7.6.1 Effects on blood lipid profile
Feeding experiments in rats have consistently demonstrated that oxidized fats cause a reduction in the concentrations of triacylglycerols and cholesterol in liver, plasma, and very low-density lipoproteins (VLDL) (Huang et al., 1988;
Eder and Kirchgessner, 1998; Eder et al., 2003b; SuÈlzle et al., 2004). The reason for the oxidized fat-induced lowering of triacylglycerol concentrations became clear only recently when it was shown that oxidized fats are strong activators of hepatic peroxisome proliferator-activated receptor (PPAR ) (Chao et al., 2001) ± an effect that could be confirmed in subsequent studies (SuÈlzle et al., 2004; Chao et al., 2004; 2005; Ringseis et al., 2007a; 2007b). PPAR as well as other PPAR subtypes, PPAR / and PPAR , are transcription factors belonging to the superfamily of nuclear receptors that can be activated by fatty acids and their metabolic derivatives. They are implicated in the regulation of lipid and lipoprotein metabolism, glucose homeostasis, cellular differentiation, and inflammation (Desvergne and Wahli, 1999; Chinetti et al., 2000; Duval et al., 2002). Since PPAR is highly expressed in tissues with high rates of fatty acid catabolism such as liver, where it controls a comprehensive set of genes that regulate most aspects of lipid catabolism (cellular fatty acid uptake, intracellular fatty acid transport, mitochondrial fatty acid uptake, and fatty acid oxidation) (Mandard et al., 2004), activation of PPAR results in decreased triacylglycerol concentrations in plasma, liver and VLDL. This suggests that activation of hepatic PPAR is probably largely responsible for the triacylglycerol lowering effect of oxidized fats.
The components of oxidized fats which are supposed to be responsible for PPAR activation are oxidized fatty acids such as hydroxy and hydroperoxy fatty acids (e.g., 9-HODE, 13-HODE, 13-HPODE) (KoÈnig and Eder, 2006;
Mishra et al., 2004; Muga et al., 2000; Delerive et al., 2000). Fats heated at moderate temperatures of below 100 ëC for a long period (several days or weeks) usually contain high amounts of these primary lipid peroxidation products, whereas fats heated at high temperatures have low levels of these compounds because hydroperoxides are relatively unstable and easily decompose at these 152 Oxidation in foods and beverages and antioxidant applications
temperatures. In addition, the studies from Martin et al. (2000) and Bretillon et al. (2003) suggest that CFAM, which are also characteristic substances of oxidized fats, are activators of PPAR too. CFAM are only significantly formed from the unsaturated 18-carbon fatty acids (C18:1n-9, C18:2n-6, C18:3n-3) in vegetable oils heated at temperatures above 200 ëC (Sebedio and Grandgirard, 1989; Rojo and Perkins, 1987; Tompkins and Perkins, 2000; Christopoulou and Perkins, 1989).
The cholesterol-lowering effect of oxidized fat is likely due to inhibition of transcription of genes involved in cholesterol homeostasis as suggested from a recent study (Koch et al., 2007b). Cholesterol homeostasis in mammalian cells is mainly regulated by sterol regulatory element-binding protein (SREBP)-2.
This transcription factor preferentially activates genes involved in cellular chol-esterol uptake (e.g., LDL receptor) and cholchol-esterol synthesis (e.g., 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase) (McPherson and Gauthier, 2004;
Horton, 2002). Koch et al. (2007b) demonstrated in rats that administration of oxidized fat inhibits activation of hepatic SREBP-2 and reduces hepatic transcript levels of LDL receptor and HMG-CoA reductase genes, both of which are important for cellular cholesterol uptake and synthesis, respectively. As a consequence of the reduced expression of these genes in the liver, the concentrations of cholesterol in liver and plasma are lowered, which explains the reduction of cholesterol concentrations in liver and plasma by oxidized fats.
Inhibition of SREBP-2 activation by oxidized fat is likely mediated by an up-regulation of insulin-induced gene (Insig)-1 in the liver which was observed in rats fed oxidized fat (Koch et al., 2007b). Koch et al. (2007b) proposed that the effect on Insig-1 is due to activation of PPAR because it was recently shown that synthetic PPAR activators also cause an up-regulation of Insig-1 in the liver (KoÈnig et al., 2007). Insig are membrane proteins that reside in the endoplasmic reticulum and play a central role in the regulation of SREBP activation, because they prevent the translocation of SREBP from the endoplasmic reticulum to the Golgi, where proteolytic activation of SREBP and subsequent release of transcriptionally active forms of SREBP occur (Yang et al., 2002; Yabe et al., 2002). As a result, the synthesis of cholesterol declines in response to PPAR activation. Therefore, up-regulation of Insig by oxidized fat inhibits proteolytic activation of SREBP-2 in the Golgi, thereby lowering hepatic cholesterol synthesis and liver and plasma cholesterol concentrations.
However, whether the stimulatory effect of oxidized fat on Insig-1 expression is indeed mediated by PPAR is uncertain, because lowering of mature SREBP-2 concentration and inhibition of cholesterol synthesis was also reported for PPAR activators (Klopotek et al., 2006), and hydroxy and hydroperoxy fatty acids from oxidized fats are able to activate PPAR (Nagy et al., 1998; Krey et al., 1997). Since the oxidized fat in the study from Koch et al. (2007b) was prepared by heating the oil for a long period (25 days) at a moderate temperature (60 ëC), which typically leads to the formation of hydroxy and hydroperoxy fatty acids, it is not unlikely that the stimulatory effect of oxidized fat on Insig-1 expression is indeed mediated by activation of PPAR .
Health aspects of oxidized dietary fats 153
Similar to SREBP-2 activation, administration of oxidized fat is also sup-posed to inhibit activation of SREBP-1c in the liver. In contrast to SREBP-2, this SREBP isoform is mainly responsible for the transcriptional activation of genes involved in fatty acid synthesis, the so-called lipogenic enzymes such as fatty acid synthase, acetyl CoA-carboxylase, glucose-6-phosphate dehydrogen-ase, malic enzyme, and ATP citrate lyase. In a previous study from Eder and Kirchgessner (1998), it could be shown that oxidized fats reduce the activities of these lipogenic enzymes in rats. These findings could be confirmed in a further study from Eder et al. (2003b). In addition, inhibition of other lipid-synthesizing enzymes, namely 9-desaturase and phosphatidate phosphohydrolase, could be demonstrated in the liver of rats administered CFAM (Martin et al., 2001). The study from Eder et al. (2003b), moreover, showed that gene expression of lipogenic enzymes in the liver is reduced by oxidized fats. Due to the funda-mental role of SREBP-1c in the transcriptional regulation of lipogenic enzymes, Eder et al. (2003b) proposed that reduction of gene transcription of lipogenic enzymes by oxidized fats involves inhibition of SREBP-1 maturation. This assumption is strongly supported by a recent study from KoÈnig et al. (2009) demonstrating a link between activation of PPAR or PPAR and reduction of nuclear SREBP-1 and fatty acid synthesis ± an effect which probably involves up-regulation of Insig-1 and Insig-2a. Due to its PPAR activating properties it is therefore very likely that oxidized fat inhibits fatty acid synthesis through the activation of either PPAR or PPAR or both of them, and subsequent reduction of SREBP-1 maturation. This indicates that the triacylglycerol-lowering effect of oxidized fats is not only mediated by stimulating fatty acid catabolism but also by inhibiting fatty acid synthesis.
7.6.2 Effects on fatty liver development
Chronic alcohol abuse is the most common reason for the development of fatty liver in humans. Fatty liver development is the result of both impaired fatty acid catabolism due to blockade of PPAR and increased lipogenesis in the liver due to activation of the lipogenic SREBP-1 pathway (You and Crabb, 2004; You et al., 2002; Fischer et al., 2003). A central role of the disturbed PPAR function in the pathogenesis of alcoholic fatty liver has been evidenced by the observa-tion that administraobserva-tion of pharmacological PPAR agonists to ethanol-fed animals prevented fatty liver by reversing PPAR dysfunction, and stimulating the rate of fatty acid -oxidation in the liver (Tsutsumi and Takase, 2001; Crabb et al., 2004).
Based on the knowledge that oxidized fats markedly activate hepatic PPAR and PPAR -regulated genes (Chao et al., 2001; SuÈlzle et al., 2004) and reduce hepatic triacylglycerol concentrations (SuÈlzle et al., 2004; Eder et al., 2003b), a study has recently been conducted investigating whether dietary oxidized fat is useful in the prevention of alcoholic fatty liver (Ringseis et al., 2007b). In this study, like in many other studies dealing with ethanol (You et al., 2002;
Tsutsumi and Takase, 2001; Tomita et al., 2004; Hong et al., 2004; Kharbanda 154 Oxidation in foods and beverages and antioxidant applications
et al., 2005) the rat was used as an animal model. For oral administration of the ethanol, the Lieber-DeCarli liquid diet was used, which provides an excellent means for reproducing early lesions of alcoholic liver disease such as steatosis (Hall et al., 2001). This study clearly demonstrated that triacylglycerol accumulation in response to ethanol-feeding is markedly reduced in rats by simultaneous administration of dietary oxidized fat when compared to a fresh fat. The observation that dietary oxidized fat resulted in similar hepatic triacylglycerol levels during ethanol-feeding as observed in rats fed fresh fat in the absence of ethanol suggests that dietary oxidized fat is indeed capable of preventing alcoholic fatty liver disease.
To elucidate the molecular mechanisms underlying these beneficial effects of oxidized fat, the transcript levels of PPAR and several genes involved in fatty acid catabolism, that are known to be reduced by ethanol administration, were determined in this study (Ringseis et al., 2007b). These investigations showed that, in agreement with previous studies (Fischer et al., 2003; Crabb et al., 2004), the transcript levels of PPAR and its target genes were reduced by ethanol-feeding (Ringseis et al., 2007b). However, administration of the oxidized fat, but not fresh fat, during ethanol-feeding resulted in the induction of PPAR target genes involved in fatty acid oxidation, which is indicative of PPAR activation, even in the presence of ethanol. Based on these results, it has been concluded that the oxidized fat-induced expression of PPAR target genes enhanced the capacity of the liver to oxidize fatty acids and, thus, counteracted the elevated levels of triacylglycerols and the diminished PPAR function during ethanol-feeding (Ringseis et al., 2007b).
Similar observations as with oxidized fat have been made using synthetic PPAR agonists such as WY-14,643 and fibrates (Fischer et al., 2003; Tsutsumi and Takase, 2001; Spritz and Lieber, 1966). Treatment with WY-14,643 restored the ability of the PPAR /RXR complex to bind its specific PPAR response element and induce transcript levels of many PPAR target genes resulting in a higher rate of fatty acid -oxidation (Fischer et al., 2003).
Consequently, excessive accumulation of triacylglycerols in the liver during ethanol-feeding is prevented by these agents (Fischer et al., 2003; Tsutsumi and Takase, 2001; Spritz and Lieber, 1966). Thus, the results of the study dealing with oxidized fat (Ringseis et al., 2007b) suggest that dietary oxidized fat prevents fatty liver development by similar mechanisms as reported for synthetic PPAR activators.
7.6.3 Effects of oxidized fats on carnitine homeostasis
Many years ago it was shown that starvation or treatment of rats with PPAR agonist clofibrate increases the concentration of carnitine in the liver (McGarry et al., 1975; Brass and Hoppel, 1978; Paul and Adibi, 1979). Carnitine is an essential metabolite, which has a number of indispensable functions in intermediary metabolism, like the transport of activated long-chain fatty acids from the cytosol to the mitochondrial matrix where -oxidation takes place Health aspects of oxidized dietary fats 155
(McGarry and Brown, 1997; Brass, 2002; Steiber et al., 2004). As both starva-tion and clofibrate treatment lead to an activastarva-tion of PPAR , a transcripstarva-tion factor belonging to the nuclear hormone receptor superfamily (Schoonjans et al., 1996), we have recently raised the hypothesis that activation of this nuclear receptor is responsible for the increased liver carnitine concentrations observed in those studies. Indeed, a study in our group revealed for the first time a marked, about 8-fold increase in the hepatic mRNA content of novel organic cation transporter 2 (OCTN2) in the liver of rats treated with the PPAR agonist clofibrate (Luci et al., 2006b). OCTN2 is the physiologically most important carnitine transporter, operating for the reabsorption of carnitine from the urine as well as playing a major role in tissue distribution. Subsequent studies in PPAR -knockout mice further demonstrated that transcriptional up-regulation of hepatic OCTN2 by fasting or treatment with PPAR agonist WY-14,643 is dependent on PPAR (van Vlies et al., 2007; Koch et al., 2008). In addition, studies in rats and pigs showed that OCTN2 is induced by fasting or clofibrate also in several other tissues with abundant PPAR expression including kidney, skeletal muscle, heart, and small intestine (Ringseis et al., 2007d; 2008a; 2008b; 2008c;
Luci et al., 2008).
Besides cellular carnitine uptake, evidence has been provided to suggest that carnitine biosynthesis is also regulated by PPAR ; e.g., studies in PPAR -knockout mice and corresponding wild-type mice showed that treatment with the PPAR agonist WY-14,643 stimulates the transcription of enzymes involved in carnitine biosynthesis, trimethyllysine dioxygenase (TMLD), 4-N-trimethyl-aminobutyraldehyde dehydrogenase (TMABA-DH) and -butyrobetaine dioxygenase (BBD) only in the liver of wild-type (van Vlies et al., 2007; Koch et al., 2008). This indicates that transcriptional regulation of those genes is also mediated by PPAR . Other studies revealed that PPAR -knockout mice had markedly lower plasma and tissue levels of methionine and -ketoglutarate, which serve as biosynthetic precursors and enzymatic cofactors (Vaz and Wanders, 2002), respectively, for carnitine synthesis, when compared to wild-type mice (Makowski et al., 2009). Collectively, these observations in PPAR -null mice clearly show that genes encoding proteins involved in carnitine uptake and carnitine biosynthesis are transcriptionally regulated by PPAR .
Since oxidized fats are also capable of activating PPAR , a recent study has investigated the effect of oxidized fat on tissue carnitine concentrations and on expression of genes involved in carnitine homeostasis (Koch et al., 2007a). The oxidized fat used in this study (Koch et al., 2007a) was prepared by heating sunflower oil at a relatively low temperature (60 ëC) for a long period. Such fats usually contain relatively high concentrations of primary lipid peroxidation products including 9-HODE, 13-HODE and 13-HPODE, which are very potent PPAR agonists (KoÈnig and Eder, 2006; Delerive et al., 2000; Mishra et al., 2004; SuÈlzle et al., 2004). The study from Koch et al. (2007a) showed that treatment of rats with such an oxidized fat causes the same alterations as observed with the administration of the PPAR agonist clofibrate (Luci et al., 2006b; Ringseis et al., 2007d; 2008b), namely increased hepatic mRNA 156 Oxidation in foods and beverages and antioxidant applications
concentrations of OCTN1 and OCTN2 and an increased hepatic carnitine concentration. Based on similar effects of pharmacological or starvation-induced activation of PPAR on carnitine homeostasis, the authors proposed that the up-regulation of OCTN in the liver by the oxidized fat was also mediated by PPAR activation (Koch et al., 2007a). Considering that OCTN2 has a higher carnitine transport activity than OCTN1 (Tamai et al., 2000), and that OCTN2 was more strongly induced by the oxidized fat than OCTN1, the authors suggested that the increased hepatic and the reduced plasma concentrations of carnitine in rats treated with oxidized fat were caused mainly by an increased uptake of carnitine from plasma into the liver via OCTN2.
Interestingly, it was also shown that dietary oxidized fat leads to an up-regulation of OCTN2 in the small intestine (Koch et al., 2007a) ± an effect which was also observed with PPAR activator clofibrate (Ringseis et al., 2007d, 2008b). Since clofibrate was recently demonstrated to markedly increase the absorption rate of carnitine in small intestine (Ringseis et al., 2008b), it is not unlikely that oxidized fats also increase intestinal carnitine absorption.
With respect to hepatic carnitine biosynthesis, the study from Koch et al.
(2007a) revealed that oxidized fat does not up-regulate rate-limiting enzymes involved in carnitine biosynthesis (Vaz and Wanders, 2002). Nevertheless, these observations do not definitely exclude the possibility that hepatic carnitine biosynthesis was increased in rats treated with oxidized fat, because the liver has a high capacity to convert -butyrobetaine, which is a good substrate for OCTN (Tamai et al., 1998; 2000), into carnitine (Vaz and Wanders, 2002). Thus, it is likely that an increased expression of OCTN2 by oxidized fat may have led to an increased uptake of -butyrobetaine from plasma into the liver which in turn may have stimulated synthesis of carnitine in the liver.
7.6.4 Effects on thyroid function and thyroid hormones
Only few studies have been carried out to investigate the effect of oxidized fat on thyroid function (Eder and Stangl, 2000; Eder et al., 2002; Skufca et al., 2003). Nevertheless, these studies performed with either rats or pigs consistently demonstrated that dietary oxidized fats increase the concentrations of free and total thyroxine (T4) in plasma.
In rats, moreover, it was shown that dietary oxidized fat causes alterations in the morphology of the thyroid gland (Skufca et al., 2003). Rats fed oxidized fat exhibited an increased height of thyroidal epithelial cells while the diameter of follicle lumen was reduced. This finding suggests that dietary oxidized fat stimulates the thyroid function because the function of the thyroid gland is well reflected by its morphology, particularly by the height of epithelial cells and the diameter of follicle lumen (Bidey and Cowin, 1995). Furthermore, the study from Skufca et al. (2003) revealed that dietary oxidized fat alters the expression of genes involved in thyroid hormone synthesis (Skufca et al., 2003); i.e. gene expression of sodium iodide symporter (NIS), which plays a key role in the formation of thyroid hormones by mediating the uptake of iodide from blood Health aspects of oxidized dietary fats 157
into the thyrocyte (Dai et al., 1996), was reduced, whereas that of thyroid peroxidase (TPO) increased in the thyroid glands of these rats (Skufca et al., 2003). TPO catalyzes the transformation of iodide to iodine and is involved in the iodination of thyroglobulin and the coupling reaction leading to thyroxine formation (Ekholm, 1981). Although NIS was down-regulated by oxidized fat, the increased gene expression of TPO is also indicative of a stimulatory effect of oxidized fat on thyroid function, because TPO but also NIS are regulated on the transcriptional level by thyrotropin (TSH), which is the most important regulator of thyroid hormone function (SchroÈder-van der Elst et al., 2001; Riedel et al., 2001; Damante et al., 1989). However, because the concentration of TSH was not different between rats fed oxidized fat and those fed fresh fat (Skufca et al., 2003), it is likely that the effect of the oxidized fat on the thyroid gland was not mediated by TSH. Since the study from Skufca et al. (2003) clearly showed that the alteration of thyroid function by oxidized fat is not due to an interaction with the metabolism of iodine, which is a key nutrient for the formation of thyroid hormones (Riesco et al., 1976), it has been suggested that lipid peroxidation products of the diet directly influence the expression of genes involved in thyroid hormone synthesis.
In an attempt to identify the components responsible for the effects of oxidized fat on thyroid function and the mechanisms underlying the alterations in thyroid function by oxidized fat, a recent study investigated the effect of a primary lipid peroxidation product, namely 13-HPODE ± the quantitatively most important primary oxidation product of linoleic acid (Niki et al., 2005), and a characteristic compound of fats heated at moderate temperatures (< 100 ëC) ± on the function of primary porcine thyrocytes (Luci et al., 2006a). Unexpectedly, the study from Luci et al. (2006a) failed to demonstrate an important role of 13-HPODE as a mediator of the alterations of thyroid function observed with oxidized fats. For instance, Luci et al. (2006a) showed that incubation of porcine thyrocytes with 13-HPODE did not lead to alterations in gene expression of NIS and TPO or iodide uptake, even in non-physiologic high concentrations of 100
M. In addition, gene expression of the TSH receptor was also not influenced by 13-HPODE suggesting that it also does not influence the effect of TSH on the function of porcine thyrocytes.
Luci et al. (2006a), moreover, showed that 13-HPODE leads to a down-regulation of dual oxidase 2 (DUOX2), which, like NIS and TPO, is involved in thyroid hormone synthesis by acting as a subunit of NADPH-oxidase (the major generator of H2O2in thyrocytes (Moreno et al., 2002)), and to a reduced release of hydrogen peroxide (Corvilain et al., 1991). Although the authors of this study did not measure the activity of NADPH oxidase, they suggested that the reduced gene expression of DUOX2 might be associated with a reduced activity of this enzyme. The authors, moreover, suggested that the reduced concentrations of H2O2in the cells and in the cell medium could be due to an increased activity of GPx observed in cells treated with 13-HPODE. GPx protects thyrocytes against a high intracellular concentration of H2O2, which for instance can lead to apoptosis (Demelash et al., 2004). It has been postulated that GPx in thyrocytes 158 Oxidation in foods and beverages and antioxidant applications
acts as a regulator of thyroid hormone biosynthesis by controlling the concen-tration of H2O2available for thyroid hormone synthesis (Howie et al., 1995). As the concentration of H2O2 in thyrocytes is the rate-limiting factor of thyroid hormone synthesis (Corvilain et al., 1991), Luci et al. (2006a) suggested that high concentrations of 13-HPODE could have led to a reduced formation of thyroid hormones. Since it has been shown that generation of ROS inhibit the formation of thyroid hormones in cultured thyroid cells (Sugawara et al., 2002), the authors pointed out the possibility that the effects observed on DUOX2 expression and release of H2O2by 13-HPODE were due, at least in part, to ROS produced or to lipid oxidation products formed during incubation in the cell.
Furthermore, the authors pointed out to the possibility that a reduction of the release of H2O2 could result in a reduced formation of thyroid hormones in thyrocytes.
It has also been noted (Eder and Stangl, 2000) that the hormonal pattern observed in pigs fed oxidized fat (increased concentrations of total and free T4, and an increased ratio of T4 to T3) is similar to that observed in selenium-deficient rats (Ruz et al., 1999). Selenium is an integral part of type I, 5-deiodinase (Arthur et al., 1990), but also of GPx, which is involved in the degradation of hydroperoxides. Since some studies reported an increased GPx activity in response to dietary sources of highly unsaturated fatty acids such as fish oil (Bellisola et al., 1992; Olivieri et al., 1988) and reduced selenium concentrations in plasma and some tissues (Bellisola et al., 1992; Smith and Isopenko, 1997), it is conceivable that dietary oxidized oils, which also promote oxidative stress by reducing the vitamin E status, could affect the selenium status of tissues and thus interfere with the metabolism of thyroid hormones.
Therefore, it is possible that the induction of oxidative stress is responsible for the alterations of thyroid hormone status induced by oxidized fat.
7.6.5 Effects on endogenous and exogenous antioxidant defense mechanisms
It has long been suggested that the pathophysiologic effects of dietary oxidized fats are mainly due to oxidative stress induced by lipid peroxidation products present in the oxidized fats (Gabriel and Alexander, 1977). This was based on the knowledge that the primary and secondary lipid peroxidation products are partially absorbed from the oxidized fat (Kanazawa et al., 1985; Oarada et al., 1986). Since oxidized fats are also known to induce microsomal cytochrome P-450 enzymes (Huang et al., 1988; Chao et al., 2001; SuÈlzle et al., 2004), which causes generation of ROS (Terelius and Ingelman-Sundberg, 1988), it is also possible that the induction of oxidative stress by oxidized fat is due to induction of microsomal enzymes. Indeed, several studies indicated that ingestion of thermally oxidized oil resulted in increased lipid peroxidation as evidenced by increased tissue concentrations of TBARS (Izaki et al., 1984; Kok et al., 1988).
In addition, these studies in rats also revealed that the vitamin E concentration in liver and serum decreased following ingestion of oxidized oil (Izaki et al., 1984;
Health aspects of oxidized dietary fats 159
Kok et al., 1988). Moreover, Huang et al. (1988) noted that administration of oxidized fat to rats was accompanied by an increased hemolysis of red blood cells which also implies that the vitamin E status was compromised by the oxidized fat. These findings provided strong evidence to suggest that ingestion of oxidized fat induces oxidative stress and causes a depletion of antioxidants.
Subsequent studies aimed to investigate whether the oxidative stress induced by oxidized fat could be alleviated by supplementation with antioxidants. For instance, the study from Liu and Huang (1995) demonstrated that reduced concentrations of -tocopherol and increased concentrations of TBARS in various tissues of rats fed oxidized fat could be alleviated by supplementation with a high concentration of dietary vitamin E. These authors, however, postulated that the reduction of -tocopherol concentrations in plasma and tissues of rats fed oxidized fat is not only due to an increased consumption of -tocopherol by lipid hydroperoxides ingested from the oxidized fat or formed in vivo but also due to a reduced absorption of vitamin E from the diet. Never-theless, a subsequent study from the same group (Liu and Huang, 1996) clearly established that ingestion of oxidized fat is accompanied by an increased -tocopherol catabolism and/or turnover due to the following reasons:
· The -tocopherol concentrations in tissues were lower in rats receiving a vitamin E-devoid diet containing oxidized fat compared to rats receiving a vitamin E-devoid diet containing fresh fat during a 9-week depletion period.
· The response of the oxidized fat group to -tocopherol repletion by intra-peritoneal injection of all-rac- -tocopherol was less than that of the control group as evidenced by reduced -tocopherol concentrations in tissues of rats fed the oxidized fat diet during the repletion period compared to those fed the fresh fat diet.
Another study in Sprague-Dawley rats demonstrated that the susceptibility of LDL to lipid peroxidation is increased by feeding oxidized fats (Eder et al., 2003a). The susceptibility of LDL to lipid peroxidation depends mainly on their PUFA contents and their concentrations of antioxidants (Esterbauer et al., 1989).
Since the percentages of PUFA in LDL total lipids were not different between rats fed the oxidized fat and those fed the fresh fat (Eder et al., 2003a), it has been suggested that the increased susceptibility of LDL to lipid peroxidation of rats fed oxidized fat was due to their lower vitamin E concentrations. As expected, supplementing the oxidized fat diets with a high vitamin E concentration prolonged the lag time before onset of lipid peroxidation during incubation of LDL with copper ions (Eder et al., 2003a), which is indicative of a decreased susceptibility of LDL to lipid peroxidation.
Similar to vitamin E, the vitamin C status is also impaired by feeding oxidized fat as demonstrated in guinea pigs (Liu and Lee, 1998), which, like humans, lack an ascorbate synthetic pathway. Guinea pigs fed oxidized fat had lower vitamin C and vitamin E concentrations in plasma and all tissues investigated than those fed oxidized fat (Liu and Lee, 1998). However, the vitamin C and vitamin E status of the guinea pigs significantly improved with 160 Oxidation in foods and beverages and antioxidant applications
increasing dietary vitamin C levels in the diet. Guinea pigs fed the oxidized fat diet supplemented with 1500 mg vitamin C per kg diet had essentially the same plasma and tissue vitamin E concentrations as guinea pigs fed the fresh fat diet supplemented with 300 mg vitamin C/kg diet. In this study, moreover, levels of TBARS were lowered in tissues of guinea pigs fed oxidized fat with increasing levels of vitamin C in the diet. These findings from Liu and Lee (1998) suggest that vitamin C reduces lipid peroxidation and has a vitamin E sparing action during oxidative stress induced by oxidized fat. The vitamin E sparing action of vitamin C is based on the ability of ascorbic acid to reduce tocopheroxyl radicals generated during oxidation of tocopherol to regenerate vitamin E (Niki et al., 1984; Niki, 1987). In another study with guinea pigs the lowering effect of oxidized fat on the vitamin E status and the vitamin E sparing effect of vitamin C could be confirmed (Keller et al., 2004). In addition, this study demonstrated that vitamin E is also able to spare ascorbic acid under conditions of oxidative stress induced by oxidized fat, because supplemental vitamin E increased plasma concentrations of ascorbic acid in guinea pigs fed an oxidized fat.
Besides exogenous antioxidants such as vitamin E and vitamin C, endogen-ous antioxidants such as glutathione (GSH) contribute to the antioxidant defense mechanisms. GSH in the reduced form is known as a free, non-protein thiol compound that exerts its antioxidant function by providing an hydrogen atom to electrophilic compounds and hydroperoxides (Ziegler, 1985). Studies in male Wistar rats revealed that administration of oxidized fats significantly decreases reduced GSH content in the liver (Saka et al., 2002). The decreased GSH content is likely explained by the reduction of lipid peroxides by reduced GSH catalyzed by GPx which leads to the formation of primary alcohols and oxidized GSH. This assumption is supported by the findings of a study in guinea pigs demonstrating an increase in the concentrations of oxidized GSH following administration of oxidized fat (Keller et al., 2004). The observation, however, that supplemental vitamin E and vitamin C did not reduce concentrations of oxidized GSH in the liver of guinea pigs fed the oxidized fat, suggests that the vitamins E and C do not interfere with GSH metabolism. Besides oxidation of reduced GSH, it has also been suggested (Saka et al., 2002) that the decreased reduced GSH concentration is due to conjugation of reduced GSH with compounds absorbed from the oxidized fat, such as CFAM, through enzymatic catalyzes by GSH-S-transferase, leading to non-toxic mercapturic acids.
7.6.6 Effects on glucose tolerance and insulin sensitivity
Whilst the effect of oxidized fat on lipid metabolism has been extensively studied, only a limited number of studies have addressed the effect of oxidized fat on glucose metabolism and insulin sensitivity. So far, only two studies dealing with this topic in rodents have been reported (Chao et al., 2007; Liao et al., 2008). The first study from Chao et al. (2007) revealed that rats fed an oxidized fat prepared by simulating a deep-frying process had increased fasting glucose levels and an impaired glucose tolerance as shown by an increased Health aspects of oxidized dietary fats 161
AUCGlucose(area under the curve for blood glucose) following an oral glucose load. These effects of oxidized fats were unexpected because rats fed the oxidized fat had a significantly smaller visceral adipose tissue mass (epididymal and retroperitoneal fat) and adipocyte cell size, and reduced leptin levels. It is generally accepted that a reduction in visceral adipose tissue mass is accompanied by an improved insulin sensitivity due to decreased plasma levels of white adipose tissue-secreted factors, e.g. leptin, resistin or TNF , which are known to mediate insulin resistance. Pharmacological PPAR activators of the fibrate class, for instance, reduce visceral adiposity and improve insulin sensitivity and glucose tolerance in rodents (Guerre-Millo et al., 2000; Mancini et al., 2001; Ye et al., 2001; Lee et al., 2002). Since oxidized fats exhibit significant PPAR activating properties, which should improve insulin sen-sitivity, the findings from Chao et al. (2007) suggest that the glucose intolerance induced by oxidized fat did not develop from insulin resistance. In line with this assumption is the finding that rats fed oxidized fat showed hypoinsulinemia rather than hyperinsulinemia (Chao et al., 2007). This suggests that the glucose intolerance induced by oxidized fat is due to insulin deficiency and not peripheral insulin resistance. Insulin deficiency leads to a decreased glucose uptake into skeletal muscle and white adipose tissue and an impaired glucose tolerance.
In contrast to the first study (Chao et al., 2007), mice of the C57BL/6J strain were used as model objects in the second study (Liao et al., 2008). The oxidized fat fed to the mice was prepared under the same conditions (deep-frying of dough sheets at 205 ëC for four 6-hourperiods) as in the rat study. The results of the mouse study (Liao et al., 2008) are largely confirmatory of those of the rat study in that administration of oxidized fat caused an impaired glucose tolerance despite reduced fasting and feeding insulin levels as evidenced by significantly higher AUCGlucosebut significantly reduced AUCInsulinand AUCC-peptidein mice fed the oxidized fat (Liao et al., 2008). Also in agreement with the rat study, visceral adipose tissue masses (epididymal and retroperitoneal fat) but also subcutaneous fat mass were significantly smaller in mice fed the oxidized fat compared to those fed the fresh fat. Again, these findings in mice indicate that the glucose intolerance induced by oxidized fat is due to an impaired insulin secretion by pancreatic -cells.
Regarding the mechanisms underlying the hypoinsulinemic effect of oxidized fat, the authors of both studies suspected that the oxidized fat might have provoked oxidative stress, which might have caused oxidative damage to the pancreatic -cells, and, thereby, an impaired insulin secretion (Chao et al., 2007;
Liao et al., 2008). Evidence for the induction of oxidative stress by oxidized fat has been clearly provided in one of those studies by increased levels of TBARS and reduced levels of -tocopherol in the liver (Liao et al., 2008). Although the authors of both studies did not directly study markers of oxidative stress in the pancreatic islets, it is very likely that the oxidized fat also caused oxidative stress in this tissue because several former studies dealing with oxidized fats have consistently demonstrated a depletion of antioxidants and induction of 162 Oxidation in foods and beverages and antioxidant applications
oxidative stress in every tissue investigated (see Section 1.4.5). In addition, it has been shown that among many tissues studied, the pancreatic islets are highly susceptible to antioxidant depletion (Asayama et al., 1986). Furthermore, it is well documented that the glucose-stimulated insulin-releasing capacity of pancreatic -cells is impaired by the induction of oxidative stress (Ammon et al., 1984). This also explains the diabetogenic actions of alloxan and streptozo-tocin which are mediated by reactive oxygen species (Heikkila et al., 1976;
Sandler and Andersson, 1982). Induction of oxidative stress, therefore, might also explain that diet-derived hydroperoxides, which are also contained in heated fats, were shown to contribute to the loss of insulin secretion activity in pancreatic -cells (Tsujinaka et al., 2005). Tsujinaka et al. (2005) reported that a diet high in lipid hydroperoxides due to a lack of vitamin E resulted in glucose intolerance in rats and that this was associated with the development of insulin resistance and an inability to secrete insulin. In response to the oxidative stress caused to -cells, activation of nuclear factor-B (NF-B) signaling pathway in islet cells from hydroperoxide-fed rats was also noticed (Tsujinaka et al., 2005).
NF-B is known to be activated by ROS generated during oxidative stress, and, thus, activation of NF-B can be used as an indicator of oxidative stress.
As a further mechanism explaining the hypoinsulinemia induced by the oxidized fat, the authors of the first study raised the hypothesis that alterations in prostaglandin metabolism might be associated with the hypoinsulinemia (Liao et al., 2008). This assumption was based on the observation that oxidized fat was shown to increase prostaglandin E2levels in plasma and urine of rats (Huang, 2003), and that transgenic induction of prostaglandin E2was reported to cause a destruction of pancreatic -cells (Oshima et al., 2006).
7.6.7 Effects on inflammation
Several reports in the literature demonstrate that oxidized fats strongly induce oxidative stress (see Section 1.4.5). Although a link between oxidative stress and inflammation has been clearly established (Schreck et al. 1991; Sen and Packer, 1996), only one study so far has investigated the effect of oxidized fat on inflammatory processes (Ringseis et al., 2007c). In that study, two groups of pigs were fed two different diets containing either fresh fat or oxidized fat prepared by heating at 200 ëC for 24 h. After 4 weeks on the diets, the pigs were sacrificed and intestinal epithelial cells were isolated and markers of inflam-mation (NF-B transactivation and NF-B target gene expression) determined.
NF-B plays a key role in inflammatory diseases due to its ability to bind specifically to NF-B-response elements in the promoters of key inflammatory genes (e.g., COX-2, iNOS, TNF , and IL-6) and induce their gene transcription (Barnes and Karin, 1997).
The results of the pig study (Ringseis et al., 2007c) show that markers of inflammation in intestinal epithelial cells were not altered by dietary oxidized fat indicating that oxidized fat does not induce an inflammatory response in the intestine. These findings were unexpected because in that study an increased Health aspects of oxidized dietary fats 163
lipid peroxidation and a depletion of antioxidants, both of which are indicative of the induction of oxidative stress, in the intestinal cells were clearly shown (Ringseis et al., 2007c). In addition, reduced activities of antioxidant enzymes in intestinal cells were observed in that study. This is also indicative of the induction of oxidative stress, because oxidative stress in the intestine is accom-panied by reduced activities of enzymatic antioxidants in enterocytes (Mehta et al., 1998; Thomas et al., 2005). However, the authors of the pig study observed only a comparatively (Mehta et al., 1998; Thomas et al., 2005) slight impair-ment of the antioxidant defense mechanisms, suggesting that the oxidative stress induced by the oxidized fat was only moderate. Hence, it is not unlikely that the antioxidant defense system of the intestinal epithelial cells of the pigs was still sufficient to cope with the oxidative burden of the ingested lipid peroxides and the oxidative stress induced was not strong enough to induce proinflammatory gene transcription. In contrast, strong induction of oxidative stress in intestinal cells leads to a marked activation of proinflammatory transcription factors and destabilization of cell integrity (Bernotti et al., 2003). Thus, the authors of the pig study proposed that extracellular detoxifying enzymes in the mucus layer of the intestine, which are involved in the protection of enterocytes from direct contact with diet-derived oxidants such as lipid peroxides in the gut lumen, might have limited the oxidative burden to the epithelial cell, and are therefore responsible for the slight impairment of the antioxidant defense system observed (Samiec et al., 2000). Thus, future studies have to investigate whether oxidized fats which cause a stronger induction of oxidative stress are able to induce inflammatory processes.
Inflammatory processes in various tissues, including the intestine (Straus et al., 2000) were shown to be attenuated by activation of PPAR (Su et al., 1999;
Tanaka et al., 2001; Sanchez-Hidalgo et al., 2005). This effect is mediated by inhibition of the NF-B pathway and other signalling pathways involved in inflammatory processes by PPAR (Takagi et al., 2002). This transrepression activity likely constitutes the mechanistic basis for the anti-inflammatory properties of PPAR and probably also explains why pharmacological PPAR -ligands markedly reduce inflammation in animal models of inflammatory colitis (Takagi et al., 2002; Su et al., 1999; Tanaka et al., 2001; Sanchez-Hidalgo et al., 2005). Because oxidized fats also contain agonists of PPAR such as oxidized fatty acids (Bull et al., 2003; Grisham et al., 1990), it has been hypothesized that oxidized fats inhibit inflammatory processes through activation of PPAR . This hypothesis, however, could not be verified, although the oxidized fat caused a moderate PPAR activation in the intestinal epithelial cells (Ringseis et al., 2007c). Presumably, the lack of effect of oxidized fat on markers of inflam-mation is due to an insufficient transrepression of NF-B by the oxidized fat due to the moderate PPAR activation. Alternatively, the lack of effect on inflam-matory gene transcription is due to the fact that the basal inflaminflam-matory state in the intestinal epithelium of the pigs was rather low, and a reduction of inflam-matory indices in normal healthy animals, as used in that study, is expected to be only marginal. Therefore, future studies should investigate whether oxidized fats 164 Oxidation in foods and beverages and antioxidant applications
exert a different effect on inflammatory gene transcription during states of acute intestinal inflammation, e.g. in animal models of experimental colitis.
Whether the impairment of the antioxidant defense system by oxidized fat is of significance for the integrity of the intestinal epithelium (barrier function) has to be investigated in future studies. It has been suggested that a progressive fall in enzymatic and nonenzymatic antioxidants as observed in the present study precedes the occurrence of damage to intestinal cell constituents (Bernotti et al., 2003). Since the intestinal epithelial cells are primarily responsible for the antioxidant defense of the epithelium against luminal oxidants (Grisham et al., 1990), it is likely that in the presence of stronger irritants, such as toxins or pathogens, the already impaired defense mechanisms of the intestinal epithelial cells in response to oxidized fat are not sufficient to cope with this additional challenge nor to preserve cellular integrity and homeostasis of the intestinal epithelium.