The Tetracycline-Controlled Transactivator (Tet-On/Off) System in ß-Cells Reduces Insulin Expression and Secretion in Mice.

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in ß-cells, transgene silencing, low inducibility, ectopic expression, and off-targets effects are persistent challenges. In this study, we investigated whether an inducible Tetracycline (Tet)-Off system with ß-cell-specific mouse insulin promoter (MIP)-itTA-driven expression of tetracycline operon (TetO)-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination, ß-cell architecture, in vitro and in vivo glucose-stimulated insulin secretion, and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g., improved itTA, reverse rtTA, or tTA) in ß-cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with ß-cells and highlights the need for consistent and comprehensive controls when using the tetracycline-controlled transactivator systems (Tet-On or Tet-Off) for genome editing.

Sustained Administration of ß-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic ß-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to ß-cells in culture and in transplanted tissue models. These technologies allow the study of candidate ß-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating ß-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on ß-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess ß-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Activated FoxM1 attenuates streptozotocin-mediated ß-cell death.

The forkhead box transcription factor FoxM1, a positive regulator of the cell cycle, is required for ß-cell mass expansion postnatally, during pregnancy, and after partial pancreatectomy. Up-regulation of full-length FoxM1, however, is unable to stimulate increases in ß-cell mass in unstressed mice or after partial pancreatectomy, probably due to the lack of posttranslational activation. We hypothesized that expression of an activated form of FoxM1 could aid in recovery after ß-cell injury. We therefore derived transgenic mice that inducibly express an activated version of FoxM1 in ß-cells (RIP-rtTA;TetO-hemagglutinin (HA)-Foxm1(Δ)(NRD) mice). This N-terminally truncated form of FoxM1 bypasses 2 posttranslational controls: exposure of the forkhead DNA binding domain and targeted proteasomal degradation. Transgenic mice were subjected to streptozotocin (STZ)-induced ß-cell ablation to test whether activated FoxM1 can promote ß-cell regeneration. Mice expressing HA-FoxM1(ΔNRD) displayed decreased ad libitum-fed blood glucose and increased ß-cell mass. ß-Cell proliferation was actually decreased in RIP-rtTA:TetO-HA-Foxm1(NRD) mice compared with that in RIP-rtTA mice 7 days after STZ treatment. Unexpectedly, ß-cell death was decreased 2 days after STZ treatment. RNA sequencing analysis indicated that activated FoxM1 alters the expression of extracellular matrix and immune cell gene profiles, which may protect against STZ-mediated death. These studies highlight a previously underappreciated role for FoxM1 in promoting ß-cell survival.

Loss of HNF6 expression correlates with human pancreatic cancer progression.

Normal pancreatic epithelium progresses through various stages of pancreatic intraepithelial neoplasms (PanINs) in the development of pancreatic ductal adenocarcinoma (PDAC). Transcriptional regulation of this progression is poorly understood. In mouse, the hepatic nuclear factor 6 (Hnf6) transcription factor is expressed in ductal cells and at lower levels in acinar cells of the adult pancreas, but not in mature endocrine cells. Hnf6 is critical for terminal differentiation of the ductal epithelium during embryonic development and for pancreatic endocrine cell specification. We previously showed that, in mice, loss of Hnf6 from the pancreatic epithelium during organogenesis results in increased duct proliferation and altered duct architecture, increased periductal fibrosis and acinar-to-ductal metaplasia. Here we show that decreased expression of HNF6 is strongly correlated with increased severity of PanIN lesions in samples of human pancreata and is absent from >90% of PDAC. Mouse models in which cancer progression can be analyzed from the earliest stages that are seldom accessible in humans support a role for Hnf6 loss in progression from early- to late-stage PanIN and PDAC. In addition, gene expression analyses of human pancreatic cancer reveal decreased expression of HNF6 and its direct and indirect target genes compared with normal tissue and upregulation of genes that act in opposition to HNF6 and its targets. The negative correlation between HNF6 expression and pancreatic cancer progression suggests that HNF6 maintains pancreatic epithelial homeostasis in humans, and that its loss contributes to the progression from PanIN to ductal adenocarcinoma. Insight on the role of HNF6 in pancreatic cancer development could lead to its use as a biomarker for early detection and prognosis.

Adult pancreatic acinar cells give rise to ducts but not endocrine cells in response to growth factor signaling.

Studies in both humans and rodents have found that insulin(+) cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin(+) cells arise de novo from ductal epithelium. We have found that insulin(+) cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated insulin(+) cells arise from the same cell of origin. Using a mouse model that develops insulin(+) cell-containing hyperplastic ducts in response to the growth factor TGFalpha, we performed genetic lineage tracing experiments to determine which cells gave rise to both hyperplastic ductal cells and duct-associated insulin(+) cells. We found that hyperplastic ductal cells arose largely from acinar cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin(+) cells adjacent to acinar-derived ductal cells arose from pre-existing insulin(+) cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of endocrine cells to reorganize in association with duct structures.

Deficiency of endothelial nitric-oxide synthase confers susceptibility to diabetic nephropathy in nephropathy-resistant inbred mice.

Recent studies have implicated dysfunctional endothelial nitric-oxide synthase (eNOS) as a common pathogenic pathway in diabetic vascular complications. However, functional consequences are still incompletely understood. To determine the role of eNOS-derived nitric oxide (NO) in diabetic nephropathy, we induced diabetes in eNOS knockout (KO) and wild-type (WT) mice on the C57BL6 background, using low-dose streptozotocin injection, and we investigated their glomerular phenotype at up to 20 weeks of diabetes. Although the severity of hyperglycemia in diabetic eNOS KO mice was similar to diabetic WT mice, diabetic eNOS KO mice developed overt albuminuria, hypertension, and glomerular mesangiolysis, whereas diabetic WT and nondiabetic control mice did not. Glomerular hyperfiltration was also significantly reduced in diabetic eNOS KO mice. Electron micrographs from diabetic eNOS KO mice revealed an injured endothelial morphology, thickened glomerular basement membrane, and focal foot process effacement. Furthermore, the anionic sites at glomerular endothelial barrier estimated by cationic ferritin binding were reduced in diabetic eNOS KO mice. In aggregate, these results demonstrate that deficiency of eNOS-derived NO causes glomerular endothelial injury in the setting of diabetes and results in overt albuminuria and glomerular mesangiolysis in nephropathy-resistant inbred mice. The findings indicate a vital role for eNOS-derived NO in the pathogenesis of diabetic nephropathy.

Characterization of diabetic nephropathy in a transgenic model of hypoinsulinemic diabetes.

Genetic mouse models provide a unique opportunity to investigate gene function in the natural course of the disease. Although diabetic nephropathy (DN) in models of type II diabetes has been well characterized, diabetic renal disease in hypoinsulinemic diabetic mice is still incompletely understood. Here, we characterized renal changes in the pdx1(PB)-HNF6 transgenic mouse that exhibits beta-cell dysfunction and nonobese hypoinsulinemic diabetes. Male transgenic mice developed hyperglycemia by the age of 7 wk and survived for over 1 yr without insulin treatment. Diabetes ensued earlier and progressed more severely in the HNF6 males than the females. The HNF6 males exhibited albuminuria as early as 10 wk of age, and the urinary albumin excretion increased with age, exceeding 150 microg/24 h at 11 mo of age. Diabetic males developed renal hypertrophy after 7 wk of age, whereas glomerular hyperfiltration was not observed in the mice. Hypertension and hyperlipidemia were not observed in the diabetic mice. Histological analysis of the HNF6 kidneys displayed diabetic glomerular changes, including glomerular enlargement, diffuse mesangial proliferation and matrix expansion, thickened glomerular basement membrane, and arteriolar hyalinosis. Mesangial matrix accumulation increased with age, resulting in nodular lesions by 44 wk of age. Immunohistochemistry showed accumulation of type IV collagen and TGF-beta1 in the mesangial area. No significant immune complex deposition was observed in the HNF6 glomeruli. Thus the HNF6 mouse exhibits diabetic renal changes that parallel the early phase of human DN. The model should facilitate studies of genetic and environmental factors that may affect DN in hypoinsulinemic diabetes.