Obesity‐Induced Diabetes in Mouse Strains Treated With Gold Thioglucose: A Novel Animal Model for Studying β‐Cell Dysfunction

An obesity‐induced diabetes model using genetically normal mouse strains would be invaluable but remains to be established. One reason is that several normal mouse strains are resistant to high‐fat diet‐induced obesity. In the present study, we show the effectiveness of gold thioglucose (GTG) in inducing hyperphagia and severe obesity in mice, and demonstrate the development of obesity‐induced diabetes in genetically normal mouse strains. GTG treated DBA/2, C57BLKs, and BDF1 mice gained weight rapidly and exhibited significant increases in nonfasting plasma glucose levels 8–12 weeks after GTG treatment. These mice showed significantly impaired insulin secretion, particularly in the early phase after glucose load, and reduced insulin content in pancreatic islets. Interestingly, GTG treated C57BL/6 mice did not become diabetic and retained normal early insulin secretion and islet insulin content despite being as severely obese and insulin resistant as the other mice. These results suggest that the pathogenesis of obesity‐induced diabetes in GTG‐treated mice is attributable to the inability of their pancreatic β‐cells to secrete enough insulin to compensate for insulin resistance. Mice developing obesity‐induced diabetes after GTG treatment might be a valuable tool for investigating obesity‐induced diabetes. Furthermore, comparing the genetic backgrounds of mice with different susceptibilities to diabetes may lead to the identification of novel genetic factors influencing the ability of pancreatic β‐cells to secrete insulin.     

Hybrid pancreatic tissue substitute consisting of recombinant insulin-secreting cells and glucose-responsive material

Insulin-dependent diabetes is a serious pathological condition, currently treated by blood glucose monitoring and daily insulin injections, which, however, do not prevent long-term complications. A tissue-engineered pancreatic substitute has the potential to provide a more physiologic, less invasive, and potentially less costly treatment of the disease. A major issue in developing such a substitute is the cells being used. Nonpancreatic cells, retrieved from the same patient and genetically engineered to secrete insulin constitutively or with some glucose responsiveness, offer the significant advantages of being immune-acceptable and relaxing the tissue availability limitations, which exist with islets from cadaveric donors. These cells, however, do not have insulin secretion dynamics appropriate for restoration of euglycemia in higher animals and, eventually, humans. In this study, we present the concept of a hybrid pancreatic substitute consisting of such cells sequestered in a material exhibiting glucose-dependent changes of its permeability to insulin. A Concanavalin A-glycogen material sandwiched between two polycarbonate membranes and exhibiting glucose-dependent sol-gel transformations was used. Rates of insulin transport through this material in gel and sol forms were characterized for both FITC-labeled insulin in solution and insulin secreted by betaTC3 mouse insulinoma cells. Effective diffusivities through sol were found to be up to 3.5-fold higher than through the gel state of the material. A mathematical model of a hybrid construct was formulated and analyzed to simulate the secretory behavior in response to step ups and downs in the surrounding glucose concentration. The experimental and modeling studies indicate that a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.     

Genetically engineered K cells provide sufficient insulin to correct hyperglycemia in a nude murine model

A gene therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate β cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. In this study, the murine enteroendocrine cell line STC-1 was genetically modified by stable transfection. Two clone cells were selected (STC-1-2 and STC-1-14) that secreted the highest levels of insulin among the 22 clones expressing insulin from 0 to 157.2 μIU/ml/10⁶ cells/d. After glucose concentration in the culture medium was increased from 1 mM to 10 mM, secreted insulin rose from 40.3±0.8 to 56.3±3.2 μIU/ml (STC-1-2), and from 10.8±0.8 to 23.6±2.3 μIU/ml (STC-1-14). After STC-1-14 cells were implanted into diabetic nude mice, their blood glucose levels were reduced to normal. Body weight loss was also ameliorated. Our data suggested that genetically engineered K cells secrete active insulin in a glucose-regulated manner, and in vivo study showed that hyperglycemia could be reversed by implantation of the cells, suggesting that the use of genetically engineered K cells to express human insulin might provide a glucose-regulated approach to treat diabetic hyperglycemia.     

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.     

Extract of Ocimum canum lowers blood glucose and facilitates insulin release by isolated pancreatic β-islet cells

Aqueous extract of Ocimum canum Sim, (Lamiaceae) is used by some Ghanaians to manage diabetes mellitus. In vivo modulation of levels of fasting blood glucose by 0. canum extract was evaluated in type-II diabetes mellitus using the C57BL/KsJ db/db genetically diabetic animal model, and its effects on glucose-stimulated insulin release in vitro were monitored using isolated rat pancreatic beta-islet cells. The results showed that fasting blood glucose levels and body weight decreased significantly (p < 0.05) in diabetic and non-diabetic C57BL/KsJ mice, which were administered aqueous extract of 0. canum. In vitro, the 0. canum extract significantly enhanced insulin release from isolated rat pancreatic beta-islet cells. Insulin release was found to be dependent on glucose concentration and increased with increasing O. canum concentration in the incubation medium up to an optimum extract concentration of 0.03 mg/ml. Release of the hormone decreased beyond this concentration of extract in the medium. Addition to the medium of Desmodium adscendens, a plant preparation used to manage inflammatory disorders, did not increase but rather inhibited insulin secretion by the pancreatic beta-islet cells. These results could explain the use of 0. canum in Ghanaian folk medicine to manage diabetes mellitus.     

New developments in the treatment and monitoring of type 1 diabetes mellitus

In recent years, insulin analogues are the benefits of the use in functional intensive insulin therapy for the treatment of diabetes. Shortacting insulin (lispro, aspart and glulisine) and long-acting insulin (glargine and detemir) have been developed for the management of diabetes. Short-acting insulin analogues are an alternative to regular human insulin before meals. These new short-acting insulin analogues show more rapid onset of activity and a shorter duration of action. As a result of these pharmacokinetic differences, an improved postprandial glycemic control is achieved, without increasing the risk of hypoglycemia. In addition, these insulin analogues can be administered immediately before a meal. The long-acting insulin analogues provide basal insulin levels for 24 h when administered once (glargine) or two (detemir) daily. Compared with previous intermediate- or long-acting conventional insulin, these insulins shows a flat profile of plasma insulin levels . The use of these long-acting insulin analogues appears to be associated with a reduced incidence of hypoglycemia, especially at night. The availability of these new insulin analogues has the potential to significantly improve long-term control over blood glucose in diabetic patients. In recent years more and more frequently the method of multiple daily injections (MDI) of insulin is being replaced by the method of continuous subcutaneous insulin infusion (CSII). It is the most physiological way to administer insulin. In recent years treatment with insulin pumps has been used more frequently in the pediatric patients and in the treatment of diabetes in pregnancy. Use of continuous glucose monitoring systems enables detection of glycemia fluctuations unrevealed by selfmonitoring of blood glucose, such as night hypoglycemias and early postprandial hyperglycemias. Real-time systems allow to reduce HbA1c levels and limit number of excursions. Non-invasive glucose measurement devices are introduced. Fully automated continuous glucose monitoring systems integrated with insulin pumps operating in closed-loop model, requiring no patient assistance, are still being researched. Commercially available systems operate in open-loop model, where the patient has to decide on administration and dose of insulin.     

Glucagon-like peptide 1: evolution of an incretin into a treatment for diabetes

Glucagon-like peptide 1 (GLP-1) is a product of proglucagon that is secreted by specialized intestinal endocrine cells after meals. GLP-1 is insulinotropic and plays a role in the incretin effect, the augmented insulin response observed when glucose is absorbed through the gut. GLP-1 also appears to regulate a number of processes that reduce fluctuations in blood glucose, such as gastric emptying, glucagon secretion, food intake, and possibly glucose production and glucose uptake. These effects, in addition to the stimulation of insulin secretion, suggest a broad role for GLP-1 as a mediator of postprandial glucose homeostasis. Consistent with this role, the most prominent effect of experimental blockade of GLP-1 signaling is an increase in blood glucose. Recent data also suggest that GLP-1 is involved in the regulation of beta-cell mass. Whereas other insulinotropic gastrointestinal hormones are relatively ineffective in stimulating insulin secretion in persons with type 2 diabetes, GLP-1 retains this action and is very effective in lowering blood glucose levels in these patients. There are currently a number of products in development that utilize the GLP-1-signaling system as a mechanism for the treatment of diabetes. These compounds, GLP-1 receptor agonists and agents that retard the metabolism of native GLP-1, have shown promising results in clinical trials. The application of GLP-1 to clinical use fulfills a long-standing interest in adapting endogenous insulinotropic hormones to the treatment of diabetes.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.     

A model for evaluation of the peroral insulin therapy: short-term treatment of alloxan diabetic rats with oral water-in-oil-in-water insulin emulsions.

Alloxan diabetic rats with fasting blood glucose levels above 300 mg/100 ml were treated with oral administration of water-in-oil-in-water (W/O/W) insulin emulsions at a dose of 50 U/100 g body weight, three times daily for 10 to 14 days. The course of diabetes was followed by determinations of glucose levels in blood and urine. During treatment with oral W/O/W insulin emulsions, daily excretion of urinary glucose decreased by about 30 to 40% (2 to 3 g/day) in all of the five rats studied, and returned to the pre-treatment levels after the treatment being discontinued. During treatment, a significant reduction in fasting blood glucose levels was observed in 4 out of 5 rats, giving the decrease by 18 to 44%. Quantitative estimates suggested that the effectiveness of 50 U/100 g of oral W/O/W insulin emulsions was comparable to that after intramuscular regular insulin at a dose of 0.5 U/100 g. Although oral W/O/W insulin emulsions are still of low efficiency, these results would indicate that diabetes can be controlled by effective oral insulin preparations.     

Targeted pancreatic beta cell imaging for early diagnosis

Pancreatic beta cells are important in blood glucose level regulation. As type 1 and 2 diabetes are getting prevalent worldwide, we need to explore new methods for early detection of beta cell-related afflictions. Using bioimaging techniques to measure beta cell mass is crucial because a decrease in beta cell density is seen in diseases such as diabetes and thus can be a new way of diagnosis for such diseases. We also need to appraise beta cell purity in transplanted islets for type 1 diabetes patients. Sufficient amount of functional beta cells must also be determined before being transplanted to the patients. In this review, indirect imaging of beta cells will be discussed. This includes membrane protein on pancreatic beta cells whereby specific probes are designed for different imaging modalities mainly magnetic resonance imaging, positron emission tomography and fluorescence imaging. Direct imaging of insulin is also explored though probes synthesized for such function are relatively fewer. The path for successful pancreatic beta cell imaging is fraught with challenges like non-specific binding, lack of beta cell-restricted targets, the requirement of probes to cross multiple lipid layers to bind to intracellular insulin. Hence, there is an urgent need to develop new imaging techniques and innovative probing constructs in the entire imaging chain of bioengineering to provide early detection of beta cell-related pathology.     

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.     

The challenge of type 1 diabetes mellitus

Diabetes mellitus is a heterogeneous group of diseases characterized by high blood glucose levels due to defects in insulin secretion, insulin action, or both. With the number of cases expected to increase rapidly in the years to come, diabetes is a growing health challenge worldwide. Of the approximately 16 million diabetics in the United States, about 1.5 million suffer from type 1 diabetes. In this catabolic disorder afflicting predominantly young individuals, blood insulin is almost completely absent, leading to hyperglycemia and alterations in lipid metabolism. Type 1 diabetes is thought to be induced by a toxic or infectious insult that occurs in genetically predisposed individuals. With recent advances in the understanding of the involved immunology and cellular and molecular mechanisms, researchers strive to battle the disease with new preventive and corrective strategies.     

Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Both an increase in reactive oxygen free radical species (ROS) and a decrease in the antioxidant defense mechanism lead to the increase in oxidative stress in diabetes. Electrolyzed reduced water (ERW) with ROS scavenging ability may have a potential effect on diabetic animals, a model for high oxidative stress. Therefore, the present study examined the possible anti-diabetic effect of ERW in two different diabetic animal models. The genetically diabetic mouse strain C57BL/6J-db/db (db/db) and streptozotocin (STZ)-induced diabetic mouse were used as insulin deficient type 1 and insulin resistant type 2 animal model, respectively. ERW, provided as a drinking water, significantly reduced the blood glucose concentration and improved glucose tolerance in both animal models. However, ERW fail to affect blood insulin levels in STZ-diabetic mice whereas blood insulin level was markedly increased in genetically diabetic db/db mice. This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The present data suggest that ERW may function as an orally effective anti-diabetic agent and merit further studies on its precise mechanism.     

Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreatic beta cell lines

Current therapy for type 1 diabetes mellitus involves a daily regimen of multiple subcutaneous or intramuscular injections of recombinant human insulin. To achieve long-term insulin delivery in vivo, we investigated the applicability of cytomedical therapy using beta TC6 cells or MIN6 cells, both of which are murine pancreatic beta cell lines that secrete insulin in a subphysiologically or physiologically regulated manner, respectively. We examined this therapy in the insulinopenic diabetic mice intraperitoneally injected with beta TC6 cells or MIN6 cells microencapsulated within alginate-poly(L)lysine-alginate membranes (APA-beta TC6 cells or APA-MIN6 cells). The diabetic mice treated with APA-beta TC6 cells fell into hypoglycemia, whereas those injected with APA-MIN6 cells maintained normal blood glucose concentrations for over 2 months without developing hypoglycemia. In addition, we also conducted an oral glucose tolerance test using these mice. The blood glucose concentrations of normal and of diabetic mice injected with APA-MIN6 cells similarly changed over time, although the blood insulin concentration increased later in the injected diabetic mice than in the former. These results suggest that cytomedicine utilizing microencapsulated pancreatic beta cell lines with a physiological glucose sensor may be a beneficial and safe therapy with which to treat diabetes mellitus.     

The effect of glucagon antibodies on plasma glucose and insulin levels

The actions of glucagon and insulin are interrelated as the two hormones have opposite physiological effects and the secretion of insulin is regulated, at least in part, by the level of glucagon. We have found that rabbits which are immunized against glucagon have normal fasting levels of blood glucose but a lowered level of insulin. These rabbits are also able to rapidly utilize intravenously injected glucose butwith a much lower plasma level of insulin. These results demonstrate that in the presence of glucagon antibodies, normal blood sugar levels can be maintained with a reduced supply of insulin. It is suggested that this finding may be useful in the treatment of diabetes.     

Insulin-secreting L-cells for the treatment of insulin-dependent diabetes

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complications associated with diabetes. An autologous cell source is especially attractive for regulatory and ethical reasons in addition to eliminating the need for immunosuppression. This study uses non-β-cells, genetically modified for physiologic insulin secretion. Enteroendocrine L-cells, exhibit regulated secretion in response to physiologic stimuli and their endogenous products are fully compatible with prandial metabolism. Murine GLUTag L-cells were transfected with a plasmid co-expressing human insulin and neomycin resistance and the stable cell line, GLUTag-INS, was established. Secretion properties of GLUTag-INS cells were investigated in vitro through induced secretion tests using meat hydrolysate or 3-isobutyl-1-methylxanthine and forskolin as secretagogues. GLUTag-INS cells rapidly co-secreted recombinant insulin and endogenous glucagon-like peptide in response to metabolic cues from the surrounding medium and demonstrated efficient processing of proinsulin to insulin.     

Two birds with one stone: novel glucokinase activator stimulates glucose-induced pancreatic insulin secretion and augments hepatic glucose metabolism

The hormones glucagon and insulin delicately regulate the concentration of blood glucose. When patients become resistant to the effects of insulin or produce too little of it to properly regulate glucose concentrations, then diabetes can result. Unfortunately, not all patients with insulin-resistant, type 2 diabetes mellitus respond to drugs that improve insulin sensitivity. However, there is reason to be hopeful. A new molecule that targets glucokinase (GK), the enzyme responsible for phosphorylating glucose in pancreatic beta cells and hepatic cells, acts to significantly reduce blood glucose concentrations in rodents. The GK activator RO-28-1675 increased the glucose affinity and Vmax of GK, and rats treated with RO-28-1675 had improved glucose tolerance and elevated glucose uptake in liver. These results provide the basis for improved drug design that may alleviate diabetes mellitus and the disorders that accompany it in patients.     

Assessment of type II diabetes mellitus using irregularly sampled measurements with missing data

Diabetes mellitus is one of the leading diseases in the developed world. In order to better regulate blood glucose in a diabetic patient, improved modelling of insulin-glucose dynamics is a key factor in the treatment of diabetes mellitus. In the current work, the insulin-glucose dynamics in type II diabetes mellitus can be modelled by using a stochastic nonlinear state-space model. Estimating the parameters of such a model is difficult as only a few blood glucose and insulin measurements per day are available in a non-clinical setting. Therefore, developing a predictive model of the blood glucose of a person with type II diabetes mellitus is important when the glucose and insulin concentrations are only available at irregular intervals. To overcome these difficulties, we resort to online sequential Monte Carlo (SMC) estimation of states and parameters of the state-space model for type II diabetic patients under various levels of randomly missing clinical data. Our results show that this method is efficient in monitoring and estimating the dynamics of the peripheral glucose, insulin and incretins concentration when 10, 25 and 50 % of the simulated clinical data were randomly removed.     

Alterations in glucose transporter expression and function in diabetes: mechanisms for insulin resistance.

Insulin resistance is a major pathologic feature of human obesity and diabetes. Understanding the fundamental mechanisms underlying this insulin resistance has been advanced by the recent cloning of the genes encoding a family of facilitated diffusion glucose transporters which are expressed in characteristic patterns in mammalian tissues. Two of these transporters, GLUT1 and GLUT4, are present in muscle and adipose cells, tissues in which glucose transport is markedly stimulated by insulin. To understand the mechanisms underlying in vivo insulin resistance, regulation of these transporters is being investigated. Studies reveal divergent changes in the expression of GLUT1 and GLUT4 in a single cell type as well as tissue specific regulation. Importantly, alterations in glucose transport in rodent models of diabetes and in human obesity and diabetes cannot be entirely explained by changes in glucose transporter expression. This suggests that defects in glucose transporter function such as impaired translocation, fusion with the plasma membrane, or activation probably contribute importantly to in vivo insulin resistance.