in molecular accessibility that allows the cleavage of the main chain of the protein. In this context, AGEs that have non-crosslinked structures, such as CMA, CML, and pyrraline, originate from the covalent alteration of residues of

arginine and lysine, and because of this change, can cause trypsin blockage in the intestinal digestion process [9].

The role of gut microbiota and sugar/AGE metabolism: Several recent studies have brought evidence that relates the imbalance of the intestinal microbiota caused by the intake of sugars with insulin resistance, diabetes, obesity [41–43], and metabolic syndrome, by interrupting the immune-mediated protection necessary for body homeostasis [43]. Leptindeficient ob/ob mice, for example, have a completely different microbial composition from normal mice, and the different taxa observed suggest the existence of a microbiota–host crosstalk that relates body composition to the metabolism of animals [42]. Although so far not many details are known about the metabolism of AGEs, there are reports regarding the probable action of the intestinal microbiota in this process, especially regarding Amadori products. This is based on a recent characterization of bacterial enzymes (fructoseamine-6-kinase) that allow the metabolism of these products by different bacteria, such as *Escherichia coli* [44].

In a study carried out by Mastrocola [45], it was reported that a diet enriched with MG-H1 can cause an increase in tissue AGEs, which culminated in an inflammatory imbalance and an alteration in the homeostasis of the intestinal microbiota. It has been proven that the faecal excretion of AGEs does not exceed 50%, and thus it is believed that AGEs are neither absorbed nor excreted and can be metabolized intraluminally by the intestinal microbiota itself. Bacteria such as *Anaerostipes*, *Candidatus Arthromitus*, *Bacteroidales*\_S24-7, *Ruminococcus*, and *Prevotella*, among others, in general (not yet fully understood) are related to the maintenance of the immune system, regulation of the intestinal inflammatory process, insulin sensitization, have a direct relationship with metabolic diseases such as obesity and diabetes, and may undergo changes in their colonies due to the presence of AGEs in the gut [45]. Finally, in another study, this one carried out by Wang, in a trial where AGEs were also supplemented in the diet of animals, the authors also identified a negative change in *Bacteroidales*\_S24-7, *Ruminococcaceae*, and related this condition with increased insulin resistance and a process of chronic inflammation [46].

#### **3. Intestinal Absorption of Glycotoxins**

Dietary AGE bioavailability: Whether dietary glycotoxins are absorbed at the intestinal level and contribute to the endogenous pool is still under debate, and several studies suggest that dicarbonyls and some AGEs may be scavenged and eliminated during digestion [47]. In studies carried out on humans, it was possible to detect mainly MG and GO after glucose intake [48]. However, in another human study carried out with a physiological intake of MG, it was not possible to detect MG or its metabolites in urine [33], suggesting that MG is possibly not absorbed into the bloodstream [32]. However, questions also remain about the methods to measure AGEs and their intermediaries in the circulation. Although several antibodies and kits are available for specific AGEs, they require further validation, because the epitope is an adduct and not an amino acid sequence. Even more complicated is the detection of intermediaries like carbonyls. For instance, MG detection is often made by highperformance liquid chromatography after derivatization. In fact, the most reliable method is mass spectrometry, as stated by Schalkwijk in his work (reviewed by [8]).

About 80% of all Amadori products are degraded by the intestinal flora and do not become AGEs. Moreover, recent evidence suggests that, of all AGEs in the diet, only about 10% are absorbed into the circulation and at least about 30% of these are excreted by urine, being this excretion percentage is higher for free AGEs than peptide-bound AGEs [16]. This was recently observed for the commonly found AGEs CEL, CML, and MG-H1 [49,50]. In animal models, CML accumulation in tissues was observed after consumption of an AGE-enriched diet. Higher concentrations were observed in the kidney, intestine, and lungs (81–320 μg CML g−<sup>1</sup> dry matter), although it was also found in cardiac tissues, muscle, liver, and tendons [9]. Nevertheless, other studies did not find any correlation between dietary AGEs and free and peptide-bound AGEs in circulation [16]. The percentage of urinary recovery varies according to the type of AGE and the individual/model studied. For instance, faecal excretion of dietary CML was shown to be related to its dietary intake in

adolescents, but its urinary excretion apparently has a progressive saturation [15]. Dietary pyrraline was described to be recovered in 50% in urine samples, while the free form of fluorescent AGE pentosine was found in up to 60% in urine after ingestion. For peptidebound pentosane, the urinary recovery was much lower [51]. Free AGEs/glycated amino acids and protein-bound AGEs have distinct bioavailability and their renal excretion may vary according to several factors, such as kidney function, which is often compromised in diabetic patients. In healthy subjects, free AGEs are excreted much more, in part because protein-bound AGEs are reabsorbed in the proximal tubule, although it is also expected that their filtration rate could also be different [16]. However, such mechanisms may be completely different in patients with chronic kidney disease. In both healthy subjects and patients, these measures do not really inform about intestinal absorption, since AGEs may suffer conversion in the gut or after absorption or may simply be retained in tissue.

Mechanisms of dietary AGE absorption: Glycated peptides in general have much less affinity to membrane transporters than native amino acids [9]. Free and peptidebound AGEs are likely to have distinct intestinal absorption mechanisms. AGEs bound to low-molecular-weight peptides like dipeptides were shown to be absorbed by the peptide transporter (PEPT1). These peptide-bound AGEs are later degraded by peptidases inside the enteric cells, appearing as free AGEs on the basolateral side [52] (Figure 1). This was observed, for instance, for pyrraline linked to alanine, but is less observed for other AGEs, suggesting that these mechanisms are AGE-specific [53]. AGEs bound to high-molecular-weight peptides, such as CML, argpyrimidine, or MG-H1, are most likely absorbed by simple diffusion, because they were shown not to inhibit the transport of endogenous ligands of the transporters, namely lysine transporters [54]. This type of AGE needs previous intestinal degradation before absorption, although hydrophobic AGEs like argpyrimidine were shown to pass the intestinal barrier more easily [52]. Transcytosis is also an alternative route of absorption for this type of AGE, although it is not expected to account for the major number of AGEs absorbed. A recent report on *C. elegans* showed that dietary CML may be effectively absorbed by endocytosis in the intestine wall [55]. Interestingly, free AGEs (single modified amino acids) were also shown to be poorly absorbed by peptide transporters and were suggested to be absorbed in low contents by simple diffusion [52]. It may be hypothesized that AGEs have different routes for absorption, considering not only their size but also their hydrophobicity and charge. It is also of note that lumen peptidase may have a role in preventing AGE absorption by cleaving peptides into single amino acids, which have been consistently shown to be poorly absorbed [37]. Furthermore, changes in protein conformation resulting, for instance, from cooking may alter these mechanisms, although further studies are necessary. One main concern about the absorption studies is that some of them are performed in cultured monolayer CACO-2 cells, which may not properly mimic in vivo conditions. Not only the transport system of these cells may be different but also the intracellular machinery responsible for AGE hydrolysis, which has major relevance for their absorption. Another major concern regarding the impact of dietary AGEs is that intestinal permeability has been shown to be increased in metabolic disorders and to be potentiated by poor dietary habits. It is possible that a significant number of dietary AGEs may be absorbed by intercellular transport due to a decrease in barrier integrity, while this is yet to be studied.

Inhibition of AGE intestinal absorption: As mentioned, foods rich in antioxidants and scavenging molecules may prevent intestinal absorption of Maillard reaction products and specific AGEs. However, specific products from the diet can directly inhibit AGE absorption. As also mentioned, pectin oligosaccharides were shown to reduce intestinal AGE formation and absorption at least in in vitro systems [20]. Other compounds, such as catechins and chlorogenic acid, were recently shown to inhibit AGE absorption in vitro [21,56]. Gut microbiota are a major player in degrading many of the diet's nutrients and components, and AGEs are no exception. Recently, it was shown that *Lactococcus lactis* bacteria can degrade dietary CML, mainly through β-galactosidase activity, significantly reducing intestinal absorption in healthy volunteers [57]. Nevertheless, the knowledge about this

topic is still scarce and the discovery of new compounds able not only to prevent gut AGE formation but also their absorption may be a promising research area toward the prevention of harmful effects. In fact, several compounds have been shown in the last few years to scavenge glycotoxins in vitro and some were tested in vivo. Of those molecules, aminoguanidine, pyridoxamine, NAC, resveratrol, and sulforaphane stand out. However, few reports suggest their action in clearing dietary glycotoxins (reviewed by [1,8,58]). Some of them are known activators of NRF2, suggesting that could have beneficial effects on gut anti-glycation defences, although no studies have been reported in this topic. Thus, more research is needed in order to identify molecules able to directly scavenge dietary glycotoxins or to promote their clearance by the gut microbiota.

#### **4. Conclusions and Future Perspectives**

Glycotoxins are a heterogeneous group of compounds that were initially observed to contribute to the development of diabetic complications due to their increased endogenous formation from glucose. Later, data revealed their increased formation and accumulation in tissues, since earlier stages of the disease result from the dysregulated activity of detoxification systems and increased dietary consumption, namely, from high-glucose and high-fructose foods processed at high temperatures.

Despite the little information obtained so far, we have brought here what we already have about the deleterious effect of AGEs and the glycation of target molecules on the whole that involves the process of metabolizing AGEs in the GUT and the triggering of metabolic diseases, immunological and inflammation and nutritional homeostasis. Additionally, the microbial fraction also has incredible importance regarding the molecules absorbed, and the presence of certain strains is often mandatory for the development of physiological and pathological processes.

Its intestinal absorption seems to be AGE-dependent and is apparently more facilitated by AGEs linked to small peptides through peptide transporters. Other routes of absorption have also been suggested for free AGEs and AGEs linked to high-molecular-weight peptides, although their true role in the in vivo absorption of dietary AGEs is still to be proven. Nevertheless, the presence of dietary AGEs in circulation and urine is constant in several studies and the mechanisms of passage through the intestinal barrier should be addressed in the future. Additionally, the identification of molecules able to prevent their absorption may be of significance to reduce their biological impact.

**Author Contributions:** Conceptualization: T.M.-A. and P.M. Methodology: T.M.-A.; and P.M. Writing—original draft preparation: T.M.-A. and P.M. Writing—review and editing: P.M.; Supervision: P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Foundation for Science and Technology (FCT), Portugal (PEst UID/NEU/04539/2013 and UID/NEU/04539/2019: CNC.IBILI; PEst UIDB/04539/2020 and UIDP/04539/2020: CIBB).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

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

#### **Abbreviations**



#### **References**

