AJP-Endo Articles in PresS. Published on July 30, 2002 as DOI 10.1152/ajpendo.00110.2002
Protection Against Diet-Induced Obesity and Obesity-Related Insulin Resistance in Group 1B Phospholipase A2 Deficient Mice
Kevin W. Huggins, Amy C. Boileau, and David Y. Hui
From the Center for Lipid and Arteriosclerosis Studies, Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
[Running title: Obesity Resistance in Phospholipase A2 Deficient Mice]
Address for correspondence: David Y. Hui, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45245-0529. Telephone: (513) 558-9152; Fax: (513) 558-2141; Email:
[email protected]
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Copyright 2002 by the American Physiological Society.
Abbreviations used: PLA2 , the Group 1B type phospholipase A2 ; PTP-1B, protein tyrosine phosphatase-1B; TNF-α, tumor necrosis factor-alpha
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Abstract Group 1B phospholipase A2 (PLA2 ) is an abundant lipolytic enzyme that is well characterized biochemically and structurally. Due to its high level of expression in the pancreas, it has been presumed that PLA2 plays a role in the digestion of dietary lipids, but in vivo data has been lacking to support this theory. Our initial study on mice lacking PLA2 demonstrated no abnormalities in dietary lipid absorption in mice consuming a chow diet. However, the effects of PLA2 deficiency on animals consuming a high fat diet have not been studied. To investigate this, PLA2 +/+ and PLA2-/- mice were fed a Western diet for 16 weeks. The results showed that PLA2 -/mice were resistant to high fat diet-induced obesity. This observed weight difference was due to decreased adiposity present in the PLA2 -/- mice. In comparison to PLA2 +/+ mice, the PLA2 -/- mice had 60% lower plasma insulin and 72% lower plasma leptin levels after high fat diet feeding. The PLA2 -/- mice also did not exhibit impaired glucose tolerance associated with the development of obesity-related insulin resistance as observed in the PLA2 +/+ mice. In order to investigate the mechanism by which PLA2 -/- mice exhibit decreased weight gain while on a high fat diet, fat absorption studies were performed. The PLA2 -/- mice displayed 50% and 35% decreased plasma [3 H]triglyceride concentrations 4 and 6 h after feeding a lipid-rich meal containing [3 H]triolein. The PLA2 -/- mice also displayed increased lipid content in the stool, thus indicating decreased fat absorption in these animals. These results suggest a novel role for PLA2 in the protection against diet-induced obesity and obesity-related insulin resistance, thereby offering a new target for treatment of obesity and diabetes.
Keywords:
Lipase, Pancreatic Enzymes, Animal Models, Lipid Absorption
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The phospholipase A2 (PLA2 ) lipolytic enzyme is an abundant protein secreted by the pancreas in response to food intake. It belongs to the Group 1 class B type of secretory PLA2 (7) and is capable of hydrolyzing the fatty acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids in the intestinal lumen. In addition to its prominent expression in the pancreas, the Group IB PLA2 is also expressed in other tissues (15, 29, 31, 32). Phospholipase A2 has been one of the most extensively studied enzymes in terms of structure and mechanism of action due to its abundant availability, stability, and ease of isolation. Despite the wealth of information known about the biochemical and structural characteristics of this enzyme (7, 8), the exact physiological function of PLA2 has not been completely delineated.
Phospholipids entering the digestive tract from the diet and bile are the second most abundant dietary lipid class found in the intestinal lumen (5). Therefore, it has been suggested that PLA2 functions in hydrolyzing these phospholipids to forms that can be absorbed by the enterocytes. Various in vitro and in vivo studies have also implicated that PLA2 hydrolysis of phospholipids in the intestinal lumen is required for the efficient absorption of cholesterol from the diet. Studies in rats and humans have shown that intraduodenal infusion of phosphatidylcholine results in decreased cholesterol absorption compared to subjects infused with lower levels of phosphatidylcholine (2, 17). Mackay et al. (24) identified PLA2 as the major protein in pancreatic extract that mediates cholesterol transport in Caco-2 cells. The addition of PLA2 relieved the phosphatidylcholine inhibition of cholesterol transport from bile salt micelles 4
to Caco-2 cells (18). In addition, our laboratory has shown that PLA2 hydrolysis of phospholipids on the surface of lipid emulsions was required before pancreatic lipase digestion of triglycerides in the core of lipid emulsions, therefore, suggesting a role for PLA2 in fat absorption (39).
In order to investigate the role of PLA2 in intestinal lipid digestion and transport, we recently generated mice lacking PLA2 . Using both lymph fistula and single-dose, dual isotope fecal recovery methods, we demonstrated that mice deficient in PLA2 had no differences in the absoprtion of dietary lipids compared to wild type mice (30). We concluded that while phospholipid digestion in the intestinal lumen is a prerequisite for efficient absorption of dietary lipids, additional enzyme(s) in the digestive tract can compensate for the lack of PLA2 in catalyzing phospholipid digestion and facilitating lipid absorption in the PLA2 knockout mice (30). However, these studies were performed under basal chow-fed dietary conditions and therefore dietary lipid absorption in the PLA2 knockout mice consuming a high fat diet is not known.
The goal of the present study was to assess the lack of PLA2 in mice fed a western type high fat/high cholesterol diet. We hypothesize that the compensatory mechanisms present to compensate for the absence of PLA2 under basal chow-fed conditions may be overcome by feeding mice a high fat diet. We report that PLA2 -deficient mice are resistant to high fat dietinduced obesity and the mechanism for this effect is most likely due to decreased dietary fat absorption.
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EXPERIMENTAL PROCEDURES Generation and Maintenance of PLA2 Deficient Mice - The strategy used to disrupt the PLA2 gene to generate PLA2 deficient mice was described previously (30). All animals used in these studies were backcrossed seven times into the C57BL/6 background and were genotyped by PCR as previously described (30). The mouse colony was maintained in a temperature and humidity controlled room with a 12-hour light/dark cycle and fed a rodent chow (LM485; Harlan-Teklad, Madison, WI) with free access to water. All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati.
Diet-Induced Obesity Study - Wild type (PLA2 +/+) and PLA2 deficient (PLA2 -/-) mice were fed a standard mouse chow (LM485) or a Western type high fat/high cholesterol diet containing 21% fat and 0.15% cholesterol by weight (TD88137, Harlan Teklad) for 16 weeks. For the insulin tolerance test studies, PLA2 +/+ and PLA2 -/- were fed a high fat-high carbohydrate diet (#F3282; Bioserve Industries, Inc., Frenchtown, NJ) which contained 35.5% (w/w) fat (primarily lard) and 36.6% carbohydrate (primarily sucrose) for 14 weeks. Mice had free access to water during the study period. At the beginning of the diet study, mice were 8-10 weeks of age. Body weights were recorded throughout the experimental feeding period.
Food consumption studies - At week 14 of the experimental diet period, individually caged mice were given preweighed food and the amount of food consumed was determined over a 24 h period for 5 days. The results are expressed as grams of food consumed per day.
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Glucose tolerance tests - Following an overnight fast, PLA2 +/+ and PLA2 -/- male mice consuming either the basal low fat or the Western type diet for 15 weeks, were injected intraperitoneally with a bolus load of glucose (2 g/kg body weight). Blood was obtained from the tail vein before and 15, 30, 60, and 120 min after glucose administration. Blood glucose was measured using an automated glucose analyzer (Elite XL, Bayer Corp., Elkhart, IN).
Insulin tolerance tests - Following a 4 hour fast, PLA2 +/+ and PLA2 -/- female mice consuming either the basal low fat or high fat/high carbohydrate diet for 14 weeks were injected intraperitoneally with bovine insulin (1 U/kg body weight; Sigma Chemical Co., St. Louis, Missouri). Blood was obtained for glucose determination from the tail vein before and 15, 30, and 60 min after insulin administration.
Animal sacrifice - Following the 16 week experimental feeding period, the mice were fasted for 4 h and then anesthetized by intraperitoneal injection with a solution composed of ketamine (80 mg/kg body weight; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and xylazine (16 mg/kg; The Butler Co., Columbus, OH) diluted in 0.9% saline. Body temperature was determined using a rectal thermometer. Blood was removed by cardiac puncture into tubes containing 1mM EDTA. Adipose tissue (epidydimal and uterine fat pads), brown adipose tissue (intrascapular), liver, heart, spleen, lungs, and kidneys were removed and wet weight recorded.
Plasma chemistries - Plasma was obtained by low speed centrifugation of the blood samples. Plasma triglyceride, cholesterol, and free fatty acid concentrations were determined by colometric assays from Wako Chemicals, Inc. (Richmond, VA). Plasma leptin and insulin 7
concentrations were measured using radioimmunoassay kits from Linco Research Inc. (St. Charles, MO). Blood glucose was measured as described above. Results from male and female mice were averaged together since there were no apparent differences based on sex of the animal. Free fatty acid concentrations in plasma were determined only from the female mice.
Postprandial Fat Absorption - Mice maintained on the basal low fat diet or Western diet for 4 weeks were fasted overnight. The following morning, the mice were injected with 12.5 mg Triton WR1339 to block lipolysis (1). Ten min later, the mice received an intragastric load of 1 µCi of [3 H]triolein (Amersham Pharmacia Biotech, Piscataway, NJ) in 50 µl olive oil. Mice were allowed access to water but no food during the course of the experiment. Blood samples were taken 1, 2, 4, and 6 h after gavage by tail bleeding. Radioactivity appearing in plasma was determined by liquid scintillation counting.
Fecal lipid analysis - Feces were collected from mice fed western diet for 4 weeks over a 24 h period. Mice in each group were housed 4 per cage and therefore results represent data from pooled fecal samples. The stool samples were dried to a constant weight, the lipids were extracted from 100 mg of dried feces as described (32) and analyzed by thin-layer chromatography as described (3, 23).
Statistical analysis - All results are presented as mean ± S.D. Differences between the two genotypes were determined by Student's t test or the Mann-Whitney Rank Sum test. Differences in the body weight growth curves, glucose tolerance tests, and insulin tolerance tests were determined by one-way ANOVA followed by the Tukey-Kramer tests. P < 0.05 was accepted as 8
statistically significant. All statistical analysis was completed using the SigmaStat software from Jandel Corporation (San Rafael, CA).
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RESULTS Mice lacking PLA2 have normal growth, lipid metabolism, and reproductive functions while maintained on a basal low fat diet (30, and unpublished observations). To assess the lack of PLA2 on mice fed a western type high fat/high cholesterol diet for an extended period of time, wild type (PLA2 +/+) and PLA2 knockout (PLA2 -/-) male and female mice were fed either a standard mouse chow or a high fat/high cholesterol (21% fat, 0.15% cholesterol) diet for 16 weeks. There was no difference in body weight between PLA2 +/+ and PLA2 -/- mice when fed the basal low fat diet (males: 25.3 ± 3.4 versus 23.8 ± 4.0 g; females: 23.9 ± 4.0 versus 21.6 ± 0.60 g, respectively). In contrast, the PLA2 +/+ male and female mice gained significantly more weight than the PLA2 -/- mice upon feeding the western diet (Fig. 1). This resulted in approximately 57% and 40% more weight gained by the male (insert in Fig. 1A) and female (insert in Fig. 1B) PLA2 +/+ mice compared to the PLA2 -/- mice after feeding the high fat diet for 16 weeks.
To account for the differences in weight gain between the PLA2 +/+ and PLA2 -/- mice, various tissues were removed and weighed. Under low fat dietary conditions there were no differences in epidydimal and uterine fat pad, brown fat, and liver weights between male and female PLA2 +/+ and PLA2 -/- mice (Fig. 2A and B). In contrast, the PLA2 -/- mice had approximately 50% lower epidydimal and uterine fat pad weight compared to PLA2 +/+ mice (Fig. 2C and D). The increased adiposity was specific for white fat since there was no difference in brown adipose mass between the two genotypes after high fat dietary treatment (Fig. 2C and D). Male PLA2 -/- mice also had approximately 36% lower liver weight compared to the PLA2 +/+ mice (Fig. 2C). However, this difference in liver weight was not apparent in the female mice (Fig. 2D). There were also no significant differences in heart, spleen, lung, and kidney weights 10
between the PLA2 +/+ and PLA2 -/- mice fed the western diet (data not shown). These data suggest that the observed weight difference among the mice fed the western diet was due to increased adiposity in the wild type mice.
In addition to differences in body weight gain and adiposity upon feeding a high fat/high cholesterol diet, the PLA2 -/- mice had decreased fasting plasma leptin and insulin concentrations compared to the PLA2 +/+ mice (Table I). The difference in leptin levels is most likely due to changes in adipose tissue observed between the two groups of mice. In contrast, no significant difference was observed in fasting plasma glucose and free fatty acid concentrations (Table I). However, there was a consistent trend toward decreased (20%) fasting glucose levels in the PLA2-/- mice. Plasma cholesterol and triglyceride levels were similar between the PLA2 +/+ and PLA2-/- mice after high fat dietary treatment (Table I).
The high fat-fed PLA2 +/+ mice appear to be more insulin resistant than the high fat-fed PLA2-/- mice because the increased fasting plasma insulin concentrations are necessary to maintain normal plasma glucose concentrations. To directly test glucose metabolism in these animals, glucose tolerance tests were performed on PLA2 +/+ and PLA2 -/- under basal low fat dietary conditions when the animals were similar in weight and adiposity and after feeding a high fat diet. There was no difference in glucose metabolism between the PLA2 +/+ and PLA2 -/mice consuming the chow diet (Fig. 3A). In contrast, after high fat feeding, the PLA2 -/- mice displayed lower blood glucose concentrations 15 and 30 min after intraperitoneal glucose administration compared to those observed in the PLA2 +/+ mice. (Fig. 3B). In addition, insulin levels 30 min post-glucose injection were 45% lower (1.15 ± 0.28 vs. 0.63 ± 0.08 ng/mL; P < 11
0.05) in the PLA2 -/- mice. These data demonstrate that high fat-fed PLA2 +/+ mice have impaired glucose tolerance due to the development of obesity-related insulin resistance, whereas high fatfed PLA2 -/- mice maintained normal glucose tolerance.
To directly test the development of obesity-related insulin resistance in these animals, insulin tolerance tests were performed. Both the PLA2 +/+ and PLA2 -/- mice were fed a low fat and high fat/high carbohydrate diet for 14 weeks. The latter diet has been shown previously to induce obesity and insulin resistance in C57BL/6 mice (36, 37). Interestingly, female PLA2 -/- mice exhibited increased glucose disposal 30 min after insulin injection when compared to PLA2 +/+ mice under low fat feeding conditions (Fig. 4A). After feeding the high fat/high carbohydrate diet for 14 weeks, the PLA2 -/- had lower blood glucose levels 15, 30, and 60 min after insulin injection when compared to PLA2 +/+ mice (Fig. 4B). These results confirmed that PLA2 -/- mice were protected against the development of high fat-induced insulin resistance. The data also suggest that PLA2 -/- mice have improved insulin sensitivity compared to PLA2 +/+ mice even under low fat feeding conditions.
To examine potential mechanisms for the resistance to diet-induced obesity in the PLA2 -/mice, we performed studies to measure food consumption, body temperature, and postprandial fat absorption. There was no difference in the amount of food consumed per day or resting body temperature between the PLA2 +/+ and PLA2 -/- mice (Table I). This data suggests that caloric intake and energy expenditure is not contributing to the resistance to diet-induced obesity in the PLA2-/- mice. To examine postprandial fat absorption, we injected mice fed the basal low fat diet or the high fat diet for 4 weeks with Triton WR1339 to inhibit lipolysis and suppress lipoprotein 12
clearance from circulation. A bolus load of olive oil containing [3 H]triolein was then fed to each mouse by gastric gavage. Lipid absorption efficiency was determined based on the appearance of [3 H]triglyceride in the plasma. Results, as shown in Fig. 5A, indicated no difference in the appearance of [3 H]triglyceride in the plasma of PLA2 +/+ and PLA2 -/- mice fed the chow diet. Interestingly, PLA2 -/- mice previously maintained on a high fat diet had decreased appearance of [3 H]triglyceride in the plasma 4 and 6 h after oil administration (Fig. 5B). In addition, there were no significant differences in [3 H]triglyceride present in the intestinal wall between PLA2 +/+ and PLA2-/- mice (data not shown). This data suggests that the decreased postprandial fat absorption observed in the PLA2 -/- mice was not due to increased intestinal retention of radiolabel, thus suggesting sub-obtimal lipid digestion and/or uptake in the intestinal lumen of PLA2 -/- mice.
Possible difference in fat absorption efficiency between PLA2 +/+ and PLA2 -/- mice was addressed directly by measuring their fecal lipid output after feeding them the western diet for 4 weeks. Fecal lipids were extracted and analyzed by TLC analysis. There was increased lipid in the form of triglycerides, fatty acids, and a lipid band migrating with cholesteryl ester in the PLA2-/- mice compared to that observed in PLA2 +/+ mice (Fig. 6). Since the diet contains relatively small amounts of cholesteryl ester, the identity of the fastest migrating band may be retinyl ester which is known to co-migrate with cholesteryl ester. These data provided additional supporting evidence to document that mice lacking PLA2 have increased fecal lipid output when maintained on a high fat diet.
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DISCUSSION Obesity has developed into a significant health problem in Westernized societies over the past twenty years due to its association with various chronic diseases such as noninsulin dependent diabetes (type 2 diabetes), cardiovascular disease, and cancer (10, 19). The increased prevelance of obesity as been attributed to increased availability and consumption of fat-rich foods and reduced physical activity (35). This has led many researchers to develop animal models that will allow for the study of the mechanisms by which diet-induced obesity contributes to various disease states.
The C57BL/6 mouse has been shown previously to be a good model for studying dietinduced obesity and diabetes. They develop obesity, insulin resistance, and hyperlipidemia resembling human type 2 diabetes after feeding a western type high fat diet (22, 36-38). Results obtained from our wild type mice are consistent with these previous reports. Interestingly, C57BL/6 mice with a PLA2 -null mutation were resistant to diet-induced obesity. This resulted in these animals being hypoinsulinemic, hypoleptinemic, and more insulin sensitive compared to their obese wild type counterparts. The phenotype of these PLA2 -/- mice are similar to that observed in mice lacking the acyl CoA:diacylglycerol transferase (Dgat) gene (34) and to animals lacking the protein tyrosine phosphatase-1B (PTP-1B) gene (7). In the Dgat -/- mice, obesity resistance was attributed to increased energy expenditure and increased activity (34). Obesity resistance and increased insulin sensitivity in the PTP-1B-/- mice was due to alteration in fat metabolism and increased energy expenditure as a consequence of alterations in insulin signaling pathway in muscle (20). Since the amount of food eaten and resting body temperature were similar between PLA2 +/+ and PLA2 -/- mice, we conclude that decreased caloric intake and 14
increased energy expenditure are not contributing to the resistance to diet induced obesity in the PLA2-/- mice. In contrast, the increased fecal lipid output observed in the PLA2 -/- mice suggests that the most likely mechanism to account for the observed difference in diet-induced obesity is reduced fat absorption in the PLA2 -/- mice after high fat feeding.
Previous studies from our laboratory has shown that PLA2 -/- mice displayed normal lipid absorption efficiency when fed a low fat diet (30), and responded to high fat feeding with increased plasma cholesterol and triglyceride levels to the same extent as that observed in the PLA2 +/+ mice. These results are consistent with previous observations that additional phospholipase(s) in the intestine can partially compensate for the lack of PLA2 in the PLA2 -/mice (30). The reduced fat absorption efficiency in these animals after chronic feeding of a high fat diet suggested that these compensatory phospholipases are not sufficient for complete fat digestion under high fat loading conditions and that PLA2 is required for optimal lipid digestion and absorption. The mechanism by which PLA2 inhibits fat absorption upon high fat feeding is likely related to the requirement for both pancreatic triglyceride lipase and PLA2 in complete fat digestion prior to its absorption by intestinal cells (18, 24, 39). Previous studies have clearly documented that inhibition of fat absorption by pancreatic triglyceride lipase inhibitors is effective in weight reduction in obese patients (25, 28). The current study revealed that reducing level and activity of PLA2 was also effective in reducing fat absorption and weight gain.
An explanation to account for this data is that PLA2 activity and/or its presence is necessary for the upregulation of fat absorption pathways that are induced by high fat feeding. One possible mechanism by which this may occur is its putative role in the release of digestive 15
enzymes and/or hormones from the pancreas. It is well established that high fat feeding results in an increase in pancreatic lipase synthesis and secretion from pancreatic acinar cells (4). This presumably occurs because more lipase is needed to digest the increased amounts of fat entering the small intestine. PLA2 may be necessary for the upregulation of lipase from pancreatic acinar cells under high fat conditions. This may occur either through hydrolysis of membrane phospholipids and thereby modifying the properties of the membranes favoring the secretory process, or alternatively, PLA2 catalyzed hydrolysis of phospholipids may generate lipid signaling molecules, such as lysophospholipids, that are mediators of the secretory process.
Another possible route by which PLA2 may influence dietary lipid absoprtion is through an indirect mechanism mediated by its regulation of secretin release. This gastrointestinal hormone is known to be important in the pancreatic adaptation to dietary fat (4). It has also been shown to stimulate insulin secretion and enhance insulin response to glucose (13). Recent studies have identified the Group 1B PLA2 as the secretin releasing factor in the intestinal lumen (6, 21). Since secretin release is stimulated by fat feeding, it is possible that PLA2 -/- mice have reduced secretin level due to their lack of this secretin releasing factor. This in turn may influence the fat stimulated release of lipase leading to reduced fat absorption in these animals. Additional studies will need to be conducted to test this hypothesis.
While the decreased level of fat absorption is the most likely cause of the protection against diet-induced obesity and obesity-related insulin resistance observed in the PLA2 -/- mice, other mechanisms may also contribute to this observed phenotype. The increased glucose disposal in response to insulin challenge observed in the PLA2 -/- mice under low fat feeding 16
conditions suggested that PLA2 gene inactivation may also alter insulin signaling pathways in a manner similar to that observed in the PTP-1B-/- mice (9, 20). Previous studies have shown that the Group 1B PLA2 is also expressed in other tissues, in addition to its prominent expression in the pancreas (15, 29, 31, 33). The peripheral PLA2 interacts with specific receptors and modulates cell functions (14). Thus, it is possible that defects in PLA2 interaction with PLA2 receptors, such as those observed in PLA2 deficient mice, may influence insulin sensitivity in a favorable manner. In support of this hypothesis is the report that PLA2 receptor-defective mice are more resistant to endotoxic shock due to reduced plasma level of tumor necrosis factor-á (TNF-á) (16). This cytokine confers insulin resistance in cells by regulating glucose transporter synthesis and by interfering with insulin signaling (26). Accordingly, Group 1B PLA2 deficiency may lower TNF-á production and increase insulin sensitivity by this manner. Also, cell culture data have suggested that PLA2 participate directly in insulin secretion from pancreatic islets (11, 12, 27). Challenging the PLA2 -/- mice with a high carbohydrate diet without fat may help to elucidate a role for PLA2 in insulin signaling.
In summary, we report that mice lacking Group 1B PLA2 are resistant to diet-induced obesity. This most likely occurs through suppression of dietary fat absorption under high fat feeding conditions. Breeding the PLA2 deficient mice to various genetic models of obesity will provide exciting tools to understand the mechanisms of PLA2 on energy metabolism. Finally, regardless of the precise mechanism by which PLA2 deficiency results in protection against diet induced obesity and obesity-related insulin resistance, these results offer a novel therapeutic strategy, i.e., the inhibition of Group 1B PLA2 activity, for the treatment of obesity and type 2 diabetes. 17
ACKNOWLEDGMENTS This research was supported by a Program Project Grant (DK54504) from the National Institutes of Health. K.W.H. was the recipient of a National Research Service Award from the NIH (#F32 DK10065), and A.C.B. received a Post-Doctoral Fellowship in Interdisciplinary Nutrition Science from the Dannon Institute.
We thank Drs. Patrick Tso, Laura Woollett, and Phillip Howles for valuable discussions and Dr. David D'Alessio for assistance with the insulin assay. Nick Schildmeyer and Tara Riddle provided excellent technical assistance to this study.
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FIGURE LEGENDS
Fig. 1. Body weights of PLA2 +/+ and PLA2 -/- mice after feeding a western type diet for 16 weeks. Male (Panel A) and female (Panel B) mice 8-10 weeks of age were placed on diet containing 21% fat and 0.15% cholesterol (w/w) for 16 weeks. Body weight of wild type (filled circles) and PLA2 -null (open circles) mice were determined every 2 weeks. The insert in each panel shows the weight gained from PLA2 +/+ (solid bars) and PLA2 -/- (hatched bars) mice after 16 weeks. The data points represent the mean ± S.D. from 4 animals in each group. * indicates significant difference between the groups at P < 0.05.
Fig. 2. Tissue weights. White adipose, brown adipose, and liver wet weights from chow-fed male (Panel A), chow-fed female (Panel B), 16 week western-fed male (Panel C) and 16 week western-fed female (Panel D) PLA2 +/+ (solid bars) and PLA2 -/- (open bars) mice. Data are expressed as mean ± S.D. from 4 animals in each group. Statistically significant differences were determined by Student's t test.
Fig. 3. Glucose tolerance tests. Glucose homeostasis in male PLA2 +/+ (filled circles) and PLA2 -/(open circles) mice fed a chow (Panel A) and western diet (Panel B) for 15 weeks. Four animals in each group were injected i.p.with a bolus load of glucose (2 g/kg body weight) following an overnight fast. Blood was obtained from the tail vein for glucose analysis. Data are expressed as mean ± S.D. * indicates significant difference from wild type animals at P < 0.05.
24
Fig. 4. Insulin tolerance tests. Insulin sensitivity in female PLA2 +/+ (filled circles) and PLA2 -/(open circles) mice fed a chow (Panel A) and high fat/high carbohydrate diet (Panel B) for 14 weeks. Animals were injected i.p. with bovine insulin (1 U/kg body weight) following a 4 h fast. Blood was obtained from tail vein for glucose analysis. Data are expressed as % of fasting glucose levels (mean ± S.D.) from 8 –10 animals in each group fed the chow diet and 5 animals in each group fed the high fat/high carbohydrate diet. * indicates significant difference from wild type animals at P < 0.05.
Fig. 5. Postprandial fat absorption. Fat absorption was determined by measuring the appearance of [3 H]triglyceride after an intragastric gavage of [3 H]triglyceride in olive oil. After an overnight fast, male PLA2 +/+ (filled circles) and PLA2 -/- (open circles) mice fed a chow (Panel A) and western diet (Panel B) for 4 weeks were injected via the retroorbital plexus with 12.5 mg of Triton WR1339. Ten min after injection, the mice received an intragastric load of 2 µCi of [3 H]triolein in 50 µl olive oil. Blood samples were taken 1, 2, 4, and 6 h after gavage by tail bleeding. Radioactivity appearing in plasma was determined by liquid scintillation counting. Data are expressed as mean ± S.D. from 7 animals in each group fed the chow diet and 4 animals in each group fed the western diet. * indicates significant difference from wild type animals at P < 0.05.
Fig. 6. Fecal lipid analysis. Lipids from feces of wild type and PLA2 -null mice fed western diet for 4 weeks were collected over a 24 h period, extracted, and analyzed by thin-layer chromatography. Results represent data from pooled fecal samples from 4 animals in each 25
group. The lipid standards are shown on the right of the panel. MG, monoacylglycerol; FFA, free fatty acid; CH, cholesterol; DG, diacylglycerol; TG, triglyceride; CE, cholesteryl ester.
26
Table I
Food Intake and Serum Chemistries in PLA2 +/+ and PLA2-/- Mice fed Western Diet
a
PLA 2 +/+
PLA 2 -/-
Body temperature (°C)
36.8 ± 0.5
36.5 ± 0.8
Food intake (g/day)
2.5 ± 0.4
2.7 ± 0.2
Triglyceride (mg/dL)
58.5 ± 7.4
61.6 ± 8.2
Cholesterol (mg/dL)
139.7 ± 12.0
136.8 ± 11.8
Leptin (ng/mL)
22.5 ± 6.8
6.3 ± 1.8a
Insulin (ng/mL)
0.92 ± 0.34
0.38 ± 0.10a
Glucose (mg/dL)
179 ± 32
140 ± 24
FFA (mM)
0.24 ± 0.02
0.36 ± 0.12
P < 0.05
27
*
*
*
*
*
50
Body Weight (g)
*
40 30 20 10
25
P = 0.02
20 15 10
B
40
*
20
15
10
5 0
0 4
6
*
*
P = 0.02
12 9 6 3 PLA2(+/+) PLA2(-/-)
0 2
*
0
PLA2 (+/+)PLA2 (-/-)
0
*
*
30
Weight gain (g)
A
Weight gain (g)
Body Weight (g)
50
8 10 12 14 16
0
Weeks on High Fat Diet
2
4
6
8 10 12 14 16
Weeks on High Fat Diet
(Fig. 1)
28
A.
3.0
Wet weight (g)
Wet weight (g)
3.0 2.5 2.0 1.5 1.0 0.5
C.
n ow Br
t Fa
2.0 1.5 1.0
P = 0.03
0.0
er Liv
P = 0.03
3.0
Wet weight (g)
Wet weight (g)
at eF t i h W
2.5 2.0 1.5 1.0 0.5 0.0
2.5
0.5
0.0
3.0
B.
at eF hit W
D.
n ow Br
t Fa
er Liv
t Fa
er Liv
P < 0.05
2.5 2.0 1.5 1.0 0.5
at eF hit W
n ow Br
t Fa
0.0
er Liv
(Fig. 2)
29
at eF hit W
n ow Br
(Fig. 3)
30
Fasting blood glucose (% of original)
A
100 80 60 40
*
20 0 0
15
30
45
60
Fasting blood glucose (% of original)
Time (min) 100
B
80 60
*
40
*
20 0 0
15
30
45
Time (min)
(Fig. 4)
31
60
[ 3H]Triglyceride (dpm/10 µL)
2000
A
1500 1000 500 0 1
2
3
4
5
6
[ 3H]Triglyceride (dpm/10 µL)
Time (h) 2000
B
1500 1000 500
*
*
0 1
2
3
4
5
Time (h)
(Fig. 5)
32
6
(Fig. 6)
33
