Influence of type-1 diabetes on viability of offspring in Long-Evans rats
Kenta Kajimoto, Department of Biology, University of Nebraska at Kearney, Kearney Nebraska 68849, USA.
Mentor: Dr. Janet Steele, Professor, Department of Biology, University of Nebraska at Kearney, Nebraska 68849, USA. Email: firstname.lastname@example.org)
To Cite This Paper:.
Kajimoto, K. 2007. Influence of type-1 diabetes on viability of offspring in Long-Evans rats. Research Thesis, Department of Biology, University of Nebraska at Kearney, Kearney, Nebraska 68849, USA.
OBJECTIVE—The purpose of this work was to examine the influence of type 1 diabetes on viability of offspring from diabetic mothers in rats.
RESEARCH DESIGN AND METHODS—Rats were made diabetic by streptozotocin injection. Four diabetic mothers and three control mothers were mated. Pups’ weight was measured daily for three weeks and glucose tolerance was evaluated four weeks after birth to determine whether there were some differences between two groups. The number of pups that died was counted for one month and mortality was measured for each group.
RESULTS—Growth rate for pups from control mothers was 1.5 times greater than pups from diabetic mothers. As predicted, baseline blood glucose level for pups from diabetic mothers was significantly higher than pups from control mothers. Interestingly, however, the blood glucose level for pups from control mothers was significantly higher than pups from diabetic mothers throughout the glucose tolerance test. The mortality rate for pups from diabetic mothers was significantly higher than for pups from control mothers.
CONCLUSION—Growth rate, glucose tolerance, and mortality of pups from diabetic mother were affected by maternal condition. Obviously, growth and mortality rate were affected negatively. The difference in glucose tolerance between two groups may be due to the influence of maternal hyperglycemic condition and difference stress responses for each group.
Recently, diabetes mellitus has become a great interest to many people. According to the American Diabetes Association, 20.8 million people in the United States have diabetes and 54 million have pre-diabetes. Diabetes is caused by beta-cell dysfunction, and these cells are located in the pancreas. As a result, insulin secretion is decreased or stopped because of the abnormal beta-cells (1). Insulin is a hormone which is secreted when blood glucose concentration is high. It regulates blood glucose levels and promotes energy storage (2). There are two types of diabetes, type-1 and type-2. Type-1 diabetes is due to insulin secretion deficiency from beta-cells and is often caused by environmental factors, such as viral infection and toxins. The environmental agents trigger processes leading to a beta-cell-destructive autoimmune response (3). Type-2 diabetes results from insufficient insulin secretion as insulin resistance is developed, and the causes of type-2 is diabetes genetic predisposition influenced by age and obesity (4). Diabetes increases the likelihood of cardiovascular disease and death because of chronically elevated blood glucose levels (5). While the exact cause(s) of type-1 diabetes are not yet fully understood, the number of type-1 diabetic children has increased over the world in the past decades.
Autoimmune attacks promote destruction of beta-cells, so beta-cell replication would not be successful in restoring beta-cell mass in type-1 diabetes because the cells are so quickly destroyed (3). The cause of type-1 diabetes is considered to have both genetic and environmental factors. Class 1 genes HLA-B and HLA-A of the major histocompatibility complex have been identified as major genetic factors of type 1 diabetes susceptibility in both humans and non-diabetic mice (6-7). The genes of the major histocompatibility complex encode proteins that play important roles in the immune response (8-9). One example of an environmental factor is a virus. Enterovirus infection induces type-1 diabetes to develop because the pancreatic beta-cells are directly destroyed by the virus (10). Another environmental factor is the maternal environment because hormones can be passed from mothers to their offspring. Therefore, the uterine environment is directly affected by the maternal environment (11).
The effects of the maternal diabetes are caused by increased transplacental lipid transport; therefore, the type-1 diabetic mother affects placental lipid metabolism. The placental triglyceride concentration is increased in placentas from diabetic mothers. That is, type-1 diabetic mothers tend to accumulate triglycerides in the placenta (12). Reduced placental lipid metabolism of diabetic mothers is one of the most significant reasons why offspring from diabetic mothers tend to have fetal macrosomia. Also, there is strong evidence that offspring from diabetic mothers have beta-cell dysfunction and diabetes or poor glucose tolerance caused by being exposed to hyperglycemic conditions in utero. Offspring showed increased birth weight when their mothers were exposed to hyperglycemia conditions, but there was no conclusive evidence of beta-cell dysfunction (13). Also, there are some differences in the size of Langerhans islets in offspring of experimental diabetic mothers compared with non-diabetic controls. On the first day after parturition, the size of the islets of offspring from diabetic mothers is smaller than those of offspring from control mothers. Five days after parturition, the size of Langerhans islets in offspring from control mothers decreases and those in offspring from diabetic mothers increase in size, and finally the size of Langerhans islets tends to be normal (14).
Many researches are trying to identify some relationships between type-1 diabetic mothers and their children. Unfortunately, due to the complicated mechanism of type-1 diabetes, these relationship and treatments are difficult to establish. Type-1 diabetes in the mothers does significantly affect for their offspring. In this study we investigated the influence of maternal condition on pups’ growth rate, glucose tolerance, and mortality. Our results demonstrate that the growth rate for pups from control mothers was greater than the rate for pups from diabetic mothers. In case of glucose tolerance test, the baseline blood glucose level for pups from diabetic mothers was higher than pups from diabetic mothers. However, the blood glucose level for pups from control mothers was significantly higher than pups from diabetic mothers throughout the glucose tolerance test. Also, the mortality rate for pups from diabetic mothers was significantly higher than for pups from control mothers. Therefore, our data suggest type-1 diabetic mother significantly affects her offspring in a negative way.
RESEARCH DESIGN AND METHODS
Animal studies. Studies with rats followed the approval of Institutional Animal Care and Use Committee, approval number 091907. If an animal became distressed it would be removed from the study. If a rat died, it would be removed immediately. Long-Evans Rats were used for this experiment. A total of 7 female Long-Evens rats and 3 male Long-Evans rats served as subjects. All rats were housed in the Bruner Hall of Science Animal Care Facility on the campus of the University of Nebraska at Kearney. Rats were housed under 12 hours light and 12 hours dark cycle (light 0700 to 1900) and received a normal diet with free access to drinking water. Before mating, four of the female rats were made diabetic by streptozotocin injection (65mg streptozotocin / kg body weight in 50mmole / L of pH 4.5 sodium citrate). Glucose level of rats was measured from tail clip using a glucose meter. Two days after the injection, diabetes was confirmed if the glucose level was higher than 200mg / dL on two consecutive measurements (15).
Growth rate. Beginning with the day of parturition, pups’ weights were measured daily for three weeks. Their weights were measured initially using an electronic balance and later using a triple beam balance.
Glucose tolerance test. Accucheck Active glucometer, manufactured by Roche, was used for this experiment. Four weeks after weaning, a glucose tolerance test was performed on all pups. Rats were fasted overnight for 16 hours. Then, blood samples to measure baseline blood glucose were collected from the tails followed by an interperitoneal injection of 2 mg of glucose / g body weight. The glucose level was measured every 30 minutes for 3 hours.
Mortality rate. The number of pups that died was counted for one month and mortality was measured for each group.
Statistical methods. Data were compared using the T-test and 0.05 was used for probability.
Determination for the differences of the growth rate between pups from diabetic mothers and pups from control mothers. The growth rate of pups from control mothers was significantly greater than pups from diabetic mothers starting at day 4 (FIG. 1.). Pups from control mothers had grown an average of 12g larger than pups from diabetic mothers after 3 weeks. Therefore, control pups grew 1.5 times larger than pups from diabetic mothers.
FIG. 1. Growth rate for both pups from diabetic mothers and control mothers. The growth rate was measured by weighing pups body mass every day for three weeks. * indicates significant differences with probability 0.05.
Difference for glucose tolerance test between the pups from diabetic mothers and pups from control mothers. “Normal” blood glucose level is about 100mg /dL. As expected, baseline blood glucose level for pups from diabetic mothers was significantly higher than pups from control mothers (FIG. 2.). Interestingly, however, the blood glucose level for pups from control mothers was significantly higher by about 20 mg/dL than for pups from diabetic mothers throughout the glucose tolerance test.
FIG. 2. Differences of glucose tolerance between pups from diabetic mothers and control mothers. Pups were tested four weeks after their birth. * indicates significant differences with probability 0.05.
Mortality rate for both groups. Mortality rate was 9% for pups from control mothers and 57% from diabetic mothers after one month. Diabetic mothers were observed to have much more cannibalistic behavior than control mothers. Eight of twenty-three pups from diabetic mothers were eaten by their mothers. Cannibalism was the greatest single cause of death in pups from diabetic mothers, and it was 62 % of all deaths. In particular, the smaller pups had a much greater chance of death in first month. In addition, diabetic mothers were not observed nursing as much as control mothers. These data indicate that the mortality rate of pups from diabetic mothers is relatively higher than pups from control mothers.
Impaired fetal growth may occur in as many as 20% of offspring from diabetic mothers and 10% of offspring from non-diabetic mothers. Maternal renovascular disease is the most common cause of impaired fetal growth by maternal diabetes (16). While I did not examine kidney function, my observation of impaired growth rate in pups from a diabetic mother could be caused by maternal diabetic nephropathy. Also, the difference in growth rate may be affected by both milk content and milk production. Increased glucose and insulin content of milk from diabetic mothers may affect growth rate, although some researchers have not found differences in major nutritional contents of milk. However, there is no difference in milk consumption between infants from type-1 diabetic mothers and non-diabetic mothers in humans (17).
A human study determined that the blood glucose level of offspring from diabetic mothers is higher than that of offspring from control mothers two hours after glucose loading (18). However, my research showed an opposite result. I may still be able to conclude that offspring from diabetic mother are somewhat affected by the hyperglycemic condition of type-1 diabetic mother because their baseline blood glucose levels were significantly higher. Blood glucose is also affected the hormones glucagon and cortisol, both of which act to raise blood glucose. Cortisol is secreted under stressful conditions, and repeated sampling of blood from the tail is certainly stressful. It could be that the two groups of pups have different stress responses and the blood glucose differences are related to differences in cortisol secretion.
Before the discovery of insulin in 1921, fetal mortality rates for offspring from diabetic mothers were higher than 65%. Today mortality rates of offspring from diabetic mother are still relatively high. In mothers with type-1diabetes, peritoneal mortality rate is five times higher and infant mortality is three times higher than normal (19). Specialized treatments are needed for diabetic women who are pregnant. In rats, cannibalism is a normal behavior in nature. In my research, cannibalism was very common in diabetic mother and pups have 8 times higher chance to be cannibalized by their mother. Also, diabetic mother limited the number of pups who could grow up. Dead pups were mostly the smallest in the all pups. The growth rate may be related to the mortality rate due to the impaired fetal growth. Therefore, some abnormal or different behaviors in the mother may be triggered by hyperglycemia and other kinds of related disease.
This work has received support from Dr. Janet Steele and the Biology Department of the University of Nebraska at Kearney.
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