Genetic and environmental factors participate in adiposity and metabolic syndrome development (1). They are considered a risk factor to develop pathologies such as cancer (2), cardiovascular disease (3), and diabetes mellitus (4). The initial steps to obesity are in most cases the surplus of nutrients and the lack of physical activity and energy expenditure. However, genetic influence is undoubtedly a strong predisposing factor and diet also plays a significant role as a prooxidant factor (5).
Cardinal signs in metabolic syndrome (MS) are hypertension, dyslipidemia, hyperinsulinemia and insulin resistance, conditions that raise the risk of developing cardiovascular problems, and type 2 diabetes mellitus (DM2). Adipose tissue is metabolically active and secretes hormones called adipokines (6). Some of them have been linked to insulin resistance and fatty liver development (7) such as leptin, tumor necrosis factor α (TNF-α), resistin, and interleukin 6 while adiponectin increases insulin sensitivity.
Pancreatic beta cells are unique because they respond to glucose increase in the extracellular medium secreting insulin. Glucose-stimulated-insulin-secretion coupling (GSISC) involves the serial activation of several proteins and the mechanisms are not completely understood; it has been divided in proximal metabolic and distal ionic apparatus. The proximal component includes the glucose/fructose transporter type 2 (GLUT2) followed by the glycolitic pathway, which results in an increase in the ATP/ADP intracellular ratio that triggers a cascade of electrochemical events (distal ionic component) that culminate in an increase of intracellular calcium ([Ca2+]i) and insulin exocytosis (8).
Diabetes mellitus (DM) is a heterogeneous disorder characterized by abnormally high blood glucose levels. Type 2 DM is the most prevalent form of this disease and has been recognized as a serious health problem, growing significantly worldwide (9). Metabolic problems develop years before type 2 DM manifests clinically. It has been suggested that the main factor that triggers diabetes is centered in beta-cell dysfunction (4).
Peripheral resistance to insulin biological actions on glucose and lipid metabolisms is a main sign in metabolic syndrome (MS) and an important warning to diabetes development. In MS, beta cells are continually stimulated, and secrete high amounts of insulin. Eventually, they become exhausted and incapable of secreting enough hormone to keep normal glucose levels and hyperglycemia develops. The cellular mechanisms that lead to hypersecretion of insulin in the early stages of MS could involve the chain of events in GSISC, such as an increase in glucose transporters, enzymes, or ionic channels.
In an effort to understand and treat MS and DM, these entities had been extensively studied in many different animal models and humans. However, it is very difficult to make comparisons among them, and the goal has not been achieved yet. The molecular features that interact to develop MS are not clear, neither beta-cell exhaustion.
Analysis of short-term changes in metabolism of islet cells by diet is interesting because MS usually starts with an increase in food intake. Understanding the first conditions that appear in MS may contribute to learn how to reverse them on time. This is a clear reason to explore metabolic syndrome animal models, where conditions can be controlled better than in humans.
MS ANIMAL MODELS
Genetically Modified Animals
There are several different genetically modified animals that develop MS and DM2. Among them the most studied are the ob/ob mice (OM) that lack functional leptin and are hyperphagic, obese, show intolerance to glucose and hyperinsulinemia. The metabolism of OM mice appears normal at birth, but at the postweaning period become hyperinsulinemic and fat. MS in adults is milder than during the growth period, but the animals remain sensitive to glucose increase (10). OM also develop hyperglucagonemia, and it has been demonstrated that endogenous glucagon neutralization improves glycemic control by reducing hepatic glucose production, plasma glucose and improving triglycerides, and A1C levels (11). OM beta cells secrete more insulin in basal state than those of normal mice and develop insulin resistance. Metabolic syndrome in OM can be reversed in adults by leptin administration or leptin gene transfection (12). Other models also related to lack of leptin receptor are db/db mice and fa/fa rats (13, 14). In these cases, leptin administration does not modify MS. These models have also been used as a model of obesity followed by DM2. Despite the leptin models have shown useful to understand some metabolic changes in obesity, leptin participates in other functions in the organism. The effects observed in this model are imprint by this fact.
Intolerance to glucose and insulin resistance have been observed in many strains of genetic models of rats, these include, Otzuka Long Evans Tokushima fatty rats (15, 16), nonobese Goto-Kakazaki (17), spontaneous hypertensive rats, hypertriglyceridemic rat (18), and Norway-derived congenic strain PD/Cub rat (19). In these genetic models, diet is also important to define metabolism. However, these models show the indentation of genetic traits that affect other behaviors, which are not related to metabolism.
MS INDUCED BY CHANGES IN DIET
Changing the amount of fat or sugar in the diet affords the possibility of developing other MS and diabetes animal models. MS development in response to different diets is remarkable in rodents. Mice develop MS faster with a high fat diet while rats are more susceptible to the increase in carbohydrates.
MS IN HIGH FAT DIET IN MICE
A high-fat diet may result in metabolic by-product accumulation, activation of intracellular stress response mechanisms, and modifications in hormonal and inflammatory signaling networks among organs (20). The physiological changes observed in high fat diets, largely depend on the amount and type of fat used in the experiments. The amount of fat used varies from 30 to 60% (21), and it comes from different fat sources. Most widely types of fat used are lard, (7) soybean oil (7, 22) olive oil, coconut fat and fish oil (23), butter, and canola (24).
Physiological changes observed in high-fat models mainly include a gain in body weight and adipose tissue mass (21, 7, 25), accompanied by increased free fatty acids, cholesterol, and triglycerides (7, 22, 25). Glucose and insulin plasma levels are increased in several of these models (22, 25), high-insulin secretion has been associated with a beta-cells mass increase. High-fat diets also induced insulin resistance, sometimes accompanied by fat liver infiltration (26), renal fat accumulation, and albuminuria (22).
High-fat diet can lead to an increase in adiposity that may trigger inflammation through adipokines and oxidative stress. The later has been proposed to decrease vasodilation of smallvessels in response to reactive oxygen species (ROS), nitric oxide (NO) scavenging, and smooth muscle decreased sensitivity to NO (27).
The stress of the endoplasmic reticulum (ER) may also play an important role in obesity development. This stress is caused by nutrient excess that overloads the ER leading to a decrease in protein folding or degradation (reviewed in REF. 28). More recently, gene expression in ER stress has been analyzed, showing that protein synthesis was downregulated, while de novo lipogenesis and phospholipid synthesis are upregulated. This altered gene expression leads to inhibition of calcium ATPase (SERCA) activity and stress (29).
Mice are more susceptible to develop obesity and metabolic syndrome with a high-fat diet, while in Wistar rats, this only induces an increase in abdominal fat (25). Finally, animal sex also appears to play an important role, as male Sprague Dawley rats are more sensitive to develop obesity in comparison with females of same strain (21).
MS IN RATS WITH HIGH-CARBOHYDRATE DIET
A direct relationship between high-carbohydrate intake and the mechanisms responsible of the pathological changes observed had not been clearly elucidated. MS in rats has been induced by different amount of high-sucrose diets (25, 30, 31, 32). A widespread model uses 30% sucrose in drinking water (33, 34), starting usually in the postweaning male rat (35, 36). Previous studies showed that during the lactation period in rats, the pancreas experiments structural and functional modifications, and these changes may determine the adult pancreatic physiology (37).
Models with 30% sucrose diet reported an increase in the weight and plasma triglycerides levels, some of them showed an increase in adipose mass tissue, and other changes in the OGTT (oral glucose tolerance test), blood pressure, cholesterol, and plasma glucose levels (35, 36, 38). However, only two of them reported this condition as metabolic syndrome (35, 36). The obese condition was tightly associated to insulin resistance, endorsed by alterations in the glucose response kinetics during an IPGTT (intraperitoneal glucose tolerance test).
Other common MS models are developed by using a high-sucrose or high-fructose diet, in spontaneously hypertensive rats, Sprague Dawley, or Wistar rats. High-fructose diet is composed of 60–80% fructose, with the rest being comprised by protein (∼ 10–20%) and 5% fat. The high-sucrose diet was given by 12–30% sucrose, dissolved in the drinking water, with a normal chow diet.
Wistar Kyoto spontaneous hypertensive rats are basally hyperinsulinemic and insulin resistant, when they are fed with a high-sucrose diet, the observed metabolic changes are not as clear as the observed in the normal Sprague Dawley rats (38).
We had been interested in MS development in normal Wistar rats, because this is a model that does not show a particular susceptibility to develop obesity and diabetes. We used 20% sucrose in drinking water and normal rat chow (Fig. 1A), during 8 weeks in young adult Wistar male rats (250–280 g), resembling to soft drink human consumption, with a normal solid diet (animal care was performed according to the “International Guiding Principles for Biomedical Research Involving Animals,” Council for International Organizations of Medical Sciences, 2010).
Figure 1B shows that body weight in high-sucrose diet group (MS rats) increased by 90% compared to the beginning of the experiment, because they presented a significant increase in the abdominal circumference (AC), related to an increase in the abdominal fat (AF) but not with changes in body length, which remained constant in both groups. This observation is correlated with the body mass index (BMI) that is significantly greater in the treated group (Fig. 1C).
Figure 2 shows the comparison between metabolic parameters in both groups. MS rats showed an increase in the plasma insulin and triglycerides levels (Figs. 2A and 2C, respectively), whereas no differences were observed in glucose and cholesterol plasma levels (Figs. 2B and 2D, respectively). Furthermore, the high-sucrose diet produced a modest increase in the systolic blood pressure (SBP, Fig. 2E), without modifying the cardiac frequency. Finally, tolerance of glucose curve showed a slower glucose clearance in MS rats, compared to controls (Fig. 3).
PANCREATIC BETA-CELL FUNCTION AND DYSFUNCTION
Hyperinsulinemia in rodent models may be due to both an increased beta-cell mass and beta-cell hyperfunction (39, 40). In the MS rats, we observed a tendency to increase insular cell mass (Fig. 4C) and also in the number of small and medium islets. A similar increase in beta-cell mass was observed in nondiabetic obese humans. The opposite was present in obese patients with impaired fasting glucose or type 2 diabetes (38, 39, 41), indicating that beta cells could no longer sustain glucose homeostasis and probably are getting exhausted.
Pancreatic beta cells are unique because they secrete insulin in response to glucose elevation in extracellular medium. The higher levels of insulin in the plasma showed by MS rats could be related to alterations in the structure of the endocrine pancreas, including number and size of islets. Figures 4A and 4B show that the immunoreactive area of islets in MS rats tends to be bigger in comparison to normal rats; however the average was not statistically different (Fig. 4C). In Fig. 4D, we evaluated beta cell size by constructing a frequency distribution. No differences were observed in this parameter between groups, although the percentage of beta cells with area between 76 and 100 μm2 was higher in MS rats. In this model, early MS changes could be more functional than anatomic.
BETA-CELL MOLECULAR MECHANISMS THAT INCREASE INSULIN SECRETION
As mentioned earlier, the most common progression pathway to the development of DM2 begins with obesity and continues with MS. We explored possible changes in the first event in GSISC. GLUT2 is a monosaccharide transporter protein member of a Solute Carrier 2A (SLC2A) gene family that facilitates diffusion of glucose across the cell membrane of pancreatic beta cells (42).
After 8 weeks with 20% sucrose, we observed an islet increase in the total amount of GLUT2 protein associated to an increase in its membrane localization (Figs. 5 and 6, respectively). These findings are interesting and represent an early MS molecular response.
Other models of obesity had shown increased mRNA and protein levels of GLUT2 in pancreatic islets (43, 44). Insulin secretion, as discussed earlier, involves a complex signaling pathway. GLUT2 increase, as well as other molecular changes could be explained by a continued stimulation of beta cells and partially explain insulin hypersecretion.
Transfection of nonbeta-cells AtT20ins with GLUT 2, but not GLUT 1, confers them glucose sensitivity, compared to the nontransfected controls. In these cells, glucose utilization is not different, transfected cells secrete insulin in response to changes in extracellular glucose (45).
Beta cells from the spontaneously hypertensive rats show insulin hypersecretion and a lower threshold to glucose than controls. Glucose utilization in these animals is higher, without changes in glucokinase levels and activity. It has been suggested from these observations that the mechanism that explains hyperinsulinemia in these rats is related to GLUT2 protein amount (46).
It has been suggested that insulin induces GLUT2 internalization in enterocytes and decreased expression in hepatocytes (47). Insulin resistance in mice causes a loss of GLUT2 trafficking, resulting in a permanently high GLUT2 level in enterocytes brush border membrane and reduced expression in the basolateral membrane (48). In addition, evidence points to a mechanism mediated by an intracellular increase in calcium concentration that controls GLUT2 insertion in the apical membrane of enterocytes (49). This mechanism could be involved in the increase of GLUT2 membrane expression in beta cells that occur in the 20% sucrose MS rat model, in which calcium current during depolarization is also increased (not shown). Finally, in the MS rat model, frequent sucrose consumption or an increase in energy intake may stimulate and depolarize pancreatic beta cells, increasing GLUT2 membrane insertion.
Interestingly, it has been observed that GLUT2 mRNA, protein and the membrane localization, in beta cells decrease in different DM2 models, (50–52), indicating that beta cells could no longer be overstimulated, although glucose levels remain high.
In the transition of MS to DM2, multiple cell-specific responses could be implicated; including, changes in protein quantities, function, or location, and thus in signal transduction, starting with GLUT2 in the early stages.
Early changes in metabolic syndrome are regulated by genetic and environmental factors. When caloric content of the diet is raised, metabolic changes appear soon in rodent models. After 8 weeks of 20% sucrose in drinking water treatment, young male adult Wistar rats developed central obesity and insulin resistance and showed higher blood pressure.
One mechanism that could explain a higher production of insulin by pancreatic beta cells include more GLUT2 transporters in the plasmatic membrane of the cells, which is followed by other dysfunction of the cells that leads to a higher insulin secretion that in a long-term treatment may exhaust beta cells, leading to decrease in GLUT2 expression and DM2 development (Fig. 7).
The authors are grateful to Alvaro Caso for reading and discussing the manuscript. The authors are also grateful to Carmen Sánchez for technical academic support, Felix Sierra for technical support; Ana Ma. Escalante and Francisco Pérez Eugenio, from the Computer Unit; Gabriel Orozco-Hoyuela from the Microscopy Unit. All of them from the Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, UNAM. This work was supported by Gobierno del Distrito FederalPICDS08-72, DGAPA-PAPIIT, and SDI.PTID. 05.6 Facultad de Medicina, UNAM.