Cafestol, a Bioactive Substance in Coffee, Stimulates Insulin

Diet and exercise intervention can delay or prevent development of type-2-diabetes (T2D), and high habitual coffee consumption is associated with redu...
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Cafestol, a Bioactive Substance in Coffee, Stimulates Insulin Secretion and Increases Glucose Uptake in Muscle Cells: Studies in Vitro Fredrik Brustad Mellbye, Per Bendix Jeppesen, Kjeld Hermansen, and Søren Gregersen* Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Tage-Hansens Gade 2, 8000 Aarhus C Denmark ABSTRACT: Diet and exercise intervention can delay or prevent development of type-2-diabetes (T2D), and high habitual coffee consumption is associated with reduced risk of developing T2D. This study aimed to test whether selected bioactive substances in coffee acutely and/or chronically increase insulin secretion from β-cells and improve insulin sensitivity in skeletal muscle cells. Insulin secretion from INS-1E rat insulinoma cells was measured after acute (1-h) and long-term (72-h) incubation with bioactive substances from coffee. Additionally, we measured uptake of radioactive glucose in human skeletal muscle cells (SkMC) after incubation with cafestol. Cafestol at 10−8 and 10−6 M acutely increased insulin secretion by 12% (p < 0.05) and 16% (p < 0.001), respectively. Long-term exposure to 10−10 and 10−8 M cafestol increased insulin secretion by 34% (p < 0.001) and 68% (p < 0.001), respectively. Caffeic acid also increased insulin secretion acutely and chronically. Chlorogenic acid, trigonelline, oxokahweol, and secoisolariciresinol did not significantly alter insulin secretion acutely. Glucose uptake in SkMC was significantly enhanced by 8% (p < 0.001) in the presence of 10−8 M cafestol. This newly demonstrated dual action of cafestol suggests that cafestol may contribute to the preventive effects on T2D in coffee drinkers and be of therapeutic interest.

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the reduced risk of T2D in coffee consumers.11−14,19 Most observational studies focus on filtered coffee.6−8 It should be noted that magnesium, cafestol, and kawheol poorly pass a cellulose paper filter.20 Relatively few studies have tried to clarify the mechanisms of action of specific components in coffee. For example, caffeic acid has been found to increase insulin secretion in rat insulinoma INS-1E cells,21 and several studies have focused on chlorogenic acid and its derivatives.22,23 Chlorogenic acid increases insulin secretion from rat islets as well as increases glucose uptake in rat L6 myocytes.24 However, more research is needed to identify and characterize the putative insulin secretory and insulin sensitizing effects of substances in coffee. This may lead to new insight that may be used to decrease the risk of T2D as well as to the development of new antidiabetic medications.

ype-2-diabetes (T2D) is characterized by impaired insulin secretion and/or reduced insulin sensitivity. Prospective, randomized intervention studies1−4 have demonstrated that life-style intervention with diet and exercise can delay or prevent T2D. The global burden of T2D makes it important to identify modifiable risk factors. The preventive capability of a healthy diet, nutritional supplements, and common stimulants such as coffee and tea needs attention. Meta-analyses of observational studies have shown that moderate coffee consumption is inversely associated with development of T2D.5,6 Subjects consuming 3−4 cups of coffee per day have about a 25% lower relative risk of developing T2D, compared to those who drink 1 or fewer cups per day. Interestingly, increasing coffee consumption over a 4-year period is associated with a lower risk of T2D, while reduction of coffee consumption is associated with a higher risk of T2D in the subsequent years.7 Coffee, Cof fea arabica Benth. (Rubiaceae) and Cof fea canephora Benth. (Rubiaceae), contains a diversity of bioactive components that varies with coffee species and the roasting and brewing methods.8−10 The large number of substances includes methylxanthines (especially caffeine), diterpenes (like cafestol and kahweol), the lignan secoisolaricresenol, phenolic acids (e.g., caffeic-, ferulic-, chlorogenic- and quinic acid), trigonelline, minerals, and degradation products. Previously, much attention has been put on the role of caffeine; however, decaffeinated coffee seems to confer similar benefits as regular coffee.5,11−14 Caffeine acutely reduces insulin sensitivity and glucose disposal,15−18 but this effect fades after continuous daily caffeine consumption. Therefore, other substances in coffee besides caffeine may be responsible for © XXXX American Chemical Society and American Society of Pharmacognosy

We hypothesize that specific substances in coffee have beneficial effects on insulin secretion. The aim was to study effects in vitro of specific bioactive substances from coffee, viz., cafestol, caffeic acid, chlorogenic acid, trigonelline, secoisolariciresinol, and oxokahweol on insulin secretion from the clonal rat INS-1E cell line. Subsequently, we tested the impact of Received: June 17, 2015

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DOI: 10.1021/acs.jnatprod.5b00481 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Acute effects on insulin secretion from INS-1E cells after 1-h incubation with bioactive substances from coffee. A 3.3 and 16.7 mM glucose control without addition of the substances was also included. All statistical comparisons are made between the 16.7 mM glucose control and each substance individually. Data are presented as mean ± SEM; *p < 0.05, ***p < 0.001.

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RESULTS AND DISCUSSION Acute Insulin Secretion Studies in INS-1E Cells. The exposure of INS-1E cells to 16.7 mM glucose compared to 3.3 mM glucose significantly stimulated insulin secretion in all experiments. The effects of the coffee related substances were tested at concentrations ranging from 10−12 to 10−6 M in the presence of 16.7 mM glucose. Incubation of INS-1E cells with cafestol showed a significantly higher insulin secretion at concentrations ranging from 10−10 to 10−6 M (Figure 1A). The insulin secretion was increased by 12% (p < 0.05) at 10−10 M, 12% (p < 0.05) at 10−8 M, and 16% (p = 0.001) at 10−6 M of cafestol, respectively. Caffeic acid increased insulin secretion significantly at concentrations ranging from 10−10 to 10−6 M (Figure 1B). The insulin secretion was increased by 9% (p < 0.05) at 10−10 M, 18% (p < 0.001) at 10−8 M, and 9% (p < 0.05) at 10−6 M of caffeic acid, respectively. In contrast, chlorogenic acid did not significantly change insulin secretion,

cafestol on the glucose uptake in isolated human skeletal muscle cells. When selecting the specific substances under investigation, the amount of each substance present in coffee played a major role. Consequently, we selected substances from each of the major chemical structure groups. Thus, diterpenes, lignans, phenolic acids, and plant alkaloids are all represented in the experiments. The methylxanthine caffeine was not included in our study, as it is already thoroughly investigated.11,12 Roasted green Arabica coffee beans consist of 4.5% chlorogenic acid, 0.5% caffeic acid, and 1% trigonelline.9 A cup of coffee (200 mL) contains 70−350 mg of chlorogenic acid and 35− 175 mg of caffeic acid. A cup of Scandinavian boiled coffee, Turkish coffee, and French press coffee contains 6−12 mg cafestol and kahweol, while a cup of filtered coffee contains only 0.2−0.6 mg cafestol and kahweol.25 While a cup of instant coffee contains 1.7 mg secoisolariciresinol, a cup of infused coffee only contains 32 μg.26 B

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although the highest dose of 10−6 M tended to stimulate insulin release (p < 0.1) (Figure 1C). No significant changes in insulin secretion from INS-1E cells were detected with trigonelline (Figure 1D), secoisolariciresinol (Figure 1E), or oxokahweol (Figure 1F). Chronic Incubation Studies in INS-1E Cells. To explore the potential chronic effects of the two insulinotropic substances, cafestol and caffeic acid, we performed 72-h incubations with cafestol and caffeic acid at concentrations ranging between 10−8 and 10−12 M followed by a 60 min stimulation with high glucose (16.7 mM). As expected, the clonal β-cells increased the release of insulin when glucose was elevated from 3.3 to 16.7 mM (Figure 2A,B).

(p < 0.05) (Figure 2B). At 3.3 mM glucose, insulin secretion was significantly lower with 10−12 M caffeic acid (p < 0.05) (Figure 2B). Glucose Uptake in Human Skeletal Muscle Cells. In the presence of 10−8 M cafestol, the glucose uptake in muscle cells was significantly higher (8%) (p < 0.001) compared to control (Figure 3). In the presence of 10−10 and 10−12 M cafestol, no

Figure 3. Chronic effects of cafestol on insulin-stimulated glucose uptake in human muscle skeletal cell line SkMC C-12580. The cells were incubated for 72 h with cafestol. A negative control group without addition of cafestol and a rosiglitazone group (10−8 M) were included. After 15 min of incubation with deoxy-D-glucose and insulin, glucose uptake was measured. “Counts per min” is a direct measure of glucose uptake. All comparisons are made between the negative control and each substance individually. Data are shown as mean ± SEM; ***p < 0.001.

significant difference in glucose uptake was seen. The positive control, rosiglitazone (10−8 M), increased the glucose uptake by 7% (p < 0.001). Dual Beneficial Action of Cafestol. In the present study, we demonstrated that two bioactive substances in coffee, cafestol and caffeic acid, acutely increased insulin secretion from the clonal rat insulinoma cell line INS-1E. Furthermore, we found that both caffeic acid and cafestol had the potential to chronically enhance glucose-stimulated insulin secretion. In addition, we demonstrated that chronic exposure to cafestol increased glucose uptake into human skeletal muscle cells to a similar extent as the antidiabetic agent rosiglitazone. Cafestol and caffeic acid both hold the potential to acutely and chronically increase insulin secretion and cafestol also to improve insulin sensitivity by increasing the glucose uptake in muscle cells. To our knowledge, this dual beneficial action of cafestol has not previously been demonstrated. Cafestol and caffeic acid both acutely increased glucosestimulated insulin secretion from INS1-E cells at concentrations as low as 10−10 M. In the acute studies, the substances seem to be equipotent. However, in the chronic experiments, cafestol appeared to be more potent than caffeic acid in terms of percentual increase in insulin secretion. At low glucose, both substances had minor and inconsistent effects on the insulin secretion. For both substances, there were no indications of acute or chronic toxic effects on insulin secretion even at the highest concentrations. In addition, we demonstrated that trigonelline, secoisolariciresinol, oxokahweol, and chlorogenic acid at concentrations of 10−6 to 10−12 M had no acute stimulatory effect on insulin release. Absence of a stimulatory effect of chlorogenic acid contrasts previous results24 that found an insulinotropic effect of chlorogenic acid in INS-1E cells.

Figure 2. Chronic effects on insulin secretion from INS-1E cells after 72-h incubation with (A) cafestol and (B) caffeic acid, and then a 60 min incubation with either 3.3 or 16.7 mM glucose. A 3.3 and 16.7 mM glucose a control without addition of the substances was also included. All statistical comparisons with 16.7 mM glucose are made between the 16.7 mM glucose control and each 16.7 mM glucose substance individually. All statistical comparisons with 3.3 mM glucose are made between the 3.3 mM glucose control and each 3.3 mM glucose substance individually. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Long-term (72-h) incubation of cafestol at 10−8 and 10−10 M resulted in a prominent increase in insulin secretion at high (16.7 mM) glucose [34% (p < 0.001) and 68% (p < 0.001), respectively] (Figure 2A). At low (3.3 mM) glucose, the insulin secretion was significantly lowered at 10−12 M cafestol (by 15% (p < 0.01)), neutral at 10−10 M cafestol, and increased at 10−8 M cafestol (by 27% (p < 0.001)) (Figure 2A). Following 72-h incubation with 10−8 M caffeic acid, glucose(16.7 mM)-stimulated insulin secretion was increased by 16% C

DOI: 10.1021/acs.jnatprod.5b00481 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and the high absorption rate29 suggest that at least the lowest concentrations used in our study may be achieved in the circulation of coffee drinkers. Potential Antidiabetic Capabilities. To our knowledge, the present study is the first to demonstrate short- and longterm effects of cafestol on insulin secretion in vitro and a beneficial long-term effect of cafestol on glucose-uptake in skeletal muscle cells. Thus, cafestol may play a role in preventing T2D in coffee consumers. Since consumption of coffee normally is on a continuous daily basis, it is especially relevant to study long-term rather than acute effects of coffee. Further research is needed to confirm our findings and to extend and characterize the mechanisms of action of bioactive substances in coffee. Our study points to the potential use of cafestol for the prevention and treatment of T2D.

There may be various explanations for this. One explanation of the diverging results of chlorogenic acid could be ascribed to differences in concentrations tested. While we used chlorogenic acid at 10−6 to 10−12 M, Tousch et al. used chlorogenic acid at the extreme high concentrations of 3 × 10−2 and 1.4 × 10−1 M. Such high concentrations are unlikely to occur in the circulation after acute or chronic coffee intake and limit the relevance of their results. Other explanations could be related to differences in incubation time (60 min in our study versus 30 min).24 Administration of trigonelline in Wistar rats resulted in reduced pancreatic islet and β-cell damage, increased GLP-1 levels, reduced plasma glucose during an oral glucose tolerance test, and inhibition of DPP-4 and α-glucosidase activity in plasma and the small intestine.27 We did not observe direct effects of trigonelline in our study. A conceivable explanation for this discrepancy is that the effects of trigonelline may be indirect e.g., via increase in GLP-1. It is noteworthy that structurally similar substances do not seem to exert equivalent effects on insulin secretion. The diterpenes cafestol and oxokahweol and phenolic acids caffeic and chlorogenic acid were expected to display similar insulin secretory capabilities. The lack of an insulinotropic effect of oxokahweol in our study was surprising, and the reason remains unknown. We have chosen only to study the chronic effects of cafestol and caffeic acid, which had rather prominent acute insulinotropic effects. In contrast, many other substances tested did not acutely affect insulin secretion in vitro. However, it cannot be ruled out that they may possess chronic effects despite not detecting any acute effects. Furthermore, we did not explore for potential additive effects of the coffee-related substances. Substances present in coffee with and without caffeine, e.g., chlorogenic acid and trigonelline, have previously been shown to acutely alter the early phase insulin response and glucose levels during oral glucose tolerance test in overweight men.13 We have chosen only to test the impact on glucose uptake in human skeletal muscle tissue in vitro for the bioactive substance, cafestol, since it had the most prominent insulinotropic effect after long-term exposure. More studies are needed to explore if other substances may alter insulin secretion and affect glucose uptake in the human muscle cell line. T2D is characterized by abnormal islet cell function28 including impaired glucose stimulated insulin secretion as well as profound insulin resistance in insulin sensitive tissues. We conducted in vitro studies in the human skeletal muscle cell line SkMC C-12580 to clarify if cafestol in addition to its potent insulinotropic effect also can improve muscle glucose uptake as a surrogate measure of insulin sensitivity. We found a clear cafestol-induced glucose uptake in vitro in the muscle cells, an effect comparable to that observed with an equimolar concentration of rosiglitazone. Rosiglitazone acts as a PPARγbinding insulin sensitizer. Even though the effect of cafestol on glucose uptake is similar to that of rosiglitazone, the mechanism of action might not be the same. Further studies could reveal this more thoroughly. As previously mentioned, cafestol poorly passes the cellulose filter applied for production of filter coffee. Since most studies claiming that coffee consumption can prevent or delay the onset of T2D are made with filtered coffee, it is unlikely that cafestol is the sole explanation for the positive effects of coffee in relation to prevention of T2D. However, the levels of cafestol in, e.g., French press coffee (1.7−3.5 mg/100 mL)20

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EXPERIMENTAL SECTION

General Experimental Procedures. Chemicals. Cafestol acetate (Sigma, Steinheim, Germany), ≥98% (Tandem Mass Spectrometry); trigonelline hydrochloride (Sigma), ≥98% (High-performance liquid chromatography (HPLC)); chlorogenic acid (Sigma), ≥95% (titration); secoisolariciresinol (Sigma), ≥95% (HPLC); caffeic acid (Sigma), ≥98% (HPLC); rosiglitazone (Sigma), ≥98% (HPLC). Cell Culture. INS-1E cells were cultured in modified RPMI 1640 (GIBCO, Life Technologies, Naerum, Denmark) (11.1 mM glucose, 10 mM HEPES (Sigma), 5 μM β-mercaptoethanol (Sigma), 10% inactivated fetal calf serum (GIBCO), 1% penicillin/streptomycin (GIBCO), and sterile filtered 0.3 μm), in a humidified atmosphere at 37 °C, 5.0% CO2 and passaged weekly. For glucose uptake studies, we used proliferating human skeletal muscle cells (SkMC C-12580) (Promocell, Heidelberg, Germany). As the cells were stored in a dimethyl sulfoxide (DMSO, Sigma) solution at −80 °C, they were thawed and centrifuged for 10 min at 1000 rpm in order to remove DMSO. Cells were then prepared as instructed in the Promocell manual, except that we washed the cells during subcultivation with Dulbecco’s phosphate buffered saline (PBS, Sigma) instead of Hanks buffered salt solution (HBSS.) Incubation Experiments. In the acute insulin secretion studies, the following substances were tested: cafestol acetate, trigonelline, chlorogenic acid, secoisolariciresinol, and caffeic acid. For the chronic insulin secretion studies, cafestol acetate and caffeic acid were tested. All substances were dissolved in DMSO at a stock concentration of 10−3 M. The stock solutions were serially diluted in a modified KrebsRinger buffer (M-KRB, Sigma) pH 7.4 (0.1 M NaOH adjusted) with 125 mM NaCl, 5.9 mM KCl, 5.9 mM MgCl, 1.2 mM CaCl2, 5.0 mM NaHCO3, 25.0 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) (Sigma), and 1% Bovine serum albumin (Sigma), added at 16.7 mM glucose, ending with 10−6, 10−8, 10−10, and 10−12 M. DMSO was added to 10−8, 10−10, and 10−12 to obtain the same DMSO concentration in all solutions. A 3.3 mM glucose and a 16.7 mM glucose control M-KRB solution were used as control. Acute Insulin Secretion Studies. Cells were seeded (1 mL/well, 0.3 × 106 cells/mL) onto a 24-well plate (Nunc A/S, Roskilde, Denmark) and pre-pre-incubated for 72 h at 37 °C, 5.0% CO2. Hereafter, the medium was removed. Cells were preincubated for 15 min with M-KRB. After preincubation, the M-KRB was removed and the wells were placed on ice. Hereafter, 1 mL of the solutions containing the above-mentioned substrates or controls was added. After 60 min of incubation at 37 °C, 5.0% CO2, the cells were placed on ice. Supernatants (300 μL) were transferred to tubes and centrifuged for 2 min at 1000 rpm. Supernatants (200 μL) were collected and stored at −20 °C for later insulin analysis. After the secretion study, the substrates were removed from the well and 1 mL of 0.1 M NaOH was added to each well for 30 min and then transferred to tubes for measurement of the protein content. Chronic Insulin Secretion Studies. Substrates of investigation were serially diluted in the cell-growth-medium to reach final concentrations of 10−8, 10−10, and 10−12 M. DMSO was added to D

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ensure same concentration in all wells. Cells were then seeded (1 mL/ well, 0.3 × 106 cells/mL) onto a 24-well plate and incubated for 72 h at 37 °C, 5.0% CO2. Medium was removed and the adhered cells were preincubated for 15 min with 1 mL of M-KRB. After preincubation, the M-KRB was removed and the wells were placed on ice. Hereafter, 1 mL of M-KRB with either 3.3 or 16.7 mM glucose was added to each well. After 60 min of incubation at 37 °C, 5.0% CO2, the cells were placed on ice. Supernatants (300 μL) were transferred to tubes and centrifuged at 1000 rpm for 3 s. Supernatants (200 μL) were collected and stored at −20 °C for later insulin analysis. Glucose Uptake in Human Skeletal Muscle Cells. Human skeletal muscle cells were seeded into 24 wells (0.3 × 106 cells/well) containing 1 mL of growth medium (Promocell, Heidelberg, Germany). Once 70−90% confluence was reached, the growth medium was replaced by 1 mL differentiation medium (Promocell). This medium was changed every second day for 2 weeks until multinucleated syncytia were visible in microscope. Then, the differentiation medium was adjusted and made into five groups: (1) control, (2) cafestol (10−8 M), (3) cafestol (10−10 M), (4) cafestol (10−12 M), and (5) rosiglitazone (10−8 M). After 72 h of incubation, cells were washed twice with PBS. Three hundred microliters of a modified-Krebs Ringer buffer with 0.1% BSA, 0.1 mM glucose, 1.5 μCi deoxy-D-glucose 2-[1,2−3H(M)] (PerkinElmer, Skovlunde, Denmark) and 100 nM of insulin (Human insulin (I2643), Sigma-Aldrich Danmark A/S, Copenhagen, Denmark) was added to each well. Cells were kept on ice while adding medium. After 15 min of incubation at 37 °C, 5.0% CO2, cells were washed twice with M-KRB supplemented with 0.1% BSA and 50 mM glucose, stopping the incubation and glucose uptake. NaOH (0.2 mL 0.1 M) was added to each well and kept for 30 min at room temperature. Hereafter, 0.1 mL was transferred from each well to a 24 well counting plate (Wallac Oy, Turku, Finland). After adding 0.9 mL of Hisafe II scintillator (PerkinElmer, Skovlunde, Denmark), plates were stored in the dark for 12 h before counted using a Trilux Micro Beta Counter (Wallac Oy, Turku, Finland). “Counts per min” is a direct measure of glucose uptake. Insulin Assay. Insulin was measured using a guinea pig antiporcine insulin antibody and mono-125I-(Tyr A14)-labeled human insulin as tracer and rat insulin as standard (all from Novo Nordisk A/ S, Bagsvaerd, Denmark). Bound and free radioactivity was separated using ethanol.30 Protein Analysis. The cells used in acute insulin secretion studies were controlled by protein amount, as the protein content of the cell suspensions was analyzed using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).31,32 Statistical Analysis. All statistical analysis was performed with GraphPad Prism Software (San Diego, CA). After confirming a Gaussian distribution of the results, comparison was made using the Students unpaired t-test. Data are presented as mean ± SEM; p-values