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The streptozotocin (STZ)-induced diabetic mouse model has been extensively used as a model for diabetes and diabetic nephropathy, but it is still influenced by many off-target toxic effects and large variation in diabetes induction. Therefore, the aim of this study was to compare different STZ dosing regimens to optimise animal welfare and minimise unwanted effects of STZ measured by acute renal toxicity, impairment of stomach emptying and weight loss. Male 129/Sv mice were injected with 1 × 50, 1 × 100, 1 × 125, 1 × 150, 1 × 200, 5 × 50, 2 × 100 and 2 × 125 mg/kg STZ or vehicle and euthanized 24 hours after the last injection. All STZ doses were found to induce significant enlargement of the stomach. All multiple doses of STZ increased the albumin:creatinine ratio significantly, and immunohistochemical staining of KIM-1 and Ki-67 was increased by 5 × 50 and 2 × 100 mg/kg STZ. Renal gene expression of Cdkn1a, KIM-1, NGAL and MCP-1 was increased by most of the STZ doses. No difference was found between the double intermediate dose of 2 × 100 mg/kg and the multiple low dose of 5 × 50 mg/kg regarding either stomach enlargement or kidney injury. However, the reduced fasting periods and injections in the 2 × 100 mg/kg STZ group could have lowered the impact on the general condition measured as change in body weight. This shows that the double intermediate dose is a good alternative to the recommended multiple low dose for diabetes induction in these mice. The STZ-induced mouse model has again proven to be a model with large variations affecting both animal welfare and model robustness.

The widely used db/db mouse as a model of diabetic nephropathy (DN) only mimics the early changes in human DN with a slow disease progression. Since high protein diet (HPD) has been reported to affect progression of nephropathy in both humans and mice, we investigated whether HPD could accelerate nephropathy in db/db mice. Diabetic (C57BLKS-Lepr db/db ) and non-diabetic (C57BLKS-Lepr db /+) mice were fed either HPD (60 kcal% protein) or control diet (22 kcal% protein), from 7 to 22 weeks of age. In db/db mice, HPD was found to significantly increase all measured readouts of renal injury including albuminuria, renal hypertrophy, mesangial expansion and expression of a panel of DN related markers, including KIM-1, Ki67 and Collagen III, which increased on both gene and protein levels. Furthermore, HPD activated the Renin-angiotensin system significantly and increased hyperfiltration, measured as reduced plasma Cystatin C. Usefulness of the HPD db/db mouse as a model for faster drug efficacy studies was investigated in a 5-week treatment study with the SGLT2 inhibitor, dapagliflozin. Expectedly, dapagliflozin normalised blood glucose levels and improved glucose intolerance in both HPD and control diet mice. Only HPD db/db mice, not the control diet db/db mice, showed clear hyperfiltration that was significantly reduced with dapagliflozin treatment at both 2 and 4 weeks of treatment. In conclusion, these studies confirm that HPD can significantly accelerate progression of nephropathy in db/db mice, and that this model could be useful for rapid evaluation of drug targets with potential to ameliorate features of DN, especially glomerular hyperfiltration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The streptozotocin (STZ)-induced diabetic mouse is a widely used model of diabetes and diabetic nephropathy (DN). However, it is a well-known issue that this model is challenged by high weight loss, which despite supportive measures often results in high euthanization rates. To overcome these issues, we hypothesized that supplementing STZ-induced diabetic mice with water-softened chow in addition to normal chow would reduce weight loss, lower the need for supportive treatment, and reduce the number of mice reaching the humane endpoint of 20% weight loss. In a 15 week STZ-induced DN study we demonstrated that diabetic male mice receiving softened chow had reduced acute weight loss following STZ treatment ( p = 0.045) and additionally fewer mice were euthanized due to weight loss. By supplementing the diabetic mice with softened chow, no mice reached 20% weight loss whereas 37.5% of the mice without this supplement reached this humane endpoint ( p = 0.0027). Excretion of corticosterone metabolites in faeces was reduced in diabetic mice on softened chow ( p = 0.0007), suggesting lower levels of general stress. Finally, it was demonstrated that the water-softened chow supplement did not significantly affect the induction of key disease parameters, i.e. %HbA1C and albuminuria nor result in abnormal teeth wear. In conclusion, supplementation of softened food is refining the STZ-induced diabetic mouse model significantly by reducing stress, weight loss and the number of animals sacrificed due to humane endpoints, while maintaining the key phenotypes of diabetes and nephropathy.

Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates., Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation., Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion., Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates., (Copyright © 2017 Novo Nordisk A/S. Published by Elsevier GmbH.. All rights reserved.)

Cell encapsulation has been shown to hold promise for effective, long-term treatment of type 1 diabetes (T1D). However, challenges remain for its clinical applications. For example, there is an unmet need for an encapsulation system that is capable of delivering sufficient cell mass while still allowing convenient retrieval or replacement. Here, we report a simple cell encapsulation design that is readily scalable and conveniently retrievable. The key to this design was to engineer a highly wettable, Ca 2+ -releasing nanoporous polymer thread that promoted uniform in situ cross-linking and strong adhesion of a thin layer of alginate hydrogel around the thread. The device provided immunoprotection of rat islets in immunocompetent C57BL/6 mice in a short-term (1-mo) study, similar to neat alginate fibers. However, the mechanical property of the device, critical for handling and retrieval, was much more robust than the neat alginate fibers due to the reinforcement of the central thread. It also had facile mass transfer due to the short diffusion distance. We demonstrated the therapeutic potential of the device through the correction of chemically induced diabetes in C57BL/6 mice using rat islets for 3 mo as well as in immunodeficient SCID-Beige mice using human islets for 4 mo. We further showed, as a proof of concept, the scalability and retrievability in dogs. After 1 mo of implantation in dogs, the device could be rapidly retrieved through a minimally invasive laparoscopic procedure. This encapsulation device may contribute to a cellular therapy for T1D because of its retrievability and scale-up potential., Competing Interests: Conflict of interest statement: L.G.G., L.W., C.I., N.S.C., J.J.F., and F.W.S. are Novo Nordisk A/S employees and are shareholders in the company.

Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Background: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm., Methodology/principal Findings: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm., Conclusion/significance: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling.