Cholecystokinin attenuates ß-cell apoptosis in both mouse and human islets.

Loss of functional pancreatic ß-cell mass and increased ß-cell apoptosis are fundamental to the pathophysiology of type 1 and type 2 diabetes. Pancreatic islet transplantation has the potential to cure type 1 diabetes but is often ineffective due to the death of the islet graft within the first few years after transplant. Therapeutic strategies to directly target pancreatic ß-cell survival are needed to prevent and treat diabetes and to improve islet transplant outcomes. Reducing ß-cell apoptosis is also a therapeutic strategy for type 2 diabetes. Cholecystokinin (CCK) is a peptide hormone typically produced in the gut after food intake, with positive effects on obesity and glucose metabolism in mouse models and human subjects. We have previously shown that pancreatic islets also produce CCK. The production of CCK within the islet promotes ß-cell survival in rodent models of diabetes and aging. We demonstrate a direct effect of CCK to reduce cytokine-mediated apoptosis in a ß-cell line and in isolated mouse islets in a receptor-dependent manner. However, whether CCK can protect human ß-cells was previously unknown. Here, we report that CCK can also reduce cytokine-mediated apoptosis in isolated human islets and CCK treatment in vivo decreases ß-cell apoptosis in human islets transplanted into the kidney capsule of diabetic NOD/SCID mice. Collectively, these data identify CCK as a novel therapy that can directly promote ß-cell survival in human islets and has therapeutic potential to preserve ß-cell mass in diabetes and as an adjunct therapy after transplant.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Successful in vitro fertilization and generation of transgenics in Black and Tan Brachyury (BTBR) mice.

The Black and Tan Brachyury (BTBR) mouse strain is a valuable model for the study of long-term complications from obesity-induced type 2 diabetes mellitus and autism spectrum disorder. Due to technical difficulties with assisted reproduction, genetically modified animals on this background have previously been generated through extensive backcrossing, which is expensive and time-consuming. We successfully generated two separate transgenic mouse lines after direct zygote microinjection into this background strain. Additionally, we developed in vitro fertilization (IVF) methods for the BTBR mouse. We found low rates of fertilization and implantation in this strain, and identified the BTBR oocyte as the primary culprit of low success with BTBR IVF. We achieved an increase in live born pups from 5.9 to 35.6 % with IVF in the BTBR strain by use of BTBR females at a younger age (18-25 days), collection of oocytes 15-17 h after superovulation, and the use of supplemented fertilization media. This method eliminates the need for time consuming assisted embryo manipulations that are otherwise required for success with BTBR oocytes. This advancement provides an exciting opportunity to directly generate BTBR transgenics and gene-edited mice using both traditional and emerging genomic editing techniques, such as CRISPR/Cas9. These methods also allow effective colony preservation and rederivation with these strains. To our knowledge, this is the first report describing embryo manipulations in BTBR mice.

Cholecystokinin expression in the ß-cell leads to increased ß-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis.

Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic ß-cells, but only in models of obesity and insulin resistance. Whole body deletion of CCK in obese mice leads to reduced ß-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from ß-cell apoptosis. To determine the specific role of ß-cell-derived CCK in ß-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the ß-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain ß-cell mass when subjected to apoptotic stress, with advanced age, and after streptozotocin treatment. Aged MIP-CCK mice have increased ß-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced ß-cell apoptosis. Directed CCK overexpression in cultured ß-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of ß-cells from apoptotic stress. Understanding the role of ß-cell CCK adds to the emerging knowledge of classic gut peptides in intraislet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect ß-cells from apoptosis.

Transgenic expression of the human growth hormone minigene promotes pancreatic ß-cell proliferation.

Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic ß-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates ß-cell proliferation. We aimed to investigate the effect of FoxM1 on ß-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased ß-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls.

Pancreatic ß-cell proliferation in obesity.

Because obesity rates have increased dramatically over the past 3 decades, type 2 diabetes has become increasingly prevalent as well. Type 2 diabetes is associated with decreased pancreatic ß-cell mass and function, resulting in inadequate insulin production. Conversely, in nondiabetic obesity, an expansion in ß-cell mass occurs to provide sufficient insulin and to prevent hyperglycemia. This expansion is at least in part due to ß-cell proliferation. This review focuses on the mechanisms regulating obesity-induced ß-cell proliferation in humans and mice. Many factors have potential roles in the regulation of obesity-driven ß-cell proliferation, including nutrients, insulin, incretins, hepatocyte growth factor, and recently identified liver-derived secreted factors. Much is still unknown about the regulation of ß-cell replication, especially in humans. The extracellular signals that activate proliferative pathways in obesity, the relative importance of each of these pathways, and the extent of cross-talk between these pathways are important areas of future study.

Endoscopic diagnosis of a pylorogastric intussusception with spontaneous resolution.

A 7 mo old intact female golden retriever was evaluated for acute vomiting. Abdominal radiographs revealed a possible gastric foreign body. Upper gastrointestinal endoscopy revealed an edematous, tubular antral mass, which on further evaluation was determined to be a pylorogastric intussusception based on radiographic, endoscopic, and surgical findings. Spontaneous resolution of the intussusception occurred upon surgical exploration of the abdomen. Histopathology of a full-thickness gastric biopsy revealed vascular congestion consistent with an intussusception, but did not indicate the primary cause. The dog recovered uneventfully from surgery and had no further vomiting during the 6 mo follow-up period. This case was significant as it was the first report of pylorogastric intussusception diagnosed using endoscopy. This description of the unique endoscopic appearance of pylorogastric intussusception will be useful for the veterinary endoscopist.

Rhinoscopic diagnosis of Eucoleus boehmi infection in a dog.

A dog presenting for chronic purulent nasal discharge was diagnosed with an Eucoleus boehmi infection based upon rhinoscopic appearance of the nasal worms in situ, identification of the adult parasites in rhinoscopic nasal biopsies, and ova in the feces. The dog was successfully treated with a 2 wk course of fenbendazole and measures preventing reinfection through coprophagia. Patients with chronic nasal discharge should have a fecal examination performed to rule out infection with E. boehmi.