Original Research Article

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1 IUBMB Life, 48: 109±113, 1999 Copyright c 1999 IUBMB /99 $ Original Research Article Effects of Genetic and Diet-Induced Obesity on Lipid Metabolism Roksan Libinaki, Mark Heffernan, Woei-Jia Jiang, Esra Ogru, Vera Ignjatovic, Robert Gianello, Lisa Trickey, Michelle Taylor, and Frank Ng Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3168, Australia Summary C57BL/6J obese (ob/ob) and lean mice fed ad libitum on a normal mouse chow diet (Normal), were compared with lean mice of the same age and strain fed ad libitum on a high-fat diet, consisting of the Normal diet with the addition of beef lard (Lard), from age 3 months for 34 days. The lard-fed mice were seen to have significantly higher (P < 0.05) body weight in this 34-day period than that of the other two groups fed on the Normal diet. Epididymal fat depot and adipocyte cell size were signi cantly larger (P < 0.05) in the Lard-fed lean mice and in the obese (ob/ob) mice than were those of the Normal-fed lean mice. Dietary Lard intake did not signi cantly affect concentrations of plasma triglyceride although those of plasma cholesterol were signi cantly increased (P < 0.05). The development of obesity in these Lard-fed mice appeared to be accelerated and signi cant. IUBMB Life, 48: 109± 113, 1999 Keywords Diet-induced obesity; genetic obesity; high-saturated fat diet; mice. INTRODUCTION Excess fat mass, characterising obesity, usually results from excessive energy storage over a prolonged period of time. This energy imbalance can theoretically result from excessive energy intake and lower energy expenditure, such as abnormal metabolic processes, impaired thermoregulation, or reduced physical activity. The development of obesity is believed to be in uenced by a number of factors, including genes and environment. Studies in animal models have clearly demonstrated that these 2 distinct types of obesity exist and that their mechanisms of excess fat mass accumulation may differ substantially (1). First is genetic obesity, as seen in rodent strains such as the Zucker fatty (fa/fa) rat and the obese (ob/ob) mouse used in this Received 20 October 1998; accepted 26 January Address correspondence to Associate Professor Frank Ng. study; these animals become obese under a wide range of experimental conditions. If food is provided ad libitum, hyperphagia will usually occur and account for most of the excess body fat gain. If food is restricted, a decrease in energy expenditure (primarily thermoregulation) is seen, and the resulting positive energy balance will again permit substantial body weight (2). The second basic type of obesity results from environmental interactions, but evidence to provide a de nitive mechanism for this type of obesity is yet to be de ned. With few exceptions, obesity can be induced by high-fat diets in several animal species, including monkeys, dogs, pigs, hamsters, squirrels, rats, and mice (1). Thesecafeteria-fed animals in previous studies have demonstrated increased body weight and metabolic changes that favour an increase of fat accumulation. Dietary fat is poorly regulated; that is, excessive intake of fat at one meal is not followed by decreased intake at the next meal (3). Fat in the diet increases the palatability of food, as well as having a higher energy density than carbohydrates and proteins. The amount of ATP produced by a given weight of fat far exceeds that produced by the same weight of carbohydrates and proteins. Support for the notion that genetic abnormalities may also contribute to obesity came with the identi cation in 1994 of the obese gene (ob) and its protein product, leptin. Leptin is produced in adipose tissue and is proposed to act as an afferent satiety signal in a feedback loop that putatively affects the appetite and satiety centers in the brain (4). In ob/ob mice, which are markedly obese, the ob gene is mutated and no leptin is produced; when these mice were given leptin, their food intake was reduced. The operation of the genetic factor in human obesity, however, is far more complicated. A resistance to leptin action, but not any lack of production, is believed to be the problem, and multiple genes responsible for obesity are also proposed (4). However, the ob/ob mice provide a convenient experimental model for investigations of genetic obesity. Changes in lipid synthesis and metabolism have been reported among diet-induced obese and genetically obese rats (5). The studies reported here were conducted to examine the 109

2 110 LIBINAKI ET AL. changes of body weight and lipid metabolism in mice; ob/ob mice were used as a model of genetic obesity and lean mice fed on a Lard diet provided a model of diet-induced obesity. This diet, which was given for 34 days, consisted of normal mouse chow pellets crushed and combined with beef lard such that the latter contributed 30% of the diet s weight. MATERIALS AND METHODS Animals and Treatments. Male C57BL/6J mice, ages 13±14 weeks, were used in this study. A total of 18 mice were used, 12 lean and 6 obese (ob/ob), divided into 3 groups of 6 mice each: 1 group of ob/ob, and 2 groups of lean mice. The animals were housed 6 per cage and maintained on a normal 12-h light/dark cycle at aconstant room temperature of 22 ± C in the animal house of the Department of Biochemistry and Molecular Biology. All 3 groups of animals were fed ad libitum and were allowed free access to water at all times. The ob/ob mice and 1 group of the lean mice were fed normal mouse chow pellets (Glen Forrest Stockfeeders, Australia), the Normal diet; the other group of lean mice were fed the Lard diet. The composition of the diets used are shown in Table 1. The treatment period for all 3 groups of mice was 34 days. Cumulative Weight Gain. Cumulative weight gain was determined at 2-day intervals by measuring the body weight of each animal. The animals were placed in a large narrow chamber to minimise movement during the weighing procedure. Determination of Adipose Tissue Weight. White adipose tissues consisting of whole intact epididymal fat pads were excised with the identical techniques previously described (6) from the mice immediately after they were killed. The tissues were washed in cold physiological (isotonic) saline, gently blotted, and weighed. This weight was divided by the total body weight of the individual mouse and expressed as a percentage of adipose fat pad weight to total body weight for comparison, between the Table 1 Diet composition Diet contents, amount in 100 g Ingredients Normal Lard Carbohydrate, g Crude protein, g Fat, g Crude fat, g (6.0) (4.2) Beef lard, g (0.0) (30.0) Crude bre, g Calcium, g Phosphorus, g Sodium chloride, g Energy, kj The Lard diet wascomposed of 70% of the Normal diet plus 30% beef lard, which essentially is 100% fat, making the lard diet > 5.5 times higher in fat content than the Normal diet. groups to be made. For ex vivo lipolytic assays and adipocyte isolation, the portions of adipose tissues without blood vessels were used. Adipocyte Isolation and Size Determination. Adipose tissues from each of the 3 groups of animals were pooled and scissor-cut into small fragments. Each gram of the tissue was placed in a 10-ml ask containing 2 ml of a solution containing Krebs±Ringer bicarbonate (KRB) buffer (ph 7.4), 80 mg of bovine albumin serum (BSA), 1 and 1 mg of collagenase type II, (Sigma, St. Louis, MO); this was incubated at 37 ± C with a shaking rate of 90 cycles/min, for 1 h. After this period the suspension was passed through 8-ply gauze and washed 3 times with fresh KRB buffer, before thecells were xed in 10% formalin and placed on microscope slides. The isolated adipocytes on the slides were then examined with a Microcomputer Imaging Device (MCID; Imaging Research, Inc., Canada) technology, measuring the diameter of 100 randomly selected adipocytes from each of the 3 groups examined. Assay for Lipolytic Activity. The rate of glycerol release from the samples of adipose tissue of the 3 groups was measured as an index of lipolytic activity. Adipose tissues from the animals were sliced into segments of 150±200 mg each. Each tissue was placed in a 10-ml ask containing 2 ml of a solution containing KRB buffer (ph 7.4), 40 mg of BSA, and 0.2 mg of glucose, under an atmosphere of carbogen (O 2 :CO 2 95:5) at a shaking rate of 90 cycles/min; the incubations were maintained at 37 ± C for 1 h. Glycerol concentration in the medium was determined by enzymatic assay, performed by mixing 1 ml of glycerol phosphate oxidase (GPO)-Trinde r reagent (Sigma) and 10 l l of sample medium in a 2-ml cuvette. After 15 min, the absorbance at 540 nm was recorded. The same operations were performed for a glycerol standard and a distilled water blank, and the tissue glycerol content was determined. Oral Glucose Tolerance Test. At the end of the prolonged treatment period (60 days) blood samples (25 l l) were withdrawn from the cornea of 1 eye of 15 mice (5 from each of the 3 groups of mice tested), collected in heparin-coated tubes, and mixed. After the zero time at which the samples were taken, a glucose load of 1 mg/g of body weight was given to the mice orally via a gavage needle (7.5 cm 0.1 cm, length diameter) connected to a tuberculin disposable 1.0-ml syringe. Blood samples were then taken 30 and 120 min later. The whole-blood glucose concentration of each sample was measured immediately with YSI Model-2300 STAT Glucose Analyzer (Yellow Springs, OH), using an immobilised glucose oxidase methodology; the results were expressed as mmol/l. Assays for Serum Triglyceride and Total Cholesterol. Animals were killed by aortic puncture and blood samples were collected by using a 2-ml syringe with no needle, thecontents of which was then transferred into 500- l l Capiject TM tubes (Termo Medical Corp., Melbourne, Australia), left at room temperature 1 Abbreviations: BSA, bovine albumin serum; FFA, free fatty acids; GPO, glycerol phosphate oxidase; KRB, Krebs±Ringer bicarbonate buffer; MCID, microcomputer imaging device.

3 for 2 h, and thencentrifuged at 2000 g for 5 min. Serum was removed from the samples and used for metabolite assays. Triglyceride and total cholesterol in serum were measured by enzymatic spectrophotometric methods. The reagents are based on either a modi ed GPO-Trinder type colour reaction (7) or a cholesterol oxidase-4-aminoantipyrine method (8). Sigma assay kits containing calibrators were used and absorbances were recorded. Statistical Analysis. The Student s t test was used to analyse the results. All data are expressed as the mean SEM. P values of < 0.05 are accepted as statistically signi cant. GENETIC AND DIET-INDUCED OBESITY AND LIPID METABOLISM 111 RESULTS AND DISCUSSION Lean mice fed a normal mouse chow diet (closed circles in Fig. 1) were used as a control for the other two experimental groups, i.e., Obese and Lard, in terms of cumulative weight gain during long-term treatment (34 days). The Lean mice were seen to have a stable body weight over the period examined, whereas that of the Obese mice increased gradually and eventually became signi cantly differentiated ( P < 0.05). The Lard mice had a considerable weight gain over the entire 34-day treatment period, the difference from that of the other groups reaching signi cance (P < 0.05) within 4 days and becoming even more signi cant (P < 0.01) after day 28. Table 1 shows the Lard diet composition contributed a 40% increased caloric intake. The weight gain of the mice fed on the Lard diet translates to just over a 5-g increase, on average, in body weight, compared with the control Lean-diet mice (Table 2). The mice in all the groups were essentially the same age (13±14 weeks), and the weight of the Obese mice, on average, is >27 g heavier than that of the control Lean mice and at least 20 g heavier than the Lard mice. The body weight gain (compared withcontrol Lean mice) was proportional to the increase in adipose tissue depots. Epididymal fat pads were excised from the mice immediately after their death and weighed. Results were expressed as a percentage of total body weight of each of the animals in the 3 groups examined (Table 2). The Obese mice, as previously mentioned, weighed Figure 1. Effect of a high-fat diet on cumulative weight gains in male C57BL/6L lean mice fed a Lard diet, compared with C57BL/6J ob/ob and lean mice fed on Normal diets, during a 34- day period. Animals were fed ad libitum on their respective diets and were allowed free access to water at all times. Body weight changes were determined at 2-day intervals and each point represents the mean SEM of 6 animals. The differences between Normal and Lard diets in terms of cumulative weight gain were statistically analysed., P < 0.05;, P < 0.01,compared with the corresponding control (in this case the lean group of mice fed on a Normal diet). at least 20 g more than both the other tested groups and had the largest epididymal fat pad depot. Both the Obese mice and the Lard mice had larger fat depots than the Lean mice did, with increases of almost 3-fold and 2-fold, respectively. Because Table 2 Effect of a Lard diet on body weight, adipose tissue mass, and size (all data represent the mean SEM of 6 animals) Parameter Obese (Normal) Lean (Lard) Lean (Normal) l Initial body wt., g Final body wt., g Body wt. gain, g a Adipose tissue, % of total body wt. b Adipocyte diameter, m c , P < 0.05;, P < a Difference between initial and nal body weight measured. b The intact pair of epididymal fat pads represented the adipose tissue weight, which was divided by the total body weight to give the percent of total body weight that was adipose tissue. c Adipocyte cell diameter was determined as the average of 100 adipocytes measured in each of the three groups.

4 112 LIBINAKI ET AL. Figure 3. Ex vivo lipolytic activity of adipose tissues of C57BL/6J obese and lean mice fed on a Normal diet compared with C57BL/6L lean mice fed on a Lard diet. Tissues were incubated at 37 ± C for 1 h in KRB (ph 7.4) containing 2% BSA and 1 mm glucose. The lipolytic rate was estimated by the time course of glycerol release., P < Figure 2. Effect of a high-fat diet on adipocyte diameter in male C57BL/6L lean mice fed a Lard diet (C), compared with C57BL/6L lean mice (A) and C57BL/6L ob/ob mice (B), after a 34-day treatment period. the Lard and Lean groups of mice differed solely in the fatty nature of the diet used, this increase in epididymal fat depot can be directly related to the high-(saturated)fat intake in the diet. Adipocyte diameter was also measured in each group; the data are shown in Table 2. Analysis of the diameters of 100 cells was used to estimate the approximate size of the adipocytes in each group analysed (see Fig. 2). The adipocyte diameters of the Lard mice were signi cantly higher ( P < 0.05) than those observed in thecontrol Lean mice, suggesting that the increased size of the epididymal fat pad depots may be the result of an enlargement of the adipocytes. However, the Obese mean adipocyte diameter was slightly smaller, although not signi cantly so, than in the Lard mice, suggesting that perhaps the increase in adipocyte number as well as size was responsible for the very large epididymal fat pad depots seen in the ob/ob mice. Fig. 3 shows the lipolytic activity in the isolated adipose tissues, which was signi cantly (P < 0.05) less in Lard mice than in the control Lean mice. This observation supports the explanation in relation to the proposed effects of a high-fat diet. The animalscan use both glucose and free fatty acids (FFA) as energy sources; although some organs, such as the brain and erythrocytes, prefer the utilization of glucose, most other organs will use whichever fuel sources are most readily available. In a highfat diet, fat has one of two fates: It may be stored as an excess in the form of adipose tissue, or it may be converted to FFA and used as the major energy source in the body. The consequence of either of these occurrences is simple: Excess storage of fat will cause the size of the adipocytes to increase; in addition, the high FFA concentrations in the circulation inhibit the enzyme hormone-sensitive lipase (HSL) (9), which is required for lipolysis to occur, and lipolysis is inhibited. Furthermore, the utilization of FFA as an energy source rather than glucose means that the concentration of glucose in the circulation may rise, therefore stimulating beta cells of the pancreas to release insulin. Over time, if this situation is prolonged and the beta cells arecontinually stimulated to release insulin, the tissue sensitivity to insulin may decrease and glucose intolerance may result. All of the aforementioned consequences of a high-fat diet were observed in the Lard-fed mice in this study. Increased

5 GENETIC AND DIET-INDUCED OBESITY AND LIPID METABOLISM 113 Table 3 Effect of a Lard diet on plasma concentrations of triglyceride and total cholesterol (blood samples were collected from mice via an aortic puncture Obese Lean Lean Item (Normal) (Lard) (Normal) Triglycerides, mmol/l Cholesterol, mmol/l, P < 0.05;, P < Figure 4. Oral glucose tolerance test. Five mice from each of the treated groups (Lean, Lard, and Obese) were fasted for 6 h, then given an oral glucose load of 1 mg/g body weight, and eye bled 0, 30, and 120 min afterwards. Blood glucose was measured with a STAT YSI Glucose Analyser (Model 2300). The results are shown as the mean SEM., P < 0.05;, P < adipose tissue size was clearly demonstrated in the 3-fold increase in epididymal fat pad size (Table 2), and a decreased lipolytic rate was observed in the Lard-fed mice (Fig. 2). Lastly, although no difference in the basal blood glucose level was evident between groups of mice, a de nite hyperglycemia and impaired glucose tolerance were seen after the glucose load was administered (Fig. 4), with high blood glucose levels persisting even > 120 min after having received the glucose load. The blood glucose in Obese and Lean mice both returned to the basal levels within 2 h, and the obese (ob/ob) mice were considered to be hyperinsulinemic (10). The high-fat Lard diet and the genetic abnormality affected the concentrations of circulating cholesterol (Table 3). The total cholesterol level in the plasma was signi cantly higher in both Obese and Lard mice than in the control Lean mice ( , , and , respectively); that in the Lard mice was signi cantly higher than in both other groups (P < 0.05 vs. Obese, P < 0.01 vs. Lard). The concentrations of plasma triglyceride were only slightly elevated in Obese and Lard mice (results not signi cant). The addition of 30% beef lard in the mice diet caused marked increases in total body weight, adipose tissue mass, plasma cholesterol, and glucose intolerance as well as a decrease in lipolytic activity. Obese mice have been used in many experi- ments as an animal model for accessing causes and treatment of obesity. The results of this study delineated the differences between two distinct types of obesity in terms of lipid metabolism. Although the type of obesity that is most prominent in humans is still a debatable matter, epidemiological studies clearly show that fat in the diet has steadily increased over the past few decades and as a result, so too has the number of obese individuals (10). The Lard mice, or diet-induced model, of obesity may provide a framework close to that in the overweight human and may prove to be useful for studies of the pharmacological treatment of obesity. REFERENCES 1. West, D. B., and York, B. (1998) Dietary fat, genetic predisposition and obesity: lessons from animal models. Am. J. Clin. Nutr. 67, 505S± 512S. 2. Triscari, J., Nauss-Karol, C., Levin, B. E., Sullivan, A. C. (1985) Changes in lipid metabolism in diet-induced obesity. Metabolism 34, 6:580± Truett, A. A., Borne, A. T., Monteiro, M. P., West, D. B. (1998) Composition of dietary fat affects blood pressure and insulin responses to dietary obesity in the dog. Obes. Res. 6, 137± Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J. M. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425± Rothwell, N. J., and Stock, M. J. (1987) In uenc e of carbohydrat e and fat intake on diet-induced thermogenesi s and brown fat activity in rats fed low protein diets. J. Nutr. 117, 1721± Ng, F., Adama o, N. A., and Graystone, J. E. (1990) Effects of exogenous growth hormone on lipid metabolism in isolated epididymal fat pad of the growth-de cient little mouse. J. Mol. Endocrinol. 4, 43± Fossati, P., and Prencipe, L. (1982) Serum triglyceride determined colorimetically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077± Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W., and Fu, P. C. (1974) Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470± Faust, I. M., Johnson, P. R., and Stern, J. S. (1978) Diet-induced adipocyt e number increases in adult rats: a new model of obesity. Am. J. Physiol. 235, E279±E Herberg, L., and Coleman, D. L. (1977) Laboratory animals exhibiting obesity and diabetes syndromes. Metabolism 26, 59±99.