Development of insulin resistant obese rat model
to evaluate the efficacy of Single Chain Insulin (SCI) analogs
Insulin is the primary regulator of glucose homeostasis that is secreted in response to elevated blood glucose levels stimulating glucose uptake by peripheral tissues including the liver, muscle and adipose tissue from the blood stream. Although current clinically used insulin analogs provide relatively adequate glycemic control to diabetic patients; the ultimate goal is the prevention of overt diabetes and its associated complications. Previous studies have shown that modifications to the structure of insulin can alter its function to produce a more favorable function such as longer action or increased potency. Active single chain insulin (SCI) analogs were developed in which the C-domain is replaced by a short, at least four amino acid residues connecting A and B chain together to confer ultra-stability. Novel single chain analogs has been shown to demonstrate a rapid onset and prolonged action compared to control insulin analogs. Previously studies of SCI’s on streptozotocin-induced type 1 diabetic rats in our lab showed that these SCI’s lowers blood glucose levels as rapidly as insulin Humalog; the first commercially available rapid-acting insulin analog. However with SCI’s the blood glucose levels remain lower for a much longer period than Humalog, mimicking the prolonged action of basal insulins like Glargine. This study proposes to analyze the mechanism for fast onset and the prolonged action of the novel single-chain insulin on an obese insulin resistance rat model. An obesity-induced model of insulin resistant rats was developed and injected with the novel SCI and a control analog to examine the protective effects of this analog on the progression of T2DM. The SCI demonstrated the same effect on the blood glucose as efficiently as Humalog but loses its prolonged action as Glargin. This finding indicates that this SCI could serve as an improved therapeutic option for diabetic patients.
Insulin, a hormone secreted by pancreatic beta cells serves as a primary regulator of glucose homeostasis; in response to increased interstitial glucose levels. Insulin contains two polypeptide chains: chain A has 21 residues and chain B has 30 residues with 3 disulfide linkages; chain A and B are connected through two inter chain disulfide linkages between A7 and B7 and A20 and B19, a third intra-chain disulfide linkages is between residues A6 and A11. The major role of insulin is its initiation of the uptake of the glucose by peripheral tissues including the liver, muscle and adipose tissue from the blood stream. Insulin suppresses gluconeogenesis in the liver and initiates glycogen synthesis in the liver and skeletal muscle.
As diabetes reaches epidemic proportions in the developed world, the role of insulin resistance and its consequences are gaining prominence. Insulin resistance is defined by an elevated insulin level required to produce a sufficient biological response. This implies impaired insulin mediated glucose disposal (1). The effects of insulin, insulin deficiency, and insulin resistance differ according to the physiological function of the target tissues and organs, and their dependence on insulin for metabolic processes (1). Insulin’s actions are not limited to glucose disposal and the manifestations of insulin resistance can be found in various tissues, however the primary tissues that are insulin dependent for intracellular glucose transport are adipose tissue and muscle. (1).
Obesity is a key feature of many metabolic disorders and it is a risk factor for cardiovascular disease and diabetes. In the past decades, obesity and associated metabolic complications have reached epidemic proportions (4). It has been suggested that environmental and behavioral factors have been the major contributors to obesity. Dietary changes is one of the main behavioral factors. High fat diets tend to be associated with insulin resistance. The fatty acid composition of the diet plays a role in the long term development of insulin resistance and affects the stimulation of insulin secretion. It is well known that consuming a high calorie diet can lead to obesity. Obesity is closely associate with insulin resistance (5). Insulin resistance is a driving factor that leads to type 2 diabetes (5).
Diabetes is a chronic condition in which the body’s regulation of blood glucose levels becomes impaired and can lead to cardiovascular disease, microvascular disease, and metabolic syndromes, and other characterized syndromes. Diabetes consists of two forms: in type 1 the pancreas fails to produce insulin because of the autoimmune destruction of pancreatic beta-cells, distinguished as absolute lack of insulin. Whereas type 2 is characterized by insulin resistance; a loss of sensitivity of peripheral tissues to insulin signaling and inability of pancreatic beta cells to synthesize and secrete the quantity of insulin required to maintain adequate blood glucose; distinguished as relative lack of insulin. Glycemic control is the main target for management of diabetes. Type 1 diabetes glycemic control is achieved by using insulin-replacement therapy. Whereas type 2 diabetes glycemic control is usually maintained by oral agents that increases insulin sensitivity or increased its production by beta cells. Insulin-based therapy of type 2 diabetes has shown effectiveness in most cases. However the overall risk of increased cardiovascular morbidity remains questionable. In addition to hyperglycemia, type 2 diabetic patients have additional risk factors. These include insulin resistance, obesity and dyslipidemia. Although current clinically used insulin analogs provide relatively adequate glycemic control; the ultimate goal is the prevention of overt diabetes and its associated complications.
In beta cells, insulin is secreted from the proinsulin precursor as a single chain. Proinsulin is translocated into the rough endoplasmic reticulum. Pre-proinsulin is processed into proinsulin after the cleavage of the N-terminal 23-amino acid signal peptide by a signal peptidase. Proinsulin folds into its native conformation in the ER lumen. The pairing of the three disulfide linkages of proinsulin represents the major kinetic barrier to the folding of the proinsulin and mature insulin will readily re-fold into their native conformation after chemical denaturation as long as the disulfide linkages remain intact. Previous studies have shown that modification of the insulin molecule through amino acid substitutions, deletions, or additions on insulin chain can alter insulin function. These modifications have been found to alter the physiological properties of the insulin. Proinsulin is a precursor to the mature hormone. Human proinsulin is more stable than the mature protein. When exposed to acidic pH and high temperature conditions, the single chain proinsulin resists fibrillation longer than the two-chain wild-type insulin. Structure- based design of insulin analogs has led to the development of single chain and heat stable insulins based on the properties of the proinsulin as a single chain. Novel single-chain insulins (SCI’s) have been designed in Dr. Weiss’s laboratory and a number of publications has suggested the clinical potential of such analogs. Active SCI analogs were developed in which the C-domain is replaced by a short, at least four amino acid residues connecting A and B chain together to confer ultra-stability.
Materials and Methods
Our materials and methods are develop in two parts.
- Developing the Insulin resistance obese rat model
24 (250-315g) Male Lewis (LEW/SsNHsd) rats were purchased from Envigo (Indianapolis, Indiana). Half of the animals were fed a diet composed of 59% Safflower oil (Dyets, Inc 112245) for 4 weeks; 12 were fed standard rat chow (LabDiet 5P76 RMH 3000). All weights and blood glucose measurements were taken after a 16 hour fast. 25mg Dexamethasone (Sigma, D1756-25MG) dissolved in 7.5ml DMSO and stored at -20C. On Day 28, 6 high fat diet fed and 6 normal diet fed rats were injected with 200ug/rat Dexamethasone (DEX) IP for 3 consecutive days. The remaining rats were kept as a control and were not injected.
On sacrifice days (Day 30): rats were anesthetized via isoflurane following an overnight fast and a ~250ul blood sample was obtained from lateral tail vein for fasting insulin levels. Blood samples were allowed to clot at room temperature (RT) for 30min. Blood sample were then centrifuged at 17,000xg for 10min at 4C and the supernatant was transferred to clean tube. Tubes were centrifuged at 17,000xg for an additional 10min at 4C and the supernatant was transferred to a clean Eppendorf tube. Tubes were stored at -80C until analysis. Also after glucose tolerance test blood samples were collected for serum during decapitation to measure insulin levels.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones (9). For measurement of insulin level; rat insulin ELISA kit from CRYSTAL Chem inc catalog # 90010 was used. ELISA was performed by Manijeh (Homa) Phillips.
Data were calculated for statistical significance using ANOVA and student’s t-test.
Glucose tolerance test (GTT)
A glucose tolerance test measures the clearance of an injected glucose load by the body. 50% stock Dextrose (Hospira, Inc) solution was diluted to 25% by mixing 6ml of 50% Dextrose with 6ml of Saline. Injection volume was 1.2ml/300g rat to give a dose of 0.5g/ 500g rat. The solution of glucose is administered by intraperitoneal injection in overnight-fasted rats and plasmatic glucose levels are measured at different time points during the two hours. Blood glucose was measured using a standard handheld blood glucose monitor.
After the GTT, rats were decapitated under isoflurane anesthesia and blood, liver, fat and mixed muscle were collected for measurement of triglyceride content. Fat was weighed and photos were taken of the liver. All tissues were frozen on dry ice and stored at -80C.
Oil Red ‘O staining
Oil red O staining is used for the detection of fat in Liver histological tissues. Oil Red O staining is performed by using the abcam Oil Red O staining kit (Lipid Stain ab150678). The assay was performed as per the supplier’s protocol for the procedure. Tissues were sliced by Case Western Reserve University core Histology facility. Pictures were taken by using Case Western Reserve University core imaging facility.
Triglyceride concentrations were determined for each sample using the Triglyceride Colorimetric assay kit (Cayman chemical, item # 10010303). The assay was performed as per the supplier’s protocol for the microplate procedure. Absorbance at 562 nm was measured on the VersaMax Microplate reader.
Insulin tolerance test
An insulin tolerance test (ITT) evaluates insulin sensitivity by observing the disappearance of endogenous glucose in response to an injection of human insulin. 6µg/300g rat of commercially available insulin Humalog (Lispro) was injected subcutaneously (SQ) to the rats. Blood samples were taken for blood glucose measurement from a tail snip. Blood glucose was measured using a standard handheld blood glucose monitor (EasyMax V).
- Evaluating the SCI’s efficacy
In this study SCI 8104 was dissolved at concentrations in Lilly® Diluent solution. The formulations contained ZnCl2 at a ratio of 3 Zn2+:1 insulin hexamer. Insulin purity was verified by C4 analytical rp-HPLC. Humalog was used as a comparison. Insulin resistance rat model (mean body mass/300 g) were utilized. Rats were injected SQ at time t = 0 with the specified dose of insulin (100 μl/300 g body mass). Following SQ, a small drop of blood was obtained from the clipped tip of the rats’ tails at t = 0 and then every 10 min for the 1st hour, every 20 min for the 2nd hour, every 30 min during the 3rd hour. Measurements of [glucose] were made using a clinical glucometer (EasyMax® V Glucose Meter).
Our results are presented in two parts. We first provide an over view of developing the obese insulin resistance rat model and next describe the evaluation of the efficacy of the single chain in this particular rat model.
High fat diet can lead to insulin resistance
To determine whether consuming high fat diet can lead to insulin resistance, male Lewis were subjected to the control or high fat diet for four weeks. We evaluated the conformation of the insulin resistance rat model by measuring weight gain, fasting blood glucose levels, insulin levels, glucose tolerance test (GTT), insulin tolerance test (ITT), liver histology, and total body insulin resistance. In this Lewis strain the high fat diet did affect the body mass. The control group has slower body mass gain than high fat diet rats. The high fat diet itself did not affect the fasting blood glucose. In fourth week we inject Dexamethasone 200ug/rat by intraperitoneal injection for three consecutive days to 6 high fat diet rat and 6 normal diet rat. Dexamethasone is a glucocorticoid; induced inhibition of glucose oxidation. Dexamethasone decreased whole body glucose uptake (6). However, hyperglycemia and hyperinsulinemia developed, indicating insulin resistance in the Dexamethasone injected group. According to our data it is established that only high fat diet cannot develop hyperglycemia, hyperinsulinemia (Fig. 1). The homeostasis model assessment-estimated insulin resistance (HOMA-IR) has been widely used for the estimation of insulin resistance in research (7). It defined as total body insulin resistance (8). HOMA-IR is a function of fasting blood glucose and serum insulin level and is a measure of total body insulin resistance. The higher the number, the more resistant to the insulin sensitivity (8). The HOMA-IR data indicate the insulin resistance in the Dexamethasone group (Fig. 1). The glucose tolerance test results represented as glucose levels (mg/dl) in the Y-axis verses time (min) in the X-axis (Fig. 2). Higher the glucose parameter compared to the groups indicates increased glucose intolerance. The smaller glucose value indicates faster glucose disappearance means improved glucose tolerance. To compare among the different rat groups plasma serum were obtained after the glucose tolerance to measure the insulin concentration by ELISA. ELISA results also depicted as insulin levels (ng/ml) in Y-axis and verses time (min) in the X-axis (Fig. 2). Higher insulin concentration in the plasma serum graph suggested glucose intolerance. Insulin tolerance test results are represented as the initial glucose levels (mg/dl) in the Y-axis verses time (min) in the X-axis (Fig. 3). For comparison among different rat group area over the curve AOC is used. Higher AOC number indicates insulin resistance in the high fat diet with dexamethasone group. Liver, mixed muscle tissues and plasma serum were analyzed for triglyceride concentration. The triglyceride concentration results for liver an mixed muscle are depicted as triglyceride concentration (mg/g)(Fig. 4).The triglyceride concentration in liver for the normal diet and normal diet with dexamethasone has a significant different (p<0.05). However there is no significant difference between high fat diet and high fat diet with dexamethasone group. The mixed muscle triglyceride results showed no significant difference between all the groups. The triglyceride concentration results for plasma serum are depicted as triglyceride concentration (mg/dl)(Fig. 4).The serum triglyceride concentration however showed the significant difference in the normal diet group with Dexamethasone group (p<0.05) then the high fat diet group with Dexamethasone. Oil Red O staining of liver tissues indicates fatty liver in the high fat diet groups (Fig. 5). The hallmark manifestation of the insulin resistance are hyperglycemia, hyperinsulinemia, abnormal glucose tolerance test, abnormal insulin tolerance test, increased HOMA-IR and fatty liver. We cannot exclude the fact that high fat diet only can contribute to the insulin resistance however high fat diet with Dexamethasone can fully capture the complete manifestation of insulin resistance in our male Lewis rats.
Evaluating efficacy of Single chain insulin in insulin resistance obese rat model
An SCI with a six linker, which includes residues that allow bending the linker into the insulin receptor has been exhibit the insulin receptor binding efficiency of that observe for two-chain insulin (2). In this study we evaluate the single chain insulin 81-04 designed by Dr. Weiss lab. SCI 81-04 has a six amino acid linker of GGGPRR connecting the threonine residue at thirtieth position of the B-chain with the glycine residue in the first position of the A-chain. In addition to the linker, 81-04 also has substitutions at A8 (Thr to His), A10 (Tyr to Glu), B28 (Pro to Asp), and B29 (Lys to Pro). Previously studies of SCI 8104 on normal rats and Streptozotocin-induced type 1 diabetic rats in our lab showed that these SCI 8104 lowers blood glucose levels as rapidly as insulin Humalog; the first commercially available rapid-acting insulin analog. However with SCI 8104 causes the blood glucose levels remain lower for a much longer period than Humalog, mimicking the prolonged action of basal insulins like Glargine compare to Humalog (Fig. 7). All Pharmacodynamics test results are represented as the initial glucose levels (mg/dl) in the Y-axis verses time (min) in the X-axis. This study analyzes the efficacy of fast onset and the prolonged action of the novel single-chain insulin 81-04. When the study conduct on insulin resistance model SCI 8104 has the same effect as compare to Humalog. SCI 8104 lowers the blood glucose as efficiently as Humalog but loses its prolonged action as Glargin (Fig. 8). In this study of insulin resistance group we used same dose as of SCI 8104 compare to the Humalog. We tested SCI 8104 with high fat diet vs high fat diet with Dex vs normal diet vs normal diet with Dex. Our further studies of comparison of SCI 8104 with the four group shows the effectiveness of the analog in insulin resistance obese rat model (Fig. 9) is less in high fat diet with Dex than the other group. We have found the optimal dose based on our normal rat study with SCI 8104 (Fig. 6). For this rat model with insulin resistance, higher doses might be used.
Insulin, a hormone secreted by pancreatic beta cells serves as a primary regulator of glucose homeostasis; in response to increased interstitial glucose levels. Type 2 diabetes is caused by impaired insulin secretion and increased insulin resistance. However the underlying causes for type 2 diabetes remains unknown. It is clear that insulin resistance leads to increased insulin demand and β-cell overstimulation (3). Exogenous insulin are the agent used to induce β-cell relaxation to normalization of blood glucose and diminishes the insulin secretory demand, thus decreasing endogenous insulin production (3). Studies have shown that the insulin therapy of the basal insulin doses early in the stage of the type 2 diabetes may have protective effects on beta cell function improving recovery from type 2 diabetes (3).
This study is focused on the efficacy of novel SCI analogs in an insulin resistance obese rat model. The results from this proposal will be very crucial to our hypothesis that SCI 8104 can be used as a basal insulin for insulin resistance. SCI’s 8104 lowers blood glucose levels as rapidly as insulin Humalog. However SCI 8104 loses the prolonged action of basal insulins like Glargine in this insulin resistance obese rat model. Insulin resistance is a condition when cells are resistant to the effects of insulin. As SCI 8104 has no prolonged action there is less conflict of hypoglycemic as other long acting basal insulins. It will only effect on hyperglycemia. However, an optimal dose can be used according to the severity of insulin resistance; which can be determine by the weight gain and fasting blood glucose measurement. Thus, SCI 8104 may delay the onset of diabetes by decreasing the beta cell over-stimulation and the metabolic syndromes associated with it; like dyslipidemia, cardiovascular disease. After a short period of using intensive insulin therapy of SCI may suggests a new paradigm for the treatment of type 2 diabetes, in which β-cell function may be preserved by the use of early insulin therapy (3). Furthermore, as the ultimate goal is to prevent diabetes and its associated complications (3). Meanwhile we could also evaluate novel SCIs designed by Dr. Wesis lab.
To further understand the mechanism of the single chain insulin analogs in obese insulin resistance rat model for better disease management we will compare the hepatic triglyceride accumulation, test insulin signaling in targeted tissues, measure glycogen synthesis, glucose uptake in liver, mixed muscle and adipose tissue.
I would like to thank my mentor, Dr. Faramarz Ismail-Beigi for his support and guidance throughout this project. I would also like to express my gratitude to Dr. Nelson Phillips, Manijeh (Homa) Phillips, Dr. Nischay Rege, Dr.Michael Glidden and Dr. Balmurugan Dhayalan for sharing their generosity with knowledge, equipment, and advice. I am thankful to my beloved co-workers Kelley Carr, Alisar Tustan, Paul Macklis, Rachel Grabowski, Leili Rahimi, Somya Sharma and Ajeet KalipuI for their contributions throughout this project and having confidence on me. Special thanks to Jenifer Mikulan for sharing her knowledge on histology towards this project. I would like to thank my committee members Dr. Snider, and Dr. Merrick for their guidance.
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Melbourne Pathology, Collingwood, VIC 3066, Monash University Department of Medicine & Clinical Nutrition & Metabolism
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Mechanisms of dexamethasone-induced insulin resistance in healthy humans.
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7. The Definition of Insulin Resistance Using HOMA-IR for Americans of Mexican Descent Using Machine Learning