The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

Overconsumption of saturated fats promotes obesity and type 2 diabetes. Excess weight gain in early life may be particularly detrimental by promoting earlier diabetes onset and potentially by adversely affecting normal development. In the present study we investigated the effects of dietary fat composition on early overnutrition-induced body weight and glucose regulation in Swiss Webster mice, which show susceptibility to high-fat diet-induced diabetes. We compared glucose homeostasis between a high-fat lard-based (HFL) diet, high in saturated fats, and a high-fat olive oil/fish oil-based (HFO) diet, high in monounsaturated and omega-3 fats. We hypothesized that the healthier fat profile of the latter diet would improve early overnutrition-induced glucose dysregulation. However, early overnutrition HFO pups gained more weight and adiposity and had higher diabetes incidence compared to HFL. In contrast, control pups had less weight gain, adiposity, and lower diabetes incidence. Plasma metabolomics revealed reductions in various phosphatidylcholine species in early overnutrition HFO mice as well as with diabetes. These findings suggest that early overnutrition may negate any beneficial effects of a high-fat diet that favours monounsaturated and omega-3 fats over saturated fats. Thus, quantity, quality, and timing of fat intake throughout life should be considered with respect to metabolic health outcomes.

Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre+LeprloxTB/loxTB) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre-LeprloxTB/loxTB). In contrast, Lyz2Cre+LeprloxTB/loxTB females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre+LeprloxTB/loxTB males had a mildly improved plasma glucose profile compared to Cre- controls while Lyz2Cre+LeprloxTB/loxTB females had a similar glucose excursion to their Cre- controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre+Leprflox/flox) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre+LeprloxTB/loxTB mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre+Leprflox/flox mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.

The mechanisms underlying zebrafish pancreatic islet vascularization have not been well characterized. We sought to determine the angiogenic factors responsible for islet vascularization and assess whether an absence of endothelial cells affects beta-cell and alpha-cell formation. We used a double transgenic zebrafish Tg(fli1:EGFP; insa:tagRFP) to label endothelial cells and beta-cells, respectively. Beta-cells developed adjacent to endothelial cells and by 72 hours post fertilization (hpf) the zebrafish pancreatic islet was highly vascularized. Zebrafish beta-cells express vascular endothelial growth factors (vegf), vegfaa and vegfab. Double knockdown of vegfaa and vegfab or the primary Vegfa receptors (Vegfr2), kdr and kdrl, resulted in vessel deficient islets. While beta-cell and alpha-cell numbers remained unchanged in vessel deficient islets, insulina expression was downregulated relative to controls. Vegfaa/Vegfab-Vegfr2 signaling is necessary for proper islet vessel development, but not for the initial formation of beta-cells and alpha-cells.

In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired β-cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient β-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of β-cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice (Ins1-/-;Ins2f/f). AAV-mediated expression of Cre in β-cells provides an effective alternative to transgenic approaches for inducible knockout studies.

The generation of functional β-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

The relative contribution of peripheral and central leptin signalling to the regulation of metabolism and the mechanisms through which leptin affects glucose homeostasis have not been fully elucidated. We generated complementary lines of mice with either leptin receptor (Lepr) knockdown or reconstitution in adipose tissues using Cre-lox methodology. Lepr knockdown mice were modestly lighter and had lower plasma insulin concentrations following an oral glucose challenge compared to controls, despite similar insulin sensitivity. We rendered male mice diabetic using streptozotocin (STZ) and found that upon prolonged leptin therapy, Lepr knockdown mice had an accelerated decrease in blood glucose compared to controls that was associated with higher plasma concentrations of leptin and leptin receptor. Mice with transcriptional blockade of Lepr (LeprloxTB/loxTB) were obese and hyperglycemic and reconstitution of Lepr in adipose tissues of LeprloxTB/loxTB mice resulted in males reaching a higher maximal body weight. Although mice with adipose tissue Lepr reconstitution had lower blood glucose levels at several ages, their plasma insulin concentrations during an oral glucose test were elevated. Thus, attenuation or restoration of Lepr in adipocytes alters the plasma insulin profile following glucose ingestion, modifies the glucose-lowering effect of prolonged leptin therapy in insulin-deficient diabetes, and may modulate weight gain.

Childhood obesity and early rapid growth increase the risk for type 2 diabetes. Such early overnutrition can be modeled in mice by reducing litter size. We investigated the effects of early overnutrition and increased dietary fat intake on β cell function in Swiss Webster mice. On a moderate-fat diet, early overnutrition accelerated weight gain and induced hyperinsulinemia in pups. Early overnutrition males exhibited higher β cell mass but reduced islet insulin content and Pdx1 expression. Males had a high diabetes incidence that was increased by early overnutrition, characterized by a progressive increase in insulin secretion as well as β cell death, indicated by histological analysis and increased circulating miR-375 levels. Females maintained normoglycemia throughout life. High-fat diet (HFD) increased diabetes incidence in males, whereas low-fat diet was completely protective. This protective effect was abolished in early overnutrition males transiently exposed to HFD in early life. Although Swiss Webster mice are not known to be diabetes-prone, the high diabetes incidence suggests an underlying genetic susceptibility that can be induced by overnutrition and increased dietary fat intake in early life. Thus, the nutritional environment in early life may impact long-term β cell function and increase diabetes risk, particularly in genetically susceptible individuals.