Background
Type 2 diabetes (T2D) in lean individuals is not well studied and up to 26% of diabetes occurs in these individuals. Although the cause is not well understood, it has been primarily attributed to nutritional issues during early development.
Objective
Our objective was to develop a lean T2D model using gestational low-protein (LP) programming.
Study Design
Pregnant rats were fed control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery. Standard diet was given to dams after delivery and to pups after weaning. Glucose tolerance test was done at 2, 4, and 6 months of age. Magnetic resonance imaging of body fat for females was done at 4 months. Rats were sacrificed at 4 and 8 months of age and their perigonadal, perirenal, inguinal, and brown fat were weighed and expressed relative to their body weight. Euglycemic-hyperinsulinemic clamp was done around 6 months of age.
Results
Male and female offspring exposed to a LP diet during gestation developed glucose intolerance and insulin resistance (IR). Further, glucose intolerance progressed with increasing age and occurred earlier and was more severe in females when compared to males. Euglycemic-hyperinsulinemic clamp showed whole body IR in both sexes, with females demonstrating increased IR compared to males. LP females showed a 4.5-fold increase in IR while males showed a 2.5-fold increase when compared to their respective controls. Data from magnetic resonance imaging on female offspring showed no difference in the subcutaneous, inguinal, and visceral fat content. We were able to validate this observation by sacrificing the rats at 4 and 8 months and measuring total body fat content. This showed no differences in body fat content between control and LP offspring in either males or females. Additionally, diabetic rats had a similar body mass index to that of the controls.
Conclusion
LP gestational programming produces a progressively worsening T2D model in rats with a lean phenotype without obesity.
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Introduction
Diabetes has reached epidemic proportions with 1 in 9 affected in the United States, with projected estimates as high as 1 in 3 by 2050 (Centers for Disease Control and Prevention). Type 2 diabetes (T2D) has been historically attributed to lifestyle and genetics, however recent studies indicate that an adverse uterine environment is associated with the development of T2D later in life. Although various aspects of T2D are well studied, the pathogenesis, progression, and mechanisms of the developmental origins of T2D are poorly understood.
A recent study on minority US populations showed that 13% of diabetic patients are of normal weight with a 5-fold higher incidence in people of Asian origin. Studies from India and the Caribbean islands also report the presence of a lean diabetic population, with 1.6-26% and 5% prevalence, respectively. This has been primarily attributed to possible in utero nutritional issues. As obesity is not required for the development of T2D, in utero nutritional issues could be a causative factor for developing insulin resistance (IR) and glucose intolerance that predispose to T2D. T2D in these lean individuals is not well understood.
Various genetic, diet, and chemically induced diabetic rodent models are utilized to study T2D. However, these models are accompanied by obesity and do not accurately mimic the lean T2D phenotype. Therefore, there is a need for a lean diabetic animal model to investigate various aspects of lean phenotype and to study the possible mechanisms of origin of lean T2D. Previous work has demonstrated that a gestational low-protein (LP) diet increases the susceptibility of offspring to the development of metabolic diseases during adulthood. Our objective was to characterize a gestational protein-restricted rat model that results in IR and glucose intolerance in offspring during adulthood, but that is not accompanied by obesity.
Materials and Methods
Animals
Timed pregnant (day 4) Wistar rats (∼200 g) were procured from Harlan Laboratories (Indianapolis, IN) and were housed in a temperature-controlled room (∼23°C) with a 14:10-hour light/dark cycle with unlimited access to food and water. Pregnant rats were fed control (20% protein Teklad diet; Harlan Laboratories) or isocaloric LP (6%) diet from gestational day 4 until delivery. Normal diet was given to mothers after delivery and to pups after weaning until the end of the study. The number of pups in the control and LP litters were culled to 8 pups per mother (pups with weights at extremes were euthanized) to ensure equal nutrient access for all offspring. Body weights and length of pups were recorded on a regular basis to calculate body mass index (BMI). Glucose tolerance test (GTT) and euglycemic-hyperinsulinemic clamp were performed in females at diestrus phase to minimize the influence of estrogens. Rats were sacrificed at 4 and 8 months to collect gonadal, perirenal, and inguinal fat pads and their weights were recorded. All experimental procedures were performed with approval by the Institutional Animal Care and Use Committee of Baylor College of Medicine.
Glucose tolerance test
GTT was performed on male and female offspring at 2, 4, and 6 months of age to identify the progression of glucose intolerance. Rats were fasted for 6 hours and were administered glucose (1 g/kg body weight intraperitoneally). Blood glucose levels were measured using ACCU-CHEK Nano (Roche, Indianapolis, IN) at 0, 15, 30, 60, 120, and 180 minutes via saphenous puncture. Blood samples were collected in heparin-coated tubes for measuring fasting plasma insulin levels.
Insulin levels
Plasma insulin levels were measured using a rat insulin enzyme-linked immunosorbent assay kit (Mercodia), following the manufacturer’s instruction as reported earlier.
Homeostatic model assessment
Homeostatic model assessment (HOMA)-IR and HOMA-insulin sensitivity (IS) were calculated to assess IR and IS of control and LP rats using the following equations :
HOMA – IR = [ fasting glucose ( mg/dL ) × fasting insulin ( mU/L ) ] ÷ 405
HOMA – IS = 10,000 ÷ [ fasting glucose ( mg/dL ) × fasting insulin ( mU/L ) ]
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