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In this thesis a number of studies are described that aim to identify genes that control the serum lipid response to dietary components. By identifying such genes it is possible to elucidate the mechanisms by which diet can affect serum lipids.
The level of serum lipids serves as a marker for the risk of cardiovascular disease (1-3), which is a major course of death worldwide (4). Therefore, it is important to understand how diet affects these risk markers. In this thesis the serum lipid response to cafestol is used as a model system for a food component that affects serum lipid levels. Cafestol is the most potent cholesterol-raising compound identified in the human diet (5). This model makes it feasible to study its effects not only in humans, but also in animal and cell culture studies.
This introduction describes how cafestol was identified as the cholesterolâ€‘raising factor in unfiltered coffee and discusses its effect on serum lipids and liver enzyme levels. Furthermore, it summarizes potential mechanisms of the cholesterolâ€‘raising effect of cafestol that have been published so far. Finally, the last paragraph gives the objective and outline of this thesis.
Differences in brewing method explain differences in the serum lipid response to coffee
In the 1980's Scandinavian studies showed that consumption of boiled coffee is associated with increased serum cholesterol levels (6). It was demonstrated that withdrawal of boiled coffee consumption lead to a reduction in serum cholesterol of 10% (7, 8). This suggested that boiled coffee causes an increase in serum cholesterol. However, the association between coffee consumption and serum cholesterol levels could not be confirmed by studies performed in the United States or Western Europe (9). Accordingly, it was hypothesized that the brewing method was responsible for the differences in observations. The main difference in the brewing method between Scandinavia versus the United States and Western Europe is the use of paper filters in the latter. In contrast to Scandinavian coffee, in paper-filtered coffee the grounds are not present in the brew.
The importance of the brewing method was confirmed by an intervention study showing that boiled coffee did raise serum cholesterol, whereas filtered coffee had no effect (10). Subsequently, intervention studies showed that boiled coffee lost its cholesterolâ€‘raising effect after passing through a paper filter (11, 12). Together these studies showed that the brewing method used determines the cholesterol-raising potential of coffee.
Identification of cafestol as the cholesterol-raising factor from coffee beans
Zock et al. showed that boiled coffee contains a lipid-rich fraction that raised serum cholesterol in an intervention trial (13). Furthermore, oil pressed from coffee beans caused elevation of serum cholesterol in humans (14, 15). Coffee oil mainly consists of triglycerides, but also contains 15% diterpene esters of fatty acids (16). When coffee oil was stripped of these diterpene esters the cholesterol-raising effect of the oil was lost (17). The most abundant diterpenes in coffee oil are cafestol and kahweol. Several intervention trials showed that for every 10 mg of cafestol per day serum cholesterol increases by 0.13 mmol/l after four weeks of consumption (5).
Cafestol raises serum cholesterol more potently than kahweol does. A mixture of cafestol (60 mg/day) and kahweol (51 mg/day) increased serum cholesterol only slightly more than pure cafestol (64 mg/day) did (18). Results with pure kahweol are not available due to difficulties with purification and stability of this diterpene.
About 80% of the rise in serum cholesterol is due to elevation of low density lipoproteins (LDL) and the remainder by elevation of very low density lipoproteins (VLDL). High density lipoproteins (HDL) are not affected or show a slight decrease (13, 17, 18). Triglycerides are raised by 0.08 mmol/l with every 10 mg of cafestol per day after 2-6 weeks (5). However, this rise in triglycerides is transient. A six-month intervention trial showed that 0.9 liter of unfiltered coffee per day raised serum triglycerides by 26% in the first month, but this effect dropped to 7% after six months of daily consumption (19). The effect of unfiltered coffee on serum cholesterol is more persistent. A 10% raise in serum cholesterol was reduced to 6% after six months of daily intake. This is in agreement with previous epidemiological studies (17, 20, 21).
Together these studies show that cafestol has a permanent effect on LDL and total cholesterol and a transient effect on serum triglycerides.
Consumption of unfiltered coffee affects health
Serum levels of LDL cholesterol are a major risk factor for atherosclerosis and consequently cardiovascular disease (1-3). Early studies already showed that consumption of boiled coffee is associated with an increased risk of cardiovascular disease (22). More recently a case-control study showed that consumption of boiled coffee appears to increase the risk of first non-fatal myocardial infarction (23). In Finland a switch from boiled to filtered coffee was associated with a decrease of 0.3 mmol/l total cholesterol between 1972 and 1992 (24). This decrease in serum cholesterol levels was also associated with a decrease in coronary heart disease.
Therefore, consumption of cafestol in unfiltered coffee increases the risk of cardiovascular disease.
Effect of cafestol on markers of liver function
Cafestol raises serum levels of alanine aminotransferase and aspartate aminotransferase
Coffee oil or unfiltered coffees not only affect serum lipids, but also serum activity of the liver enzymes alanine aminotransferase (ALAT) and to a lesser extent aspartate aminotransferase (ASAT) (15, 17, 19, 25). On average every 10 mg of cafestol or kahweol per day raised serum ALAT by 8-12% (5). Elevation of these liver enzyme activities is indicative of liver damage (26-28). However, the effect of cafestol is not compliant with cholestasis. In cholestasis ASAT activities often exceed ALAT activities and activities of ï§-glutamyltranspeptidase and alkaline phosphatase are elevated, whereas cafestol even reduces serum activities of these enzymes (15, 17, 19, 25). ALAT is predominantly located in the cytosol of hepatocytes and ASAT is predominantly present in mitochondria. Elevation of ALAT indicates damage to the membranes of hepatocytes. A larger increase in ASAT indicates more severe damage to liver cells (26-28). Therefore, cafestol seems to compromise membrane integrity of hepatocytes.
Consumption of unfiltered coffee does not cause liver disease in life-long consumers
Although ALAT activity is still elevated in the serum of subjects that consumed unfiltered coffee for six months, life-long consumers do not have elevated ALAT activities (17, 29). Also, mortality rates of liver cirrhosis have been typically low in Scandinavian countries (30). Furthermore, an inverse correlation exists between coffee consumption and risk of alcohol-induced liver cirrhosis (31, 32). This even suggests a protective effect of coffee consumption on development of alcoholic cirrhosis. Together this suggests that the effect of cafestol and kahweol on liver enzyme activities is transient and only causes subclinical damage to hepatocytes.
Possible mechanisms for the effect of cafestol
Cafestol suppresses bile acid synthesis in APOE3Leiden mice
Cafestol and kahweol affect both serum lipids and liver enzymes. Therefore, it seems likely that the liver is the target organ for these diterpenes. Indeed, a number of studies have described effects of cafestol in hepatocytes. The most striking effect of cafestol was the inhibition bile acid synthesis in rat hepatocytes and livers of APOE3â€‘Leiden mice by downregulation of expression and activity of cholesterol 7ï¡â€‘hydroxylase (33, 34). Cholesterol 7ï¡-hydroxylase is the rate-limiting enzyme in the conversion of cholesterol into bile acids in the liver. In addition, the amount of bile acid was reduced by 41% in APOE3-Leiden mice (34). Suppression of bile acid synthesis will lead to an increased pool of hepatic cholesterol, causing downregulation of the LDL-receptor and thereby elevation of serum LDL. Downregulation of the LDL-receptor by cafestol was confirmed in vitro (33, 35, 36).
Cafestol affects activity transfer proteins in the liver
Besides raising LDL levels, cafestol also causes a transient rise in serum triglyceride levels. This is probably due to increased production of VLDL1 particles (37).
Furthermore, cafestol increases activity of cholesteryl ester transfer protein (CETP) and phospholipids transfer protein (PLTP) and decreases activity of lecithin:cholesterol acyltransferase (LCAT) (38, 39). However, it remains unclear whether the elevated activities of CETP and PLTP are a cause or a consequence of the LDL elevation after cafestol consumption (39). The decrease in LCAT activity might account for the slight decrease in HDL levels after cafestol and kahweol consumption observed in some studies (17). However, because LCAT is only synthesized in the liver the decrease in activity could be caused by impairment of liver function (39).
In summary cafestol and kahweol affect bile acid synthesis, production of VLDL, and activities of lipid transfer proteins in the liver. However, it remains unclear how diterpenes regulate these processes and how modulation of these processes contributes to the effects of cafestol and kahweol on serum lipids.
The role of genetic variation and the serum cholesterol response to cafestol
The serum cholesterol response to dietary changes differs considerably between subjects, while the response is to some extent reproducible within subjects (40-44). The between-subjects variation in cholesterol response can possibly be attributed to genetic variation. Accordingly, an alternative strategy to identify genes that are involved in the regulation of the response of serum lipids to cafestol and other dietary compounds is via genetic studies. Indeed, it has been shown that polymorphisms in certain genes may affect the response (45-47). One polymorphism in the apolipoprotein A-I (apoA-I) gene was identified that is associated with the response of serum cholesterol to cafestol. Subjects with the apoA-I 83-CC genotype show a larger increase in serum cholesterol than subjects with the apoA-I 83-CT genotype (48). This shows that genetic variation can at least partly explain differences in the response of serum lipids to cafestol between individuals.
Objective and outline of this thesis
The objective of our studies was to identify genes that regulate the response of serum lipids to diet. In these studies we used cafestol as a model substance.
Cafestol is the most potent cholesterol-raising compound identified in the human diet and can be administered easily in the form of coffee oil. In addition, cafestol can also be administered to animals and added to the medium of cultured cells.
We first assessed the within-subject reproducibility of the serum lipid response to coffee oil in healthy volunteers (Chapter 2). Before a specific response can be linked to a certain genotype the reproducibly of the response within a person has to be measured. Although the effect of cafestol on serum lipids is highly reproducible on a group level, measurement of the individual response might be hampered by variation in environmental factors that are not stable during the treatment period.
During this reproducibility study we encountered larger effects of coffee oil on the liver enzymes ALAT and ASAT than expected from previous studies. For the reproducibility study we used Arabica coffee oil that contains both cafestol and kahweol. Two previous studies suggested that cafestol is mainly responsible for the effect on serum lipids, whereas kahweol is mainly responsible for the effect on liver enzymes (15, 18). Therefore, we performed a study to assess whether coffee oil from Robusta beans that contain minute amounts of kahweol affects liver enzyme levels in healthy volunteers (Chapter 3). This study showed that coffee oils rich or poor in kahweol have similar effects on liver enzyme levels. Because of the adverse effects in a number of subjects we decided to arrest the trials with coffee oil or cafestol in human volunteers. We continued our investigations into the mechanism by which cafestol raises serum lipids in animal and in vitro studies. Chapter 4 shows the combined results of these studies that indicate interaction of cafestol with two nuclear receptors: the farnesoid X receptor and the pregnane X receptor. Upon activation these receptors can downregulate expression of cholesterol 7ï¡-hydroxylase, which is in line with the observation that cafestol downregulates expression and activity of cholesterol 7ï¡-hydroxylase in rat hepatocytes and livers of APOE3-Leiden mice. Chapter 5 describes the results of the measurement of 7ï¡-hydroxy-4-cholesten-3-one in samples from the reproducibility study. This metabolite is present in plasma and reflects activity of cholesterol 7ï¡-hydroxylase in the liver and therefore of bile acid synthesis. Finally, the results of the studies above are discussed in Chapter 6.