Sulfonylurea induction of caffeine-enhanced insulin secretion and reduction of glycemic levels in diabetic rats

Abstract

Context: Caffeine can stimulate insulin secretion by attenuating hyperglycemia in diabetes models with significant reduction of pancreatic functional β cells. Knowledge of these mechanisms could contribute to new strategies for treating diabetes.

Objective: This study evaluated the effects of caffeine and physical exercise on glycemic and insulin responses in diabetic rats.

Materials and methods: The diabetes model was induced by intraperitoneal administration of 60 mg/kg of streptozotocin (STZ). Animals were divided into six groups: control, caffeine, STZ control, STZ caffeine, STZ sulfonylurea, and STZ caffeine + sulfonylurea. Acutely, control animals received 6 mg of caffeine and 10 mg/kg sulfonylurea or 10 mg/kg saline. Animals were sacrificed after physical exercise; blood samples were collected for glucose, glycerol, lactate, and insulin analyses. Cardiovascular responses were recorded before and after treatments. A one-way ANOVA and the post hoc Student–Newman–Keuls test were used to analyze statistical differences between treatments (p < 0.05).

Results: About 6 mg/kg of caffeine did not alter cardiovascular responses, but promoted blood glucose reduction after 60 min of exercise when compared to animals in the control groups (387–187 mg/dL; p < 0.05). Insulin levels increased significantly (0.6–10 µU/mL; p < 0.05) in rats that received acute caffeine treatment associated with sulfonylurea compared to rats in the other groups.

Discussion and conclusion: Acute caffeine intake with exercise can increase glucose uptake enhancing insulin secretion stimulated by sulfonylurea in β cells-deficient pancreas. The results indicate the potential use of caffeine as a strategy for glycemic and insulin control in diabetes.

• Blood glucose
• diabetes mellitus
• exercise
• streptozotocin

Introduction

Streptozotocin (STZ, 2-deoxy-2-[3-methyl-3-nitrosourido]-d-glucopyranose) is a glucose analogue that has a diabetogenic effect, damaging pancreatic β cells (Szkudelski, 2012). Evidences suggest that the glucose transporter 2 (GLUT-2) transports STZ into cells and causes necrosis by oxidative damage (Waer & Helmy, 2012). Moreover, STZ inhibits the ligation between insulin and its receptor in the cell periphery causing alterations of up to 84% in the cell membrane (Takada et al., 2007).

Caffeine is an alkaloid in the methylxanthines group (1,3,7-trimethylxanthine), with a half-life of 2.5–4.5 h, that is rapidly absorbed by the gastrointestinal tract and metabolized in the liver (Graham, 2001). The main mechanisms exerted by caffeine are linked to intracellular Ca²⁺ mobilization, increase of catecholamines, and antagonism to adenosine receptors (Paluska, 2003).

It has been reported that caffeine can stimulate insulin secretion through pancreatic β cells due to an increase in intracellular Ca²⁺ (Park et al., 2009). Caffeine can increase the expression of glucose transporter 4 (GLUT-4) in the skeletal muscle, as a result of increased concentration of intracellular Ca²⁺ and enzyme expression activated by AMP (AMPK) (Conde et al., 2012; Park et al., 2009). In addition, caffeine is involved in antagonism to adenosine receptors in the cell membrane of hepatocytes, which are involved in glycogenolysis and gluconeogenesis (Yasuda et al., 2003).

Free fatty acids are important for ATP re-synthesis in skeletal muscle during prolonged exercise of moderate intensity (Mougios et al., 2003). Caffeine has an important effect on the increase of fatty acids (Acheson et al., 2004) and glycerol (Murosaki et al., 2007), which are substrates for muscle and liver metabolisms, respectively. Caffeine inhibits cAMP degradation via the phosphodiesterase enzyme (PDE), resulting in the accumulation of cAMP and stimulating the release of catecholamines, which are responsible for increasing fatty acids (Yasuda et al., 2003).

Exercise causes important changes in glucose homeostasis and can rapidly lower blood glucose (BG) levels in diabetics. Monitoring glycemic status and consumption of drugs before, during, and after exercise is fundamental to the safety of diabetics engaging in physical activities (ADA, 2006). The intake of energy substrates or substances that aid in glycemic control is considered good strategies to avoid cardiovascular overloads or hypoglycemic rebounds during and after physical activity (ACSM, 2010).

Few studies evaluating the effects of caffeine intake associated with physical exercise in experimental diabetes models are found in the literature (Greenberg et al., 2006; Lee et al., 2005). Some evidence suggests possible effects of caffeine or aerobic exercise on insulin resistance (Egawa et al., 2009); however, the effect of both components on the metabolic control of diabetes is still unknown. Thus, the development of a study to evaluate the potential effect of caffeine on lipid metabolism and glycemic and insulin control, together with the insulin sensitizer effect provoked by physical exercise, could contribute to new strategies for the control of diabetes.

The objective of the present study is to determine the effects of caffeine supplementation associated with sulfonylurea and physical exercise on insulin and glycemic responses in diabetic rats.

Materials and methods

Animals

Forty-eight male Wistar rats with average weights of 116 ± 3.0 g and at 60 d of age were used in the study. The animals were kept in cages with controlled temperature (23 ± 2 °C) and humidity (55 ± 10% humidity), and a light/dark cycle of 12 h.

This study was approved by the Ethics Committee of research studies using animals (026/2011 Protocol).

Experimental design

The animals were divided into six groups: (1) control, (2) caffeine, (3) STZ control, (4) STZ caffeine, (5) STZ sulfonylurea, and (6) STZ caffeine + sulfonylurea. STZ (Sigma, St. Louis, MO) dissolved in citrate buffer (0.01 M, pH 4.5) was administered intraperitoneally (i.p.) (60 mg/kg), after 12 h of fasting. The rats with stabilized diabetes having fasting BG values between 150 and 250 mg/dL were considered as diabetic.

The animals received 6 mg/kg of caffeine (Sigma, St. Louis, MO) or 10 mg/kg of sulfonylurea glibenclamide (Sigma, St. Louis, MO) diluted in water, or placebo (0.9% saline) by gavages after 8 h of fasting.

Exercise protocol

All animals were adapted to an aquatic environment to be able to swim during the test, through one daily session of 10 min, for 7 d prior to the experiment. After treatment and on testing days, the animals performed a 60-min session of predominantly aerobic exercises by carrying an extra weight equivalent to 6% of their body weight in a swimming tank with 40 cm in depth, 70 cm in diameter, and water heated to 30 ± 1 °C, according to the protocol proposed by Gobatto et al. (2001).

Cardiovascular analyses

The heart rate (HR) values, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were obtained using a tail plethysmograph that transmitted data to software that codified the results (Insight®, Ribeirão, Preto, Brazil). In order to adapt animals to this device, the device was attached to their tails three times a day for 5 d before the test. On the day of the test, HR, SBP, and DBP were obtained in triplicate for all animals before and after treatments and prior to exercise.

Biochemical and hormonal analyses

The BG dosing was performed in a glucometer (ACCU-CHEK® Active®, ACCU-CHEK, Mainland, China) using approximately 25 µL of blood collected through caudal puncture, before and after intake of the testing substances.

The animals were sacrificed after the exercise protocol. Blood (5 mL) was collected from each animal and stored in tubes containing fluoride for the analysis of glucose and lactate and tubes without anticoagulant for the analysis of glycerol and insulin. Samples were centrifuged at 1500 rpm for 10 min to separate the serum and analyzed in a semi-automatic biochemical analyzer (DIAGLOBE CA-2006®, Diamond Diagnostics, Holliston, MA) for glucose, lactate, and glycerol using a BioSystems kit (Biosystems, Foster City, CA). Plasma insulin was measured by chemiluminescence (DPC immulite 2000R, Diagnostic Products Corporation, Los Angeles, CA).

Statistical analysis

All results are presented as mean ± EPM. Statistical analysis was performed using a one-way ANOVA. Values were considered statistically significant based on p < 0.05. The post hoc Student–Newman–Keuls test was used, when appropriate, to identify differences between groups.

Results

Cardiovascular analyses

Caffeine intake did not lead to significant changes in the cardiovascular variables (Table 1).

Table 1. Cardiovascular responses before and after treatments and before physical exercise (n = 48).

	HR (bpm)		SBP (mm/Hg)		DBP (mm/Hg)
	Fasting	Post-treatment	Fasting	Post-treatment	Fasting	Post-treatment
Control	375 ± 5	336 ± 6	124 ± 2	126 ± 3	79 ± 4.0	80 ± 2.5
Caffeine	450 ± 24	359 ± 28	125 ± 1	126 ± 2	83 ± 3.4	81 ± 3.0
STZ	434 ± 17	433 ± 17	126 ± 1	120 ± 5	82 ± 2.5	81 ± 2.5
STZ caffeine	436 ± 19	433 ± 25	123 ± 2	123 ± 1	81 ± 2.6	82 ± 3.8
STZ sufonylurea	436 ± 19	433 ± 25	123 ± 2	123 ± 1	81 ± 2.0	82 ± 3.8
STZ caffeine + sufonylurea	427 ± 17	417 ± 22	125 ± 12	124 ± 3	83 ± 4.0	82 ± 5.0

HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure. Data are presented as means ± EPM (one-way ANOVA with the post hoc Student–Newman–Keuls test).

Biochemical and hormonal analysis

The STZ caffeine group showed reduction in BG levels compared to the control STZ group after caffeine supplementation (p < 0.05). The BG levels decreased after exercise in the STZ caffeine group when compared to the STZ group (p < 0.05) (Figure 1).

Figure 1. Blood glucose behavior during treatments. The values are presented as mean ± EPM (n = 48). (§) Statistically different compared to the pre-treatment state in the same group (p < 0.05); (*) statistically different compared to the pre-exercise state in the same group (p < 0.05); (a, b, c) different letters indicate statistically significant difference between groups (p < 0.05; Student–Newman–Keuls after one-way ANOVA).

The effects of caffeine and sulfonylurea on plasma insulin, lactate, and glycerol levels are presented in Table 2.

Table 2. Effects of caffeine and sulfonylurea on plasma insulin, lactate, and glycerol levels after the exercise protocol was applied.

	Insulin (µU/mL)	Glycerol (mg/dL)	Lactate (mmol/L)
Control	7.4 ± 0.2^a	87 ± 10^a	5.6 ± 0.03^a
Caffeine	4.7 ± 0.09^a,b	158 ± 13^b	5.9 ± 2^a
STZ	0.6 ± 0.01^b	76 ± 42^a	5.70 ± 1.2^a
STZ caffeine	0.7 ± 0.01^b	165 ± 95^b	6.35 ± 0.9^a
STZ sulfonylurea	0.6 ± 0.04^b	174 ± 19^b	5.92 ± 0.6^a
STZ caffeine + sulfonylurea	10 ± 0.22^a	162 ± 18^b	8.12 ± 0.1^b

The values are presented as mean ± EPM. ^a,bDifferent letters indicate statistically significant difference between groups (p < 0.05; Student–Newman–Keuls after a one-way ANOVA).

Plasma insulin levels of rats in the STZ caffeine + sulfonylurea group were similar to the levels in the control group; however, they differed significantly from the plasma insulin levels of rats in the STZ, STZ sulfonylurea, and STZ caffeine groups after caffeine intake and exercise [F_(3.29) = 5.7; p < 0.05].

Plasma glycerol levels increased in the caffeine, STZ caffeine, STZ sulfonylurea, and STZ caffeine + sulfonylurea groups after their respective treatments and exercise, and showed significantly increased levels compared to the control and STZ groups [F_(3.29) = 5.7; p < 0.05].

Blood lactate was observed at the highest level in the STZ caffeine + sulfonylurea group after treatment and exercise when compared to the other treatments [F_(3.29) = 5.7; p < 0.05].

Discussion

The present study showed that caffeine intake, pre-, and post-exercise significantly reduced the BG levels in diabetic rats. In addition, an increase in plasma insulin concentration was observed after caffeine supplementation associated with sulfonylurea in the STZ-induced diabetic rats.

A weak metabolic response to circulating insulin levels contributes to disorders in peripheral tissues (muscle, liver, and adipose tissue) and in the central nervous system (hypothalamic neurons involved in food control) (Gallagher et al., 2008). In obese and sedentary individuals with diabetes type 2 or pre-diabetes type 2, the resistance presented by cells to sensitize their insulin receptors is increased 75% in the skeletal muscle and 2–3% in fat tissue (Defronzo, 2004).

Previous studies showed increased expression of GLUT-2 and glucokinase in the liver after caffeine ingestion, an event that could affect glycogenesis (Park et al., 2007). Chu et al. (2011) showed that the administration of 0.05 mg/mL of coffee could increase glucose uptake in isolated adipocytes because of a possible increase in GLUT-4. A 25% reduction in the BG values was observed in the STZ caffeine group after caffeine intake without exercise when compared to the fasting period for the same group. The comparison between the STZ and STZ caffeine groups after caffeine ingestion and exercise showed 42% reduction in glycemic levels.

In the present study, the effect of increased insulin, provided by sulfonylurea before stress, does not necessarily lead to increased glucose consumption, especially in muscle cells. However, the use of caffeine, regardless of exercise, can stimulate an increased consumption of glucose via increase of GLUT-4 in muscle cells, as shown in previous studies (Conde et al., 2012; Park et al., 2009). The concomitant use of sulfonylurea and caffeine results in less glycemia reduction compared to the use of caffeine only, and does not promote an inhibitory effect on muscle glycolysis (Larsen et al., 1999) caused by the presence of insulin. In addition, caffeine is well known for its stimulatory adrenergic effect, which stimulates muscle glycolysis and consequently could reduce glycemic levels, even at rest.

In a situation of stress, i.e., during exercise, caffeine proved to be more effective than when used in association with sulfonylurea. Significant increases in muscle glucose consumption via glycolysis are needed during exercise. It is well known that the adrenergic effect of exercise promotes the acceleration of muscle metabolic pathways involved in glucose consumption, and thus caffeine could be amplifying this signal. The presence of sulfonylurea associated with caffeine significantly increases insulin levels. However, the diabetes model used in this study is deficient in receptors and presents a reduction of up to 80% in the number of receptors compared to the streptozotocin model (Takada et al., 2007). Thus, during exercise, the presence of sulfonylurea may not necessarily result in higher glucose consumption and could influence in reducing the glycolytic speed due to high insulinemia, according to the already well-discussed inhibitory glycolytic effect of insulin (Alves-Wagner et al., 2009; Luciano et al., 2002).

Park et al. (2007) report that caffeine intake improved the glucose homeostasis through an increased insulin release, possibly through insulin growth factor 1 signaling (IGF-1), which is a factor present during the insulin release cascade in β cells. Wang et al. (2013) demonstrated that the administration of caffeine (120 mg/kg) stimulates the expression of IGF-1 and its respective pathways in 75% over the normal expression level. In the present study, caffeine intake with sulfonylurea led to a significant increase in blood insulin concentration in diabetic rats; insulin plasma levels were equivalent to that of rats without diabetes. This finding suggests that caffeine intake with sulfonylurea could potentially be used in glycemic control because of the increase in insulin release in patients with diabetes type 1. However, it should be noted that the number of pancreatic β-cells is decreased in this pathology and insulin overproduction could lead to a long-term failure in the remaining pancreatic β cells, it should be noted that the number of pancreatic β-cells is decreased by 10–30% in this pathology and insulin overproduction could lead to a long-term failure in the remaining pancreatic β cells. Therefore, the survival of pancreatic β-cells could be evaluated through the analysis of the effect of chronic administration of caffeine associated with sulfonylurea in the experimental diabetes model developed in the current study.

The data obtained indicate that glycerol increases in the groups that received caffeine compared to the other groups showing 90% difference between their means. Zheng et al. (2008) demonstrated that caffeine intake increased glycerol and free fatty acids levels in mice. According to these authors, this effect may occur through a sympathetic stimulation elevating the oxidation of triglycerides in fat cells. Larsen et al. (1999) report greater release of catecholamines when sulfonylurea was associated with exercise compared to the control group. This could explain the 100% increase in glycerol concentration in the STZ, sulfonylurea group when compared to its control. Therefore, further studies comparing levels of catecholamines could determine if this increase in serum glycerol would have an influence on catecholamine levels after isolated or combined intake of caffeine or sulfonylurea.

Larsen et al. (1999) observed increased lactacidemia when the use of glibenclamide sulfonylurea was associated with exercise, leading to enhanced stored muscle glucose, greater glucose degradation, and increased blood lactate levels. However, in this study, STZ sulfonylurea + caffeine group data showed increased lactacidemia but failed to show a difference in the serum lactate levels in the STZ sulfonylurea group. Nevertheless, caffeine and sulfonylurea could be stimulating the reduction of pyruvate in the skeletal muscle cells increasing serum levels of lactate through adrenergic stimulation. Further studies dosing plasma levels of catecholamines after supplementation with sulfonylurea and caffeine during and post-exercise could provide answers to this question.

Noordzij et al. (2005) demonstrated that the influence of caffeine on cardiovascular stimulation is directly related to the amount of caffeine ingested. The effect of caffeine on cardiovascular responses results from the classic caffeine effect of increasing the release of catecholamines, which directly influence the sympathetic nervous system and consequently elevates blood pressure (Nurminen et al., 1999; Paluska, 2003). Therefore, the lack of significant differences among the groups studied could be related to the 6 mg/kg dose of caffeine as being insufficient to produce definitive differences.

Conclusion

Caffeine significantly decreased BG levels and enhanced insulin secretion stimulated by sulfonylurea without the presence of cardiovascular alterations. In addition, caffeine intake increased triglyceride mobilization during exercise, which was observed through the elevated levels of serum glycerol. These results indicate the potential use of caffeine as an interesting strategy for glycemic and insulin response controls in diabetic patients. Further studies elucidating the dose–response effects of caffeine intake before and after exercise may contribute to validating this therapeutic strategy as important for the management of diabetes.

Declaration of interest

The authors are thankful to CAPES (Brazil) and Fundação Araucaria of Paraná (Brazil) for the financial support to this study. There are no issues to disclose. There is no potential conflict of interest with the mentioned trademarks. This manuscript has been reviewed by a professional science editor and a native English-speaking science editor.