Popejessen9179

Insulin resistance in humans and mice is an important hallmark of metabolic diseases. Therefore, assessment of insulin sensitivity/resistance in animal models provides valuable information in the pathophysiology of diabetes and obesity. Depending on the nature of the information required, we can choose between direct and indirect techniques available for the determination of insulin sensitivity. Thus, the complex hyperinsulinemic-euglycemic glucose clamps and the insulin suppression test assess insulin-mediated glucose utilization under steady-state conditions, whereas less complex methods, such as the insulin tolerance test (ITT), rely on measurements of blood glucose levels in animals subjected to intraperitoneal insulin loading. Finally, surrogated indexes derived from blood glucose and plasma insulin levels are also available for assessment of insulin sensitivity/resistance in vivo. In this chapter, we focus on the intraperitoneal insulin tolerance test (IPITT) protocol for measuring insulin resistance in mice.Type 2 diabetes is characterized by glucose intolerance, caused by insulin resistance in peripheral metabolic tissues and by impaired glucose-stimulated insulin secretion, the hallmark of beta-cell dysfunction. The glucose tolerance test is used in clinic and research to identify individuals with impaired glucose tolerance and overt type 2 diabetes. It is the most routinely used physiological test for first pass assessment of glucose homeostasis in rodents because of its simplicity. The GTT measures changes in blood glucose concentration over a 2-h period following the administration of a bolus of glucose. However, this simplicity belies several important considerations which need to be addressed, to aid reproducibility and produce interpretable data. α-D-Glucose anhydrous chemical structure Here, we describe in detail how to perform a GTT using four different routes of glucose administration intraperitoneal, oral, voluntary oral, and intravenous.Beta-cell-specific transgenic mice provide an invaluable model for dissecting the direct signaling mechanisms involved in regulating beta-cell structure and function. Furthermore, generating novel transgenic models is now easier and more cost-effective than ever, thanks to exciting novel approaches such as CRISPR.Here, we describe the commonly used approaches for generating and maintaining beta-cell-specific transgenic models and some of the considerations involved in their use. This includes the use of different beta-cell-specific promoters (e.g., pancreatic and duodenal homeobox factor 1 (Pdx1), rat insulin 2 promoter (RIP), and mouse insulin 1 promoter (MIP)) to drive site-specific recombinase technology. Important considerations during selection include level and uniformity of expression in the beta-cell population, ectopic transgene expression, and the use of inducible models.This chapter provides a guide to the procurement, generation, and maintenance of a beta-cell-specific transgene colony from preexisting Cre and loxP mouse strains, providing methods for crossbreeding and genotyping, as well as subsequent maintenance and, in the case of inducible models, transgenic induction.During embryogenesis, beta-cells arise from the dorsal and ventral bud originating in the endoderm germ layer. As the animal develops to adulthood, the beta-cell mass dramatically increases. The expansion of the beta-cell population is driven by cell division among the embryonic beta-cells and supplanted by neogenesis from post-embryonic progenitors. Here, we describe a protocol for multicolor clonal analysis in zebrafish to define the contribution of individual embryonic beta-cells to the increase in cell numbers. This technique provides insights into the proliferative history of individual beta-cells in an islet. This insight helps in defining the replicative heterogeneity among individual beta-cells during development. Additionally, the ability to discriminate individual cells based on unique color signatures helps quantify the volume occupied by beta-cells and define the contribution of cellular size to the beta-cell mass.Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.Streptozotocin (STZ) selectively destroys beta cells and is widely used to induce experimental diabetes in rodents. Rodent beta cells are very sensitive to the toxic effects of STZ, while human beta cells are highly resistant to STZ. Taking advantage of this characteristic, here, we describe two protocols for the induction of STZ-diabetes. In the first model, hyperglycemia is induced prior to islet transplantation, whereas in the second model, STZ is injected after islet transplantation. The former model has many applications and thus is the most commonly used method. However, when implanting human islets into mice, there are clear benefits to administering STZ after the transplantation. It reduces the cost and burden of experiments and the number of human islets needed for transplantation and improves the welfare and survival of animals used in the experiments. In both methods, a key step in the experimental protocol is to remove the graft-bearing kidney at the end of the experiment and monitor onset of hyperglycemia. This can be used to demonstrate that the glycemic control of the animal is due to the engrafted islets and not regeneration of endogenous beta cells. This chapter outlines protocols of administering streptozotocin pre- and post-islet transplantation in mice as well as nephrectomy to remove the graft-bearing kidney.