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ILAR Journal V34(4) 1991
The Cdb:BHE Rat--A Model for Non-Insulin-Dependent, Nonobese Diabetes Mellitus
Carolyn D. Berdanier, Ph.D.
| Dr. Berdanier is a professor of nutrition at the University of Georgia, Department of Foods and Nutrition, Athens, Georgia |
The Cdb:BHE stock is a subline of the parent BHE stock now housed in the Genetic Resource Unit, National Center for Research Resources, National Institutes of Health (NIH)a. The parent stock was developed some 50 years ago by scientists at what was then called the Bureau of Home Economics (Adams, 1964). The stock was named BHE in honor of the bureau, which was an early predecessor of the present day Beltsville Human Nutrition Research Center of the U. S. Department of Agriculture (USDA). The parental stock is the result of a cross between hooded Osborne-Mendel rats, called Yale at that time, and rats of the now extinct albino Pennsylvania State College stock. Progeny of this cross were albino, all black, all brown, black and white, brown and white, or agouti. Once the cross was made, the colony was maintained as a closed colony. Full- and half-sibling matings were avoided, as were backcrosses.
In the mid-1960s, M. W. Marshall et al. (Marshall and Lehmann, 1967; Marshall et al., 1969a, b, 1971, 1976) developed a number of sublines. Backcrosses and full-sibling matings were used to produce lines of BHE rats that were lean, obese, and/or had renal disease due to a variety of lesions. One of these lines was hyperglycemic and developed a renal pathology similar to that observed in humans with diabetes mellitus. None of these sublines now exist; however, they are of interest because they document the presence of genes for each of these characteristics in the parental BHE stock.
In 1975, the development of the Cdb:BHE subline began. Breeding stock was selected for the presence of hyperlipemia and hyperglycemia and the absence of obesity and hydronephrosis. Full-sibling matings and back-crosses were used to strengthen the appearance of the hyperlipemic/hyperglycemic trait. Following 36 generations of selection, 75 percent of the rats showed hyperlipemia and hyperglycemia at 300 days of age, but not at 100 days of age. For the last 10 generations, full-sibling matings have been avoided, and the colony is maintained by random breeding. A full genetic history back to 1975 is kept on each breeding rat of the Cdb:BHE stock. Except for the addition of 12 breeders donated by the Virginia Polytechnic Institute, Blacksburg, Virginia, in 1979, the colony has been maintained as a closed colony. Since 1975, glucose tolerance has been determined in all rats at 300 days of age. Table summarizes the breeding patterns followed to produce the present Cdb:BHE colony.
GLUCOSE TOLERANCE
Glucose tolerance in 16-hour fasted rats is determined after a glucose challenge. A tail blood sample is drawn, glucose is administered by gavage (lg/kg body weight), and subsequent samples are drawn at 30, 60, and 120 minutes. Typical glucose tolerance data (or intolerance) are shown in Table 2. Tolerance can be influenced by both age and diet. Gestational diabetes (glucose tolerance normal before and after gestation but not during the last third of gestation) can be shown in 200-day-old female rats if they are fed a 22 percent fat diet, which approximates the composition of the average diet consumed by people in the United States (Berdanier et al., 1979; Bue et al., 1989).
Although breeding stock was selected for the lipemic/ glycemic trait, avoiding massive obesity and hydronephrosis, Cdb:BHE rats, like rats of the parent strain, have slightly more carcass fat (12-14 percent) than SD (Sprague-Dawley) or WI (Wistar) rats (8-10 percent) when all are fed the same diet and examined at the same age (Adams, 1964; Allen Durand et al., 1964, 1968; Marshall et al., 1967, 1969a, b, 1971).
RENAL LESIONS
Cdb:BHE rats are prone to glomerulonephropathy. In a recently completed longevity study using male rats of the thirty-sixth generation, renal tissue was harvested both from rats that died spontaneously and from rats that were killed at 300,500,600, and 700 days of age (Berdanier et al., 1992). Varying degrees of glomerulonephrosis or "old rat nephropathy" were observed in these tissues. The microscopic features included hyaline casts, glomerulosclerosis, tubular degeneration, and fibrosis. These lesions were similar to those reported previously for the parent stock (Adams, 1964; Allen Durand et al., 1964, 1968) and to those reported in young (100-day-old) female rats (Noll-Herndon et al., 1986). Some dystrophic calcification of other tissues (e.g., aorta, heart) secondary to renal failure and uremia were also observed, as were renal stones. As the rats in this study aged, the kidneys enlarged to weigh between 5 and 8 grams despite the fact that there was very little change in body weight. Renal enlargement was not caused by age differences in food intake because food intake was controlled such that all rats were fed the same number of calories per 100 grams of body weight per day throughout the study. The increase in kidney weight correlated (r=0.98) with the increase in severity of renal lesions. It also correlated with an increase in mean arterial pressure, which was assessed by an indwelling catheter placed in either the femoral or carotid artery. Mean arterial blood pressure ranged from 98 (normal) to 147 (abnormal).
BLOOD LIPIDS
Fasting serum triglycerides and cholesterol are influenced by age, diet composition, and the duration of feeding purified diets. Early in the development of the subline, rats fed the stock diet were more lipemic and more glucose tolerant than are rats presently in this line. Apparently, selecting for the glucose intolerance feature also affected the lipemic characteristic. Whereas the fasting triglyceride level in 300-day-old, fifth generation rats was 755 + 58 mg/dl, the fasting triglyceride level in 300-day-old, thirty-eighth generation rats was 315 + 60 mg/dl. This level is, however, much higher than that observed in normal SD and WI rats at this age (50-80 mg/dl). The values for fasting serum cholesterol in Cdb:BHE rats range from 94 to 115 mg/dl at 300 days of age. These values are only slightly higher than what one might anticipate for normal rats of this age. Fasting serum cholesterol values seem to be unaffected by diet composition, whereas, fasting serum triglycerides are elevated sometimes by as much as 50 percent when purified diets containing 5 percent saturated fat and/or 65 percent sucrose are fed (Berdanier et al., 1979; Kim et al., 1989).
EARLY PHENOTYPE DETECTION
One of the challenges in working with this rat stock has been to detect the genetic trait before it is phenotypically expressed as abnormal glucose tolerance. To that end we have conducted numerous studies to determine the existence of subtle metabolic differences between these and normal rats. Because the most consistent feature of this rat is its early (50 days of age) development of hepatic hyperlipogenesis we have focused on the liver with the hope of learning why it is metabolically abnormal. Other tissues, such as adipose, pancreatic, adrenal, and muscle tissues, appear to be normal at this age.
Lakshmanan et al. (1977) reported that in stock-diet-fed (see Table 2, footnote a for composition of stock diet) 50-day-old rats, de novo fatty acid synthesis (using tritiated water) was 200 percent greater in BHE hepatic tissue than in WI hepatic tissue. This increase in de novo synthesis was accompanied by a 40 percent increase in the activities of the rate-limiting enzymes for fatty acid synthesis. This observation was expected; however, they also reported an increase in fatty acid oxidation. The latter observation is consistent with our finding of a tenfold elevation in serum free glycerol, a product of peripheral tissue lipolysis, and an increase in hepatic glycerol use for glucose synthesis and fatty acid reesterification (Berdanier et al., 1978, 1982). Detailed studies of frozen-clamped hepatic tissue from stock-diet-fed rats, (a process in which large clamps, immersed in liquid nitrogen, smash the excised liver to the thinness of a potato chip) revealed that liver cells had elevated phosphorylation and redox states, findings consistent with elevated rates of hepatic lipogenesis (Berdanier et al., 1979). Berdanier et al. (1979) also reported slower mitochondrial respiratory rates, and subsequently (Ber-danier and McNamara, 1980) reported that as these rats aged, there were further decreases in mitochondrial respiration rates. If the rats were fed a diet consisting of 65 percent sucrose; 20 percent 1:1 casein:lactalbumin; 5 percent corn oil; and adequate amounts of vitamins, minerals, and fiber (65 percent sucrose diet), there was not only a decrease in respiration, but also evidence of looser coupling of respiration to ATP synthesis (McCusker et al., 1983). Furthermore, detailed studies of lipogenesis, mitochondrial respiration, and ATP synthesis in starch(65 percent starch substituted for sucrose in 65 percent sucrose diet) or sucrose-fed progeny of starch- or su-crose-fed dams revealed a negative correlation between fatty acid synthesis and mitochondrial respiratory activity (Bouillon and Berdanier, 1983). As one would expect, there was no significant correlation between de novo fatty acid synthesis and ATP synthesis. Fatty acid synthesis requires very little ATP compared to that required by protein synthesis. In circumstances where there might be a reduction in ATP synthesis (such as in sucrose feeding), one might expect to observe a reduction in protein synthesis, and because the body must dispose of all the excess amino acids not used in protein synthesis, one might anticipate observing an increase in fatty acid synthesis when there is a decrease in protein synthesis. Indeed, one of the hallmarks of protein malnutrition, whether caused by problems in protein intake or protein synthesis, is a fatty liver.
Feeding BHE rats a 65 percent sucrose diet results in the development of a fatty liver (Berdanier et al., 1979; Bouillon and Berdanier, 1983; McCusker et al., 1983) and also in more saturated membrane phospholipid fatty acids (Wander and Berdanier, 1985). This finding may explain the decrease in mitochondrial coupling efficiency reported by McCusker et al. (1983). To test this hypothesis, BHE rats were fed a 65 percent sucrose diet in which the 5 percent corn oil was replaced with 5 percent hydrogenated coconut oil (Deaver et al., 1986). This dietary treatment resulted in even more saturated fatty acids in the mitochondrial membrane phospholip-ids and uncoupled respiration. Subsequent studies using a variety of substrates to support respiration revealed that this dietary effect was unique to the BHE rat and did not affect the oxidation of fatty acids (Kim, 1988). This in turn suggested that the genetic error in these rats has to do, in part, with the coupling of respiration to ATP synthesis. Since the inner mitochondrial membrane must be intact in order for coupling to take place, and since the composition of the lipid portion can be influenced by diet in many rat strains, one is led to conclude that the error is in the proteins whose function is determined by the lipid milieu in which it must operate. Preliminary studies by Jordan and Berdanier (1991) indicate a genetic difference in two mitochondrial proteins. One is a matrix protein, and one is an inner mitochon-drial protein. Work is underway to describe these proteins and their function in mitochondrial metabolism more completely.
Because mitochondrial function can determine the activities of a number of cytosolic pathways, we have sought to characterize these pathways in young rats as well. As mentioned previously, gluconeogenesis by isolated hepa-tocytes from rats starved for 40 hours is 20 percent greater in BHE rats than in WI or SD rats fed a stock diet ad libitum (Berdanier, 1982). Gluconeogenesis can be increased by feeding a 65 percent sucrose diet (Park et al., 1983, 1986) and increased even further by substituting hydrogenated coconut oil for corn oil in the sucrose diet (Wander and Berdanier, 1986). Note here the parallel between the mitochondrial respiration/ATP synthesis response to the dietary manipulations responses. Lipogen-esis increases as does gluconeogenesis while losses in control of mitochondrial respiration occur when sucrose is substituted for starch and coconut oil is substituted for corn oil. Diabetics in the human population have been observed to have gluconeogenic rates in excess of normal, and it is thought that this process contributes significantly to their elevated blood glucose levels. Our rats have these elevated gluconeogenic rates before they become glycemic, so it is likely that humans genetically programmed for diabetes do also. The young rat probably disposes of the excess glucose it produces by increasing the rate of glucose turnover. Indeed, measurements of glucose turnover by BHE rats fed 5 percent corn oil or 5 percent coconut oil indicate that this is so (Klm et al., 1989). Increased peripheral tissue glucose uptake, oxidation, and conversion to lipid is probably part of the compensatory process. Detailed studies of adipocyte and hepatocyte insulin receptor numbers and affinity revealed that these cells are not different in BHE rats from those in normal rats (Pan and Berdanier, 1991b). Diet can affect glucose uptake and metabolism, and as the storage cells enlarge, their insulin binding affinity decreases. This contributes to the gradual age-related impaired glucose tolerance. Nonetheless, one must argue that this feature of non-insulin-dependent diabetes mellitus in the Cdb:BHE rat is probably secondary to the aforementioned derangement in hepatic metabolism. There appears to be a genetically determined cascade of metabolic events that occurs and that explains why both diet and age are essential (and interacting) components of the development of the impaired glucose tolerance observed in non-insulin-dependent diabetes mellitus. We hypothesize that there exists an error in one or more matrices or inner mitochondrial proteins that accounts for a small decrease in respiratory efficiency. This error is probably quite minor and the cells in the young rats probably compensate for this small defect. However, the defect causes a limitation in ATP production, which in turn affects cellular synthetic processes such as protein synthesis. Protein synthesis, mole for mole, requires far more ATP to sustain optimal synthetic rate than does fatty acid synthesis. Hence, the animal will likely reduce its synthesis of protein. Those amino acids not used for protein synthesis will be deaminated and the carbon skeletons used to make fatty acids or glucose. Fatty acids synthesized de novo from either amino acids or glucose are usually saturated fatty acids. Since the fatty acids in the membranes are in constant exchange with those fatty acids around them, one might expect to see a more saturated fatty acid profile in the mitochondrial membrane when de novo fatty acid synthesis is high. Indeed, we have reported that this occurs in BHE rats fed a 65 percent sucrose diet (Wander and Berdanier, 1985). If the membranes are more saturated, the mitochondria will be affected (Deaver et al., 1986) and less ATP will be synthesized, with the result that even less protein and more fatty acids will be synthesized. As respiration using pyruvate and succinate becomes more inefficient, fatty acids will become the fuel of choice, causing an increase in ketones in the blood and, possibly, the urine. This finding has been reported (Berdanier et al., 1979). The carbon skeletons from amino acids will also be used for glucose synthesis and raise the blood glucose level. Increases in amino acid degradation results in an increased demand for urea cycle activity and a rise in blood ammonia levels. The rising ketone levels must be counterbalanced, and ammonium ion may serve this purpose. Both place a burden on the kidney. Increased glucose production, as well as an increased need to store de novo synthesized fatty acids, causes enlarged fat cells that become insulin resistant (Pan and Berdanier, 1991a). Lastly, as the renal burden increases, glomerulonephropathy develops and, secondarily, the micro- and macrovascular changes typical of non-insulin-dependent, nonobese diabetes mellitus. Much of this scenario has been documented; however, the aberration in mitochondrial protein has not been documented completely. Furthermore, we do not know whether other tissues also contain the error. In view of the fact that the manifestation of the trait for non-insulin-dependent, nonobese diabetes mellitus takes half a lifetime to appear, one suspects that only one cell type, the liver cell, is affected. Much remains to be learned before such a hypothesis can be accepted.
REFERENCESb
Adams, M. 1964. Diet as a factor in length of life and in structure and composition of tissues of the rat with aging. Home Economics Research Report No. 24. (Available from the U.S. Government Printing Office, Washington, DC 20402. Tel: 202/783-3238.)
Allen Durand, A. M., M. Fisher, and M. Adams. 1964. Histology in rats as influenced by age and diet. Arch. Pathol. 77:268-277.
Allen Durand, A. M., M. Fisher, and M. Adams. 1968. The influence of type of dietary carbohydrate: Effect on histological findings in two strains of rats. Arch. Pathol. 85:318-324.
Berdanier, C. D. 1982. Rat strain differences in gluconeogenesis by isolated hepatocytes. Proc. Soc. Exp. Biol. Med. 169:74-79.
Berdanier, C. D., R. B. Tobin, V. DeVore, and R. Wurdeman. 1978. Studies on the metabolism of glycerol by hyperlipemic and normal-ipemic rats. Proc. Soc. Exp. Biol. Med. 157:5-11.
Berdanier, C. D., and S. McNamara. 1980. Aging and mitochondrial activity in BHE and Wistar rats. Exp. Gerontol. 15:519-525.
Berdanier, C. D., R. B. Tobin, and V. DeVote. 1979. Studies on the control of lipogenesis: Strain differences in hepatic metabolism. J. Nutr. 109:247-260.
Berdanier, C. D., B. J. Johnson, D. K. Hattie, and W. A. CroweIl. 1992. Longevity in BHE/cdb rats is affected by dietary fat. J. Nutr. (tn press).
Bouillon, D. J., and C. D. Berdanier. 1983. Effect of maternal carbohydrate intake on mitochondrial activity and on lipogenesis by the young and mature progeny. J. Nutr. 113:2205-2216.
Bue, J. M., D. B. Hausman, and C. D. Berdanier. 1989. Gestational diabetes in the BHE rat: Influence of dietary fat. Am. J. Obstet. Gynecol. 161:234-24.
Deaver, O. E., C. Wander, R. H. McCusker, and C. D. Berdanier. 1986. Diet effects on membrane phospholipid fatty acids and mitochon-drial function in BHE rats. J. Nutr. 116:1148-1155.
Jordan, B. L., and C. D. Berdanier. t991. NIDDM in the BHE/cdb rat is due to a genetic aberration in hepatic mitochondrial matrix proteins. Pp. 229-237 in Frontiers in Diabetes Research. Lessons from Animal Diabetes Ill, E. Shafrir, ed. London: Smith Gordon.
Kim, M-J. C. 1988. The role of hepatic mitochondria in the regulation of glucose metabolism in BHE rats. Ph.D. dissertation, directed by C. D. Berdanier. Athens: University of Georgia. (Dissertation copy available from University Microfilms, Ann Arbor, Michigan.)
Kim, M-J. C., J-S. Pan, and C. D. Berdanier. 1989. Glucose turnover in BHE rats fed EFA deficient hydrogenated coconut oil. Diabetes Res. 10:1-5.
Lakshmanan, M. R., C. D. Berdanier, and R. L. Veech. 1977. Comparative studies on lipogenesis and cholesterogenesis in lipemic BHE rats and normal Wistar rats. Arch. Biochem. Biophys. 183:355-360.
Marshall, M. W., and R. P. Lehmann. 1967. Influence of heredity on response of inbred rats to diet: 1. Differences in body size, food intakes, incidence of spontaneous kidney defects, kidney weights, urine pH and protein. Metabolism 16:763-774.
Marshall, M. W., B. P. Smith, A. W. Munson, and R. P. Lehmann. 1969a. Prediction of carcass fat from body measurements made on live rats differing in age, sex and strain. Br. J. Nutr. 23:353-369.
Marshall, M. W., M. Womack, H. E. Hildebrand, and A. W. Munson. 1969b. Effects of types and levels of carbohydrates and proteins on carcass composition of adult rats. Proc. Soc. Exp. Biol. Med. 132:227-232.
Marshall, M. W., A. M. Allen Durand, and M. Adams. 1971. Different characteristics of rat strains: Lipid metabolism and response to diet. Pp. 381-413 in Defining the Laboratory Animal. Proceedings of the Fourth Symposium, International Committee on Laboratory Animals (ICLA), organized by ICLA and the Institute of Laboratory Animal Resources, National Research Council. Washington D.C.: National Academy of Sciences.
Marshall, M. W., M. Haubrich, V. A. Washington, M. W. Chang, C. W. Young, and M. A. Wheeler. 1976. Biotin status and lipid metabolism in adult obese hypercholesterolemic inbred rats. Nutr. Metab. 20:41-61.
McCuskcr, R. H., O. E. Deaver, and C. D. Berdanier. 1983. Effect of sucrose or starch feeding on the hepatic mitochondrial activity of BHEandWistarrats. J. Nutr. 113:1327-1334.
Noll-Herndon, J. A., C. D. Berdanier, W. A. CroweIl. 1986. Influence of dietary casein and sucrose levels on urea cycle enzyme activities and renal histology in young BHE rats. Nutr. Rep. Int. 34:403-411.
Pan, J-S, and C. D. Berdanier. 1991a. Dietary fat saturation affects glucose metabolism without affecting insulin receptor number and affinity in adipocytes from BHE ration. J. Nutr. 121:1811-1819.
Pan, J-S, and C. D. Berdanier. 1991b. Effect of dietary fat on adypocyte insulin binding and glucose metabolism in BHE rats. J. Nutr. 121:1811-1819.
Park, J. H. Y., C. D. Berdanier, and B. Szepei. 1983. Effects of dietary sucrose on the gluconeogenic capacity of isolated hepatocytes from BHE rats. Nutr. Rep. Int. 28:287-293.
Park, J. H. Y., C. D. Berdanier, O. E. Deaver, and B. Szepesi. 1986. Effects of dietary carbohydrate on hepatic gluconeogenesis in BHE rats. J. Nutr. 115:190-199.
Wander, R. C., and C. D. Berdanier. 1985. Effects of dietary carbohydrate on mitochondrial composition and function in two strains of rats. J. Nutr. 115:190-199.
Wander, R. C., and C. D. Berdanier. 1986. Effects of type of dietary fat and carbohydrate on gluconeogenesis in isolated hepatocytes from BHErats. J. Nutr. 116:1156-1164.
NOTES
a Researchers desiring these animals should contact Dr. Carl Hansen of the NIH, Bethesda, Maryland.
bThis reference list is representative of the more than 100 citations on BHE rats in the literature. A complete list can be obtained from Dr. Carloyn D. Berdanier, Department of Foods and Nutrition, University of Georgia, Athens, GA 30602.
TABLE 1 Protocols Followed for th Development of the Cdb:BHE Rat Colony
| 1975 | Rats were obtained from Flow Laboratories, which obtained its colony from USDA |
| 1975-77 | Rats were mated on the basis of the presence of hyperinsulinemia at 50 days of age. Six generations were produced. |
| 1977-79 | Generation 7-10: full-sibling matings. |
| 1979 | Generation 11: random mating. |
| 179 | Generation 12: introduction of breeding stock from Virginia Polytechnic Institute (VPI), which obtained its breeding stock from Flow Laboratories. |
| 1980-84 | Generation 13-25: random mating. |
| 1985-89 | Generation 26-38: random matings using males of the previous generation but avoiding father-daughter matings. |
| 1990 | Generation 39: random matings within generation. |
TABLE 2 Influence of age and diet on glucose tolerance in BHE/cdb rats.
| Blood glucose before and after glucose challenge (mg/dl) | ||||||
| Dieta | Age | N | Fasting | 30 min. | 60 min. | 120 min. |
| Stock | 300b | 12 | 113±4c | 149±4 | 156±4 | 154±3 |
| 6% Corn Oil | 300d | 6 | 93±2 | 176±12 | 168±6 | 161±19 |
| 600d | 6 | 110±17 | 245±32 | 235±9 | 193±40 | |
| 5% Beef Fat and | 300d | 6 | 110±10 | 174±10 | 159±10 | 124±10 |
| 0.1% Corn Oil | 600d | 6 | 92±6 | 233±15 | 278±24 | 223±24 |
| 5% Fish Oil and | 300d | 6 | 98±8 | 166±19 | 151±19 | 118±9 |
| 1% Corn Oil | 600d | 6 | 101±8 | 206±22 | 241±27 | 199±9 |
a Stock diet, Purina Laboratory Animal Chow. The proximate composition of this diet (5-6% fat, 64-65% carbohydrate, 20% protein) was used as the basis for the refined diets in which the carbohydrate was sucrose and the protein was a 1:1 mix of casein and lactalbumin and the fat was corn oil or the mixtures of fats shown.
b Generation 38
c Mean SEM
d Generation 36
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