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Timing of caffeine ingestion alters postprandial metabolism in rats
Nutrition 30 (2014) 107–111
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Basic nutritional investigation
Timing of caffeine ingestion alters postprandial metabolism in rats Sara Farhat Jarrar M.Sc., Omar Ahmad Obeid Ph.D. * Department of Nutrition and Food Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2013 Accepted 16 July 2013
Objective: The association between caffeine intake and the risk for chronic diseases, namely type 2 diabetes, has not been consistent, and may be influenced by the timing of caffeine ingestion. The aim of this study was to investigate the acute effect of caffeine administered in different scenarios of meal ingestion on postprandial glycemic and lipidemic status, concomitant with changes in body glycogen stores. Methods: Forty overnight-fasted rats were randomly divided into five groups (meal-ingested, caffeine-administered, post-caffeine meal-ingested, co-caffeine meal-ingested, post-meal caffeineadministered), and tube-fed the appropriate intervention, then sacrificed 2 h later. Livers and gastrocnemius muscles were analyzed for glycogen content; blood samples were analyzed for glucose, insulin, triglycerides, and non-esterified fatty acid concentrations. Results: Postprandial plasma glucose concentrations were similar between groups, while significantly higher levels of insulin were witnessed following caffeine administration, irrespective of the timing of meal ingestion. Triglyceride concentrations were significantly lower in the caffeineadministered groups. Regarding glycogen status, although caffeine administration before meal ingestion reduced hepatic glycogen content, co- and post-meal caffeine administration failed to produce such an effect. Muscle glycogen content was not significantly affected by caffeine administration. Conclusions: Caffeine administration seems to decrease insulin sensitivity as indicated by the sustenance of glucose status despite the presence of high insulin levels. The lower triglyceride levels in the presence of caffeine support the theory of retarded postprandial triglyceride absorption. Caffeine seems to play a biphasic role in glucose metabolism, as indicated by its ability to variably influence hepatic glycogen status. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Caffeine Meal ingestion Glycogen Glucose Insulin Triglycerides Fatty acids
Introduction Coffee has attracted considerable attention in the field of nutritional sciences, where an increasing body of evidence supports a protective role of habitual consumption on risk factors of non-communicable diseases, namely type 2 diabetes [1,2]. There has been much debate, however, over the effect of coffee on glucose and insulin responses, with some studies suggesting no postprandial changes [3–6], whereas others report significant increases in postprandial glycemic and insulinemic responses [7, 8]. Coffee contains numerous bioactive compounds that may influence glucose and insulin homeostasis, including chlorogenic
OAO designed the study and performed the statistical analysis. SFJ performed the biochemical analytical methods. Both authors conducted the research experiment and wrote the paper. * Corresponding author. Tel.: þ961 1 350000; fax: þ961 1 744460. E-mail address: [email protected] (O. A. Obeid). 0899-9007/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2013.07.015
acid [9] and quinides [10], as well as the mineral magnesium [11]. Coffee, however, also contains caffeine, a methylxanthine that has been found to impair glucose tolerance and insulin sensitivity among healthy individuals and those with type 2 diabetes in short-term metabolic studies [12–14], as well as longer-term studies [15,16]. Liver and muscle glycogen are the main reservoirs of stored glucose in the body, acting as the key suppliers of glucose to maintain homeostasis in the body [17]. Caffeine has the ability to influence body stores of glycogen, although results have been conflicting given caffeine’s simultaneous ability to prevent and induce glycogen breakdown through its varying role on different enzymatic systems in the liver and muscle. On the one hand, caffeine has been found to be a strong inhibitor of hepatic and skeletal muscle glycogen phosphorylase, the enzyme responsible for glycogenolysis [18]; competing for the binding of glycogen phosphorylse a in a synergistic manner [19,20]. On the other hand, caffeine has been documented to stimulate the
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sympathetic nervous system [21], leading to increased release of catecholamines (epinephrine and norepinephrine), which are known to stimulate glycogenolysis [22]. There is controversy surrounding the effects of caffeine on glycemic control and glycogen sparing, and there is limited evidence of the effect of caffeine on glycemic changes concomitant with changes in body glycogen stores. The aim of this study was to examine the effect of a moderate dose of caffeine on blood glucose and insulin levels, concomitant with changes in liver and skeletal muscle glycogen content, as well as blood triglyceride (TG) and non-esterified fatty acid (NEFA) levels in different scenarios of the ingestion of a control meal. Because most dietary sources of caffeine are habitually consumed in circumstances where food may be present, investigating the effect of caffeine with, before, and after a meal may, therefore, shed some light on whether caffeine leads to different physiological effects on glycemic control, while explaining the results in the context of changes in hepatic and muscle glycogen stores and other biochemical markers. Materials and methods Animal housing Forty 6-wk-old male Sprague-Dawley rats (Animal House, American University of Beirut, Lebanon) were individually housed in separate wire-bottomed cages in a temperature-controlled (22 C 1 C) and light-controlled room (12:12 dark/light cycle; light on at 0700 h) for 1 wk. During this period, rats were allowed free access to tap water and ad libitum intake of a semi-synthetic powder control meal adapted from an earlier study [23] (Table 1), and both body weight and food intake was monitored. Experimental protocol The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the medical school at the American University of Beirut. On the experimental day, overnight-fasted rats (to eliminate the effect of residual food in the stomach on postprandial metabolism), with a mean body weight of 222 3.09 g, were randomly divided into five groups (n ¼ 8) and tubefed the appropriate intervention as follows: the meal-ingested group (ML) received 1 g of the control meal; the caffeine-administered group (CAF) received 10 mg caffeine; the post-caffeine meal ingested group (CAF-MLþ1) received 10 mg caffeine followed 1 h later by 1 g of the control meal; the co-caffeine mealingested group (MLþCAF) simultaneously received 1 g of the control meal and 10 mg of caffeine; and the post-meal caffeine-administered group (ML-CAFþ1) received 1 g of the control meal followed 1 h later by 10 mg of caffeine. The 1 g of the control meal and 10 mg of caffeine were individually mixed in 3 mL of water before each administration to each of the rats for better dissolution, providing equal volumes of feedings. The 10 mg of caffeine supplemented is equivalent to around 45 mg/kg body weight in rats. Two hours after the experimental intervention, rats were sacrificed and blood was drained from the neck vessels into tubes containing ethylenediaminetetraacetic acid (EDTA) and regular plasma tubes with no EDTA. Blood samples were kept on ice until they were centrifuged for plasma collection within 2 h then were subsequently frozen at –80 C until time of analysis. Livers and gastrocnemius muscles were removed and frozen in liquid nitrogen then stored at –70 C until analysis [23].
Table 1 Composition of the semisynthetic control meal Ingredients
Quantity (g/kg)
Sucrose Cornstarch Casein Corn oil Alphacel (cellulose) Mineral mix (AIN-93G)* Vitamin mix (AIN-76A)* DL-methionine
300 300 198 100 55 35 10 2
* Purchased from Dyets Inc. experimental diets & ingredients for laboratory animals.
Analytical methods Blood was collected at the time of sacrifice and kept on ice, then centrifuged at 3000g for 15 min at 4 C. Plasma was collected and frozen at –80 C until analysis. Glucose and TG concentrations were determined using an enzymatic colorimetric method on the Vitros DT 60 II Chemistry System (Ortho-Clinical Diagnostics, Johnson and Johnson, New York, NY, USA). Insulin concentration was determined using the Rat/Mouse Insulin Elisa Kit 96-well plate for the quantification of non-radioactive insulin (Millipore, Billerica, MA, USA), whereas NEFA concentration was determined using an in vitro enzymatic colorimetric method assay via the NEFA-HR(2) diagnostic kit (Wako diagnostics, Neuss, Germany). Hepatic glycogen extraction took place following standard methods described in detail elsewhere [24,25]. Gastrocnemius muscle glycogen extraction followed the process whereby whole muscles were weighed and mechanically homogenized in a Bench-Top PRO Scientific 300 homogenizer (Oxford, CT, USA) with 5 mL 0.5 M perchloric acid at high speed for a few minutes. Extracts (5 mL) were precipitated with 0.8 mL 0.2M potassium hydroxide. Samples were then centrifuged at 3000g for 10 min. Muscle extracts of 5 mL were then transferred to individual conical tubes where 10 mL ethanol (96%) was added on ice. The obtained solutions were vortexed and then placed overnight in the refrigerator to allow for precipitation. The steps that followed were similar to those for hepatic glycogen extraction. Statistical analysis Data were analyzed using the statistical program Mini-Tab 16.1 for Windows. Statistical treatment of the data included unpaired t test for comparing results of group ML with the CAF group and the CAF-MLþ1 group, while oneway analysis of variance (ANOVA) was used to compare results of the ML group with the MLþCAF and ML-CAFþ1 groups because all retained the meal for 2 h. A probability of P < 0.05 was considered statistically significant. Results are displayed as mean SEM.
Results Body weights and food intake of rats were similar among the different groups, indicating consistent growth and weight gain before the experimental day. Effect of post-caffeine meal ingestion The effect of caffeine administration alone on postprandial metabolism was assessed by comparing the ML and CAF groups (Table 2). Plasma glucose concentration was similar between the two groups, whereas plasma insulin concentration of the CAF group was significantly higher than that of the ML group (P ¼ 0.005). Plasma NEFA levels were similar between groups, although the CAF group showed slightly, but not significantly, higher levels than the ML group. Moreover, the CAF group showed significantly lower plasma TG levels than the ML group (P ¼ 0.005). Mean liver weights of the ML group were higher than that of the CAF group (P ¼ 0.03). Likewise, hepatic glycogen contents per gram in the ML group were significantly higher than the CAF group (P ¼ 0.002). No significant differences were observed in terms of muscle weight and glycogen contents between the two groups. The effect of meal ingestion 1 h after caffeine administration (post-caffeine meal ingestion) on postprandial metabolism was assessed by comparing groups CAF and CAF-MLþ1. Measured plasma metabolites, organ weights, and glycogen contents were similar between groups. Thus, meal ingestion after caffeine administration failed to alter postprandial metabolism. Effect of co- and post-meal caffeine administration The effect of caffeine administration co- and post-meal ingestion on post-prandial metabolism was assessed by comparing groups ML, MLþCAF, and ML-CAFþ1 (Table 3). Plasma glucose concentration was similar between groups, indicating that caffeine administration co- and post-meal failed to alter
S. F. Jarrar, O. A. Obeid / Nutrition 30 (2014) 107–111
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Table 2 Glucose and glycogen status of rats 2 h after caffeine administration: Effect of post-caffeine meal ingestion Biological marker
Experimental group ML (n ¼ 8)
Plasma Glucose (mg/dL) Insulin (ng/mL) NEFA (mmol/L) TG (mg/dL) Liver Weight (g) Glycogen (mg/g liver) Muscle Weight (g) Glycogen (mg/g muscle)
126.4 0.56 0.81 94.13
Significance CAF (n ¼ 8)
4.6 0.10 0.07 8.76
122.5 0.97 1.17 59.75
6.7 0.10 0.17 4.23
CAF-MLþ1 (n ¼ 8)
114.9 0.94 1.24 58.25
t test P-value (ML vs. CAF)
t test P-value (CAF vs. CAF-MLþ1)
5.4 0.12 0.19 3.48
0.64 0.005 0.08 0.005
0.39 0.83 0.78 0.79
7.76 0.41 7.59 1.08
6.61 0.21 2.32 0.69
6.97 0.23 2.43 0.46
0.030 0.002
0.27 0.9
1.38 0.05 4.62 0.32
1.35 0.04 4.35 0.40
1.38 0.04 3.99 0.33
0.63 0.62
0.52 0.5
CAF, caffeine-administered group; CAF-MLþ1, post-caffeine meal-ingested group; ML, meal-ingested group; NEFA, non-esterified fatty acids; TG, triglycerides Values are presented as mean SEM P-values resulted from unpaired t test. Those in bold indicate statistical significance (P < 0.05)
glucose levels. Caffeine administration (whether co- or postmeal ingestion) led to a significantly higher insulin concentration compared with the ML group (P ¼ 0.036), but no statistical difference was found between the two caffeine-administered groups. Plasma NEFA levels were found to be similar between groups, although levels of groups MLþCAF and ML-CAFþ1 were slightly, although not significantly, higher than the ML group. Plasma TG levels followed the same pattern observed when analyzing the effects of post-caffeine meal ingestion (Table 1), being significantly higher in the ML group (P ¼ 0.000) compared with the two caffeine-administered groups (with no observed difference among these two). ANOVA of liver weights showed marginal significance between the different groups (P ¼ 0.05). However, post hoc analysis was able to detect lower liver weights in the MLþCAF group compared with the ML group. Liver glycogen content per gram Table 3 Glucose and glycogen status of rats 2 h after meal ingestion: Effect of co- and post-meal caffeine administration Biological marker
Experimental group ML (n ¼ 8)
MLþCAF (n ¼ 8)
Significance ML-CAF (n ¼ 8)
þ1
Plasma Glucose 126.4 4.6 116.4 3.7 122.1 2.3 (mg/dL) y 0.80 0.06y Insulin 0.56 0.10* 0.78 0.09 (ng/mL) NEFA 0.81 0.07 1.03 0.13 1.02 0.08 (mmol/L) TG (mg/dL) 94.13 8.76* 53.38 4.96y 67.13 3.74y Liver Weight (g) 7.76 0.41* 6.76 0.19y 7.18 0.16*,y Glycogen 7.59 1.08 7.55 1.38 9.82 0.43 (mg/g liver) Muscle Weight (g) 1.38 0.05 1.31 0.09 1.33 0.05 Glycogen 4.62 0.32 4.87 0.56 6.043 0.75 (mg/g muscle)
ANOVA P-value 0.18 0.036 0.21 0.000 0.05 0.23
0.76 0.19
ANOVA, analysis of variance; CAF, caffeine-administered group; CAF-MLþ1, postcaffeine meal-ingested group; CI, confidence interval; ML, meal-ingested group; MLþCAF, co-meal caffeine administered group; ML-CAFþ1, post-meal caffeine administered group; NEFA, non-esterified fatty acids; TG, triglycerides Values are presented as mean SEM P-values resulted from one-way analysis of variance (ANOVA). Those in bold indicate statistical significance (P < 0.05) ,y * Values in the same row with different superscripts are significantly different based on Fisher’s pairwise comparison 95% CI.
was similar between groups, with a trend toward a higher content in the ML-CAFþ1 group. No significant differences were observed between groups regarding muscle weight and glycogen content. However, differences in muscle glycogen content showed a similar trend to differences in hepatic glycogen among groups, where higher muscle glycogen content was observed in the ML-CAFþ1 group. Discussion The effects of caffeinated coffee on glycemic and insulinemic changes following a carbohydrate meal have found conflicting results, with some studies suggesting no postprandial effects [3,5], whereas others find significant glycemic and insulinemic responses to caffeinated coffee [8,26]. Other human studies investigating the acute effect of caffeine administration on glucose and insulin changes following a glucose solution have found similar adverse glycemic and insulinemic responses [12–14]. A recent meta-analysis of randomized controlled trials evaluating the effects of green tea catechins with or without caffeine on glycemic control showed significant reductions in fasting blood glucose concentrations (1.48 mg/dL; 95% confidence interval, 2.57 to 0.4 mg/dL; P ¼ 0.008), with greater reductions (although not significant) in trials using caffeine-containing formulations compared with trials using green tea catechins alone [27]. Following an overnight fast, glycogenolysis contributes to about only half the production of hepatic glucose, whereas the other half is supplied from the process of gluconeogenesis [28], which is mainly triggered by glucagon [22], and similarly by caffeine [29]. Thus, the similar blood glucose concentrations observed between when caffeine was administered on its own compared with the meal-ingested group, are most probably due to enhanced gluconeogenic processes in the presence of caffeine. High levels of insulin, which are normally known to suppress gluconeogenesis [22], in the caffeine-administered groups, may indicate that caffeine possibly overrode the gluconeogenic inhibitory effect of insulin. Moreover, the similarity in plasma glucose among the caffeine groups persisted despite the higher levels of insulin, indicating the presence of insulin insensitivity. The similarity in insulin levels between the caffeine groups, regardless of meal ingestion or its timing, may be due to the capacity of caffeine to acutely increase pancreatic b-cell stimulation [30]. This stimulatory effect seems to be independent of the presence of a meal, unlike what others have found. It has been reported that caffeinated coffee administered 1 h
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before a carbohydrate meal [8] and coadministered with a meal [26] elicited greater glycemic and insulinemic responses compared with decaffeinated coffee. It seems that in the presence of caffeine, the involvement of a meal (exogenous glucose) in plasma glucose sustenance is not clear and may imply that several mechanisms are involved. When caffeine was administered 1 h before meal ingestion, the low hepatic glycogen might be the consequence of inhibited glycogen synthesis, despite the availability of exogenous glucose. It was expected that glycogen content following meal ingestion in the presence of caffeine would be higher than when caffeine was administered on its own, as postprandial glycogen synthesis in the presence of insulin is normal, even in a 1-h interval following meal ingestion [24,31]. However, the development of insulin resistance after caffeine administration may have influenced hepatic glycogen synthesis, because elevated insulin from infusion [32] in the presence of obesity [31] has been shown to reduce glycogenesis. Moreover, it has been found that the effect of caffeine supplementation (400 mg) on glucose metabolism is highly pronounced starting at about 60 to 90 min after supplementation, as indicated by the increase in plasma lactate [33]. Caffeine is known to be a potent antagonist of adenosine receptors, which can influence insulin binding and signaling, as well as translocation of glucose transporters [12]. Given that adenosine receptor antagonists can inhibit glucose metabolism even in the presence of insulin [34,35], caffeine is likely to influence the metabolism of carbohydrates [36]. Moreover, caffeine’s adenosine receptor–antagonistic effect leads to increased levels of norepinephrine [37], which in turn stimulates the production of lactate from the skeletal muscle [38], implying that such an effect may be initiated 1 h after caffeine ingestion. In line with this, the low hepatic glycogen content observed in our study when the meal was ingested 1 h after caffeine might be the result of the initiation of caffeine’s adenosine receptor–antagonistic effect before meal ingestion. Bearing in mind the findings of a study [33] regarding the delayed onset of caffeine’s adenosine receptor–antagonistic effect of around 1 h, the increase in insulin during the hour following caffeine administration, in the case when caffeine was given 1 h after the meal, may have led to anabolic effects, where such an effect would be suppressed later on. In fact, while caffeine administration with and after meal ingestion did not produce significant differences in hepatic glycogen compared with meal ingestion, there appeared to be a trend toward higher glycogen content when caffeine followed meal ingestion, allowing for the assumption of possible glycogen synthesis. Thus, caffeine seems to have a biphasic action on glycogen synthesis, in which glycogen synthesis is stimulated in the first hour and inhibited subsequently. Regarding NEFAs, the observed levels in the caffeineadministered groups were found to be higher, although not significantly, regardless of meal ingestion and its timing. This is in line with others, where caffeine administration is known to increase circulating NEFA levels [39–41], which is a consequence of caffeine’s known lipolytic [42] and TG–fatty acid cycling effects in the liver [30,33], and possibly the adipose tissue [43]. NEFAs are an indicator of adipose tissue and muscle lipolysis, and are well known to reduce insulin sensitivity [44,45], consequently decreasing glucose disposal by the skeletal muscle [12]. Thus, it is probable that the higher NEFA levels following caffeine administration contributed to decreased insulin sensitivity that was further exacerbated by caffeine’s antagonistic effect on adenosine receptors. Thus, the inhibitory effect of insulin on lipolysis [46] may have been partially blunted by the lipolytic effect of caffeine.
Regarding serum TGs, the observed lower levels in the caffeine groups, especially in the cases when caffeine was administered before or with the meal, raises a question as to whether absorption of TGs from the meal was retarded. A green tea extract containing 5% caffeine, among other polyphenols, has been reported to inhibit in vitro activity of both gastric and pancreatic lipase (the main enzymes involved in the postprandial breakdown of TGs) [47], whereas caffeine supplementation in amounts of 6.6 and 13.2 mg did not seem to affect pancreatic lipase [48]. Moreover, serum TG levels are inversely related to increasing caffeine doses (5, 10, and 20 mg) in rats fed a high-fat diet, in which higher fecal TG excretion was described at the highest caffeine dose [40]. Caffeine also has been found to decrease mesenteric lymph flow and inhibit the absorption of fatty acids, by possibly influencing the intracellular packaging and secretion of lipids via chylomicrons [49]. As for muscle weight and glycogen content, the absence of significant differences between groups indicates that caffeine did not exert an effect on muscle glycogen. Interestingly, mean muscle glycogen content appeared to be the highest, although not significantly, in the post-meal caffeine administration scenario, likely demonstrating peripheral glucose uptake by the skeletal muscle during the 1-h period before initiation of the physiological sequelae of caffeine. However, why mean levels of glycogen were even higher than that of the ML group remains to be explained. Conclusions Postprandial plasma glucose status was maintained in all caffeine-administered groups regardless of meal ingestion and timing, whereas insulin concentration in these groups was elevated, implying a possible state of reduced insulin sensitivity. Additionally, postprandial TG levels were decreased, likely due to reduced fat absorption. Although caffeine administration pre-meal prevented hepatic glycogen synthesis, administration with and after meal ingestion seems to have overridden this inhibitory effect; eliciting a possible biphasic action on postprandial glucose metabolism. Acknowledgments This study was supported by a grant from the University Research Board of the American University of Beirut. References [1] Huxley R, Lee CM, Barzi F, Timmermeister L, Czernichow S, Perkovic V, et al. Coffee, decaffeinated coffee, and tea consumption in relation to incident type 2 diabetes mellitus: a systematic review with meta-analysis. Arch Intern Med 2009;169:2053–63. [2] von Ruesten A, Feller S, Bergmann MM, Boeing H. Diet and risk of chronic diseases: results from the first 8 years of follow-up in the EPIC-Potsdam study. Eur J Clin Nutr; 2013:1–8. [3] Aldughpassi A, Wolever TMS. Effect of coffee and tea on the glycaemic index of foods: no effect on mean but reduced variability. Br J Nutr 2009;101:1282–5. [4] Battram DS, Arthur R, Weekes A, Graham TE. The glucose intolerance induced by caffeinated coffee ingestion is less pronounced than that due to alkaloid caffeine in men. J Nutr 2006;136:1276–80. [5] Hatonen KA, Virtamo J, Eriksson JG, Sinkko HK, Erlund I, Jousilahti P, et al. Coffee does not modify postprandial glycaemic and insulinaemic responses induced by carbohydrates. Eur J Nutr 2012;51:801–6. [6] Johnston KL, Clifford MN, Morgan LM. Coffee acutely modifies gastrointestinal hormone secretion and glucose tolerance in humans: glycemic effects of chlorogenic acid and caffeine. Am J Clin Nutr 2003;78:728–33. [7] Lane JD, Hwang AL, Feinglos MN, Surwit RS. Exaggeration of postprandial hyperglycemia in patients with type 2 diabetes by administration of caffeine in coffee. Endocr Pract 2007;13:239–43.
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Basic nutritional investigation
Timing of caffeine ingestion alters postprandial metabolism in rats Sara Farhat Jarrar M.Sc., Omar Ahmad Obeid Ph.D. * Department of Nutrition and Food Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2013 Accepted 16 July 2013
Objective: The association between caffeine intake and the risk for chronic diseases, namely type 2 diabetes, has not been consistent, and may be influenced by the timing of caffeine ingestion. The aim of this study was to investigate the acute effect of caffeine administered in different scenarios of meal ingestion on postprandial glycemic and lipidemic status, concomitant with changes in body glycogen stores. Methods: Forty overnight-fasted rats were randomly divided into five groups (meal-ingested, caffeine-administered, post-caffeine meal-ingested, co-caffeine meal-ingested, post-meal caffeineadministered), and tube-fed the appropriate intervention, then sacrificed 2 h later. Livers and gastrocnemius muscles were analyzed for glycogen content; blood samples were analyzed for glucose, insulin, triglycerides, and non-esterified fatty acid concentrations. Results: Postprandial plasma glucose concentrations were similar between groups, while significantly higher levels of insulin were witnessed following caffeine administration, irrespective of the timing of meal ingestion. Triglyceride concentrations were significantly lower in the caffeineadministered groups. Regarding glycogen status, although caffeine administration before meal ingestion reduced hepatic glycogen content, co- and post-meal caffeine administration failed to produce such an effect. Muscle glycogen content was not significantly affected by caffeine administration. Conclusions: Caffeine administration seems to decrease insulin sensitivity as indicated by the sustenance of glucose status despite the presence of high insulin levels. The lower triglyceride levels in the presence of caffeine support the theory of retarded postprandial triglyceride absorption. Caffeine seems to play a biphasic role in glucose metabolism, as indicated by its ability to variably influence hepatic glycogen status. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Caffeine Meal ingestion Glycogen Glucose Insulin Triglycerides Fatty acids
Introduction Coffee has attracted considerable attention in the field of nutritional sciences, where an increasing body of evidence supports a protective role of habitual consumption on risk factors of non-communicable diseases, namely type 2 diabetes [1,2]. There has been much debate, however, over the effect of coffee on glucose and insulin responses, with some studies suggesting no postprandial changes [3–6], whereas others report significant increases in postprandial glycemic and insulinemic responses [7, 8]. Coffee contains numerous bioactive compounds that may influence glucose and insulin homeostasis, including chlorogenic
OAO designed the study and performed the statistical analysis. SFJ performed the biochemical analytical methods. Both authors conducted the research experiment and wrote the paper. * Corresponding author. Tel.: þ961 1 350000; fax: þ961 1 744460. E-mail address: [email protected] (O. A. Obeid). 0899-9007/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nut.2013.07.015
acid [9] and quinides [10], as well as the mineral magnesium [11]. Coffee, however, also contains caffeine, a methylxanthine that has been found to impair glucose tolerance and insulin sensitivity among healthy individuals and those with type 2 diabetes in short-term metabolic studies [12–14], as well as longer-term studies [15,16]. Liver and muscle glycogen are the main reservoirs of stored glucose in the body, acting as the key suppliers of glucose to maintain homeostasis in the body [17]. Caffeine has the ability to influence body stores of glycogen, although results have been conflicting given caffeine’s simultaneous ability to prevent and induce glycogen breakdown through its varying role on different enzymatic systems in the liver and muscle. On the one hand, caffeine has been found to be a strong inhibitor of hepatic and skeletal muscle glycogen phosphorylase, the enzyme responsible for glycogenolysis [18]; competing for the binding of glycogen phosphorylse a in a synergistic manner [19,20]. On the other hand, caffeine has been documented to stimulate the
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sympathetic nervous system [21], leading to increased release of catecholamines (epinephrine and norepinephrine), which are known to stimulate glycogenolysis [22]. There is controversy surrounding the effects of caffeine on glycemic control and glycogen sparing, and there is limited evidence of the effect of caffeine on glycemic changes concomitant with changes in body glycogen stores. The aim of this study was to examine the effect of a moderate dose of caffeine on blood glucose and insulin levels, concomitant with changes in liver and skeletal muscle glycogen content, as well as blood triglyceride (TG) and non-esterified fatty acid (NEFA) levels in different scenarios of the ingestion of a control meal. Because most dietary sources of caffeine are habitually consumed in circumstances where food may be present, investigating the effect of caffeine with, before, and after a meal may, therefore, shed some light on whether caffeine leads to different physiological effects on glycemic control, while explaining the results in the context of changes in hepatic and muscle glycogen stores and other biochemical markers. Materials and methods Animal housing Forty 6-wk-old male Sprague-Dawley rats (Animal House, American University of Beirut, Lebanon) were individually housed in separate wire-bottomed cages in a temperature-controlled (22 C 1 C) and light-controlled room (12:12 dark/light cycle; light on at 0700 h) for 1 wk. During this period, rats were allowed free access to tap water and ad libitum intake of a semi-synthetic powder control meal adapted from an earlier study [23] (Table 1), and both body weight and food intake was monitored. Experimental protocol The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the medical school at the American University of Beirut. On the experimental day, overnight-fasted rats (to eliminate the effect of residual food in the stomach on postprandial metabolism), with a mean body weight of 222 3.09 g, were randomly divided into five groups (n ¼ 8) and tubefed the appropriate intervention as follows: the meal-ingested group (ML) received 1 g of the control meal; the caffeine-administered group (CAF) received 10 mg caffeine; the post-caffeine meal ingested group (CAF-MLþ1) received 10 mg caffeine followed 1 h later by 1 g of the control meal; the co-caffeine mealingested group (MLþCAF) simultaneously received 1 g of the control meal and 10 mg of caffeine; and the post-meal caffeine-administered group (ML-CAFþ1) received 1 g of the control meal followed 1 h later by 10 mg of caffeine. The 1 g of the control meal and 10 mg of caffeine were individually mixed in 3 mL of water before each administration to each of the rats for better dissolution, providing equal volumes of feedings. The 10 mg of caffeine supplemented is equivalent to around 45 mg/kg body weight in rats. Two hours after the experimental intervention, rats were sacrificed and blood was drained from the neck vessels into tubes containing ethylenediaminetetraacetic acid (EDTA) and regular plasma tubes with no EDTA. Blood samples were kept on ice until they were centrifuged for plasma collection within 2 h then were subsequently frozen at –80 C until time of analysis. Livers and gastrocnemius muscles were removed and frozen in liquid nitrogen then stored at –70 C until analysis [23].
Table 1 Composition of the semisynthetic control meal Ingredients
Quantity (g/kg)
Sucrose Cornstarch Casein Corn oil Alphacel (cellulose) Mineral mix (AIN-93G)* Vitamin mix (AIN-76A)* DL-methionine
300 300 198 100 55 35 10 2
* Purchased from Dyets Inc. experimental diets & ingredients for laboratory animals.
Analytical methods Blood was collected at the time of sacrifice and kept on ice, then centrifuged at 3000g for 15 min at 4 C. Plasma was collected and frozen at –80 C until analysis. Glucose and TG concentrations were determined using an enzymatic colorimetric method on the Vitros DT 60 II Chemistry System (Ortho-Clinical Diagnostics, Johnson and Johnson, New York, NY, USA). Insulin concentration was determined using the Rat/Mouse Insulin Elisa Kit 96-well plate for the quantification of non-radioactive insulin (Millipore, Billerica, MA, USA), whereas NEFA concentration was determined using an in vitro enzymatic colorimetric method assay via the NEFA-HR(2) diagnostic kit (Wako diagnostics, Neuss, Germany). Hepatic glycogen extraction took place following standard methods described in detail elsewhere [24,25]. Gastrocnemius muscle glycogen extraction followed the process whereby whole muscles were weighed and mechanically homogenized in a Bench-Top PRO Scientific 300 homogenizer (Oxford, CT, USA) with 5 mL 0.5 M perchloric acid at high speed for a few minutes. Extracts (5 mL) were precipitated with 0.8 mL 0.2M potassium hydroxide. Samples were then centrifuged at 3000g for 10 min. Muscle extracts of 5 mL were then transferred to individual conical tubes where 10 mL ethanol (96%) was added on ice. The obtained solutions were vortexed and then placed overnight in the refrigerator to allow for precipitation. The steps that followed were similar to those for hepatic glycogen extraction. Statistical analysis Data were analyzed using the statistical program Mini-Tab 16.1 for Windows. Statistical treatment of the data included unpaired t test for comparing results of group ML with the CAF group and the CAF-MLþ1 group, while oneway analysis of variance (ANOVA) was used to compare results of the ML group with the MLþCAF and ML-CAFþ1 groups because all retained the meal for 2 h. A probability of P < 0.05 was considered statistically significant. Results are displayed as mean SEM.
Results Body weights and food intake of rats were similar among the different groups, indicating consistent growth and weight gain before the experimental day. Effect of post-caffeine meal ingestion The effect of caffeine administration alone on postprandial metabolism was assessed by comparing the ML and CAF groups (Table 2). Plasma glucose concentration was similar between the two groups, whereas plasma insulin concentration of the CAF group was significantly higher than that of the ML group (P ¼ 0.005). Plasma NEFA levels were similar between groups, although the CAF group showed slightly, but not significantly, higher levels than the ML group. Moreover, the CAF group showed significantly lower plasma TG levels than the ML group (P ¼ 0.005). Mean liver weights of the ML group were higher than that of the CAF group (P ¼ 0.03). Likewise, hepatic glycogen contents per gram in the ML group were significantly higher than the CAF group (P ¼ 0.002). No significant differences were observed in terms of muscle weight and glycogen contents between the two groups. The effect of meal ingestion 1 h after caffeine administration (post-caffeine meal ingestion) on postprandial metabolism was assessed by comparing groups CAF and CAF-MLþ1. Measured plasma metabolites, organ weights, and glycogen contents were similar between groups. Thus, meal ingestion after caffeine administration failed to alter postprandial metabolism. Effect of co- and post-meal caffeine administration The effect of caffeine administration co- and post-meal ingestion on post-prandial metabolism was assessed by comparing groups ML, MLþCAF, and ML-CAFþ1 (Table 3). Plasma glucose concentration was similar between groups, indicating that caffeine administration co- and post-meal failed to alter
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Table 2 Glucose and glycogen status of rats 2 h after caffeine administration: Effect of post-caffeine meal ingestion Biological marker
Experimental group ML (n ¼ 8)
Plasma Glucose (mg/dL) Insulin (ng/mL) NEFA (mmol/L) TG (mg/dL) Liver Weight (g) Glycogen (mg/g liver) Muscle Weight (g) Glycogen (mg/g muscle)
126.4 0.56 0.81 94.13
Significance CAF (n ¼ 8)
4.6 0.10 0.07 8.76
122.5 0.97 1.17 59.75
6.7 0.10 0.17 4.23
CAF-MLþ1 (n ¼ 8)
114.9 0.94 1.24 58.25
t test P-value (ML vs. CAF)
t test P-value (CAF vs. CAF-MLþ1)
5.4 0.12 0.19 3.48
0.64 0.005 0.08 0.005
0.39 0.83 0.78 0.79
7.76 0.41 7.59 1.08
6.61 0.21 2.32 0.69
6.97 0.23 2.43 0.46
0.030 0.002
0.27 0.9
1.38 0.05 4.62 0.32
1.35 0.04 4.35 0.40
1.38 0.04 3.99 0.33
0.63 0.62
0.52 0.5
CAF, caffeine-administered group; CAF-MLþ1, post-caffeine meal-ingested group; ML, meal-ingested group; NEFA, non-esterified fatty acids; TG, triglycerides Values are presented as mean SEM P-values resulted from unpaired t test. Those in bold indicate statistical significance (P < 0.05)
glucose levels. Caffeine administration (whether co- or postmeal ingestion) led to a significantly higher insulin concentration compared with the ML group (P ¼ 0.036), but no statistical difference was found between the two caffeine-administered groups. Plasma NEFA levels were found to be similar between groups, although levels of groups MLþCAF and ML-CAFþ1 were slightly, although not significantly, higher than the ML group. Plasma TG levels followed the same pattern observed when analyzing the effects of post-caffeine meal ingestion (Table 1), being significantly higher in the ML group (P ¼ 0.000) compared with the two caffeine-administered groups (with no observed difference among these two). ANOVA of liver weights showed marginal significance between the different groups (P ¼ 0.05). However, post hoc analysis was able to detect lower liver weights in the MLþCAF group compared with the ML group. Liver glycogen content per gram Table 3 Glucose and glycogen status of rats 2 h after meal ingestion: Effect of co- and post-meal caffeine administration Biological marker
Experimental group ML (n ¼ 8)
MLþCAF (n ¼ 8)
Significance ML-CAF (n ¼ 8)
þ1
Plasma Glucose 126.4 4.6 116.4 3.7 122.1 2.3 (mg/dL) y 0.80 0.06y Insulin 0.56 0.10* 0.78 0.09 (ng/mL) NEFA 0.81 0.07 1.03 0.13 1.02 0.08 (mmol/L) TG (mg/dL) 94.13 8.76* 53.38 4.96y 67.13 3.74y Liver Weight (g) 7.76 0.41* 6.76 0.19y 7.18 0.16*,y Glycogen 7.59 1.08 7.55 1.38 9.82 0.43 (mg/g liver) Muscle Weight (g) 1.38 0.05 1.31 0.09 1.33 0.05 Glycogen 4.62 0.32 4.87 0.56 6.043 0.75 (mg/g muscle)
ANOVA P-value 0.18 0.036 0.21 0.000 0.05 0.23
0.76 0.19
ANOVA, analysis of variance; CAF, caffeine-administered group; CAF-MLþ1, postcaffeine meal-ingested group; CI, confidence interval; ML, meal-ingested group; MLþCAF, co-meal caffeine administered group; ML-CAFþ1, post-meal caffeine administered group; NEFA, non-esterified fatty acids; TG, triglycerides Values are presented as mean SEM P-values resulted from one-way analysis of variance (ANOVA). Those in bold indicate statistical significance (P < 0.05) ,y * Values in the same row with different superscripts are significantly different based on Fisher’s pairwise comparison 95% CI.
was similar between groups, with a trend toward a higher content in the ML-CAFþ1 group. No significant differences were observed between groups regarding muscle weight and glycogen content. However, differences in muscle glycogen content showed a similar trend to differences in hepatic glycogen among groups, where higher muscle glycogen content was observed in the ML-CAFþ1 group. Discussion The effects of caffeinated coffee on glycemic and insulinemic changes following a carbohydrate meal have found conflicting results, with some studies suggesting no postprandial effects [3,5], whereas others find significant glycemic and insulinemic responses to caffeinated coffee [8,26]. Other human studies investigating the acute effect of caffeine administration on glucose and insulin changes following a glucose solution have found similar adverse glycemic and insulinemic responses [12–14]. A recent meta-analysis of randomized controlled trials evaluating the effects of green tea catechins with or without caffeine on glycemic control showed significant reductions in fasting blood glucose concentrations (1.48 mg/dL; 95% confidence interval, 2.57 to 0.4 mg/dL; P ¼ 0.008), with greater reductions (although not significant) in trials using caffeine-containing formulations compared with trials using green tea catechins alone [27]. Following an overnight fast, glycogenolysis contributes to about only half the production of hepatic glucose, whereas the other half is supplied from the process of gluconeogenesis [28], which is mainly triggered by glucagon [22], and similarly by caffeine [29]. Thus, the similar blood glucose concentrations observed between when caffeine was administered on its own compared with the meal-ingested group, are most probably due to enhanced gluconeogenic processes in the presence of caffeine. High levels of insulin, which are normally known to suppress gluconeogenesis [22], in the caffeine-administered groups, may indicate that caffeine possibly overrode the gluconeogenic inhibitory effect of insulin. Moreover, the similarity in plasma glucose among the caffeine groups persisted despite the higher levels of insulin, indicating the presence of insulin insensitivity. The similarity in insulin levels between the caffeine groups, regardless of meal ingestion or its timing, may be due to the capacity of caffeine to acutely increase pancreatic b-cell stimulation [30]. This stimulatory effect seems to be independent of the presence of a meal, unlike what others have found. It has been reported that caffeinated coffee administered 1 h
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before a carbohydrate meal [8] and coadministered with a meal [26] elicited greater glycemic and insulinemic responses compared with decaffeinated coffee. It seems that in the presence of caffeine, the involvement of a meal (exogenous glucose) in plasma glucose sustenance is not clear and may imply that several mechanisms are involved. When caffeine was administered 1 h before meal ingestion, the low hepatic glycogen might be the consequence of inhibited glycogen synthesis, despite the availability of exogenous glucose. It was expected that glycogen content following meal ingestion in the presence of caffeine would be higher than when caffeine was administered on its own, as postprandial glycogen synthesis in the presence of insulin is normal, even in a 1-h interval following meal ingestion [24,31]. However, the development of insulin resistance after caffeine administration may have influenced hepatic glycogen synthesis, because elevated insulin from infusion [32] in the presence of obesity [31] has been shown to reduce glycogenesis. Moreover, it has been found that the effect of caffeine supplementation (400 mg) on glucose metabolism is highly pronounced starting at about 60 to 90 min after supplementation, as indicated by the increase in plasma lactate [33]. Caffeine is known to be a potent antagonist of adenosine receptors, which can influence insulin binding and signaling, as well as translocation of glucose transporters [12]. Given that adenosine receptor antagonists can inhibit glucose metabolism even in the presence of insulin [34,35], caffeine is likely to influence the metabolism of carbohydrates [36]. Moreover, caffeine’s adenosine receptor–antagonistic effect leads to increased levels of norepinephrine [37], which in turn stimulates the production of lactate from the skeletal muscle [38], implying that such an effect may be initiated 1 h after caffeine ingestion. In line with this, the low hepatic glycogen content observed in our study when the meal was ingested 1 h after caffeine might be the result of the initiation of caffeine’s adenosine receptor–antagonistic effect before meal ingestion. Bearing in mind the findings of a study [33] regarding the delayed onset of caffeine’s adenosine receptor–antagonistic effect of around 1 h, the increase in insulin during the hour following caffeine administration, in the case when caffeine was given 1 h after the meal, may have led to anabolic effects, where such an effect would be suppressed later on. In fact, while caffeine administration with and after meal ingestion did not produce significant differences in hepatic glycogen compared with meal ingestion, there appeared to be a trend toward higher glycogen content when caffeine followed meal ingestion, allowing for the assumption of possible glycogen synthesis. Thus, caffeine seems to have a biphasic action on glycogen synthesis, in which glycogen synthesis is stimulated in the first hour and inhibited subsequently. Regarding NEFAs, the observed levels in the caffeineadministered groups were found to be higher, although not significantly, regardless of meal ingestion and its timing. This is in line with others, where caffeine administration is known to increase circulating NEFA levels [39–41], which is a consequence of caffeine’s known lipolytic [42] and TG–fatty acid cycling effects in the liver [30,33], and possibly the adipose tissue [43]. NEFAs are an indicator of adipose tissue and muscle lipolysis, and are well known to reduce insulin sensitivity [44,45], consequently decreasing glucose disposal by the skeletal muscle [12]. Thus, it is probable that the higher NEFA levels following caffeine administration contributed to decreased insulin sensitivity that was further exacerbated by caffeine’s antagonistic effect on adenosine receptors. Thus, the inhibitory effect of insulin on lipolysis [46] may have been partially blunted by the lipolytic effect of caffeine.
Regarding serum TGs, the observed lower levels in the caffeine groups, especially in the cases when caffeine was administered before or with the meal, raises a question as to whether absorption of TGs from the meal was retarded. A green tea extract containing 5% caffeine, among other polyphenols, has been reported to inhibit in vitro activity of both gastric and pancreatic lipase (the main enzymes involved in the postprandial breakdown of TGs) [47], whereas caffeine supplementation in amounts of 6.6 and 13.2 mg did not seem to affect pancreatic lipase [48]. Moreover, serum TG levels are inversely related to increasing caffeine doses (5, 10, and 20 mg) in rats fed a high-fat diet, in which higher fecal TG excretion was described at the highest caffeine dose [40]. Caffeine also has been found to decrease mesenteric lymph flow and inhibit the absorption of fatty acids, by possibly influencing the intracellular packaging and secretion of lipids via chylomicrons [49]. As for muscle weight and glycogen content, the absence of significant differences between groups indicates that caffeine did not exert an effect on muscle glycogen. Interestingly, mean muscle glycogen content appeared to be the highest, although not significantly, in the post-meal caffeine administration scenario, likely demonstrating peripheral glucose uptake by the skeletal muscle during the 1-h period before initiation of the physiological sequelae of caffeine. However, why mean levels of glycogen were even higher than that of the ML group remains to be explained. Conclusions Postprandial plasma glucose status was maintained in all caffeine-administered groups regardless of meal ingestion and timing, whereas insulin concentration in these groups was elevated, implying a possible state of reduced insulin sensitivity. Additionally, postprandial TG levels were decreased, likely due to reduced fat absorption. Although caffeine administration pre-meal prevented hepatic glycogen synthesis, administration with and after meal ingestion seems to have overridden this inhibitory effect; eliciting a possible biphasic action on postprandial glucose metabolism. Acknowledgments This study was supported by a grant from the University Research Board of the American University of Beirut. References [1] Huxley R, Lee CM, Barzi F, Timmermeister L, Czernichow S, Perkovic V, et al. Coffee, decaffeinated coffee, and tea consumption in relation to incident type 2 diabetes mellitus: a systematic review with meta-analysis. Arch Intern Med 2009;169:2053–63. [2] von Ruesten A, Feller S, Bergmann MM, Boeing H. Diet and risk of chronic diseases: results from the first 8 years of follow-up in the EPIC-Potsdam study. Eur J Clin Nutr; 2013:1–8. [3] Aldughpassi A, Wolever TMS. Effect of coffee and tea on the glycaemic index of foods: no effect on mean but reduced variability. Br J Nutr 2009;101:1282–5. [4] Battram DS, Arthur R, Weekes A, Graham TE. The glucose intolerance induced by caffeinated coffee ingestion is less pronounced than that due to alkaloid caffeine in men. J Nutr 2006;136:1276–80. [5] Hatonen KA, Virtamo J, Eriksson JG, Sinkko HK, Erlund I, Jousilahti P, et al. Coffee does not modify postprandial glycaemic and insulinaemic responses induced by carbohydrates. Eur J Nutr 2012;51:801–6. [6] Johnston KL, Clifford MN, Morgan LM. Coffee acutely modifies gastrointestinal hormone secretion and glucose tolerance in humans: glycemic effects of chlorogenic acid and caffeine. Am J Clin Nutr 2003;78:728–33. [7] Lane JD, Hwang AL, Feinglos MN, Surwit RS. Exaggeration of postprandial hyperglycemia in patients with type 2 diabetes by administration of caffeine in coffee. Endocr Pract 2007;13:239–43.
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