Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells.

BACKGROUND: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield. METHODS: We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis). RESULTS: Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose. CONCLUSION: This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture.

Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures. Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells. Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: -0.01-0.022 h-1) than cells growing as a monolayer (range: 0.039-0.045 h-1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260 mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system.

Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

The generation of functional ß-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 Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells In Vivo.

Few studies have examined the differentiation of human embryonic stem cell (hESC)-derived pancreatic endoderm cells (PECs) in different implantation sites. Here, we investigate the influence of implantation site and recipient sex on the differentiation of hESC-derived PECs in vivo. Male and female mice were implanted with 5 × 106 hESC-derived PECs under the kidney capsule, in the gonadal fat pad, or subcutaneously within macroencapsulation (TheraCyte) devices. PECs implanted within TheraCyte devices developed glucose-stimulated human C-peptide secretion faster than cells implanted under the kidney capsule or in the gonadal fat pad. Interestingly, hESC-derived PECs implanted under the kidney capsule in females developed glucose-stimulated human C-peptide faster than in males and secreted higher levels of arginine-stimulated glucagon and glucagon-like peptide 1 than other implantation sites. Furthermore, hESC-derived grafts collected from the kidney capsule and gonadal fat pad sites displayed a mix of endocrine and ductal cells as well as contained cysts, whereas TheraCyte device grafts displayed mostly endocrine cells and cysts were not observed. Here we demonstrate that the macroencapsulated subcutaneous site and the female recipient can promote faster differentiation of hESC-derived PECs to endocrine cells in mice. ARTICLE HIGHLIGHTS: Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors in vivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

Insulin Null ß-cells Have a Prohormone Processing Defect That Is Not Reversed by AAV Rescue of Proinsulin Expression.

Up to 6% of diabetes has a monogenic cause including mutations in the insulin gene, and patients are candidates for a gene therapy. Using a mouse model of permanent neonatal diabetes, we assessed the efficacy of an adeno-associated virus (AAV)-mediated gene therapy. We used AAVs with a rat insulin 1 promoter (Ins1) regulating a human insulin gene (INS; AAV Ins1-INS) or native mouse insulin 1 (Ins1; AAV Ins-Ins1) to deliver an insulin gene to ß-cells of constitutive insulin null mice (Ins1-/-Ins2-/-) and adult inducible insulin-deficient mice [Ins1-/-Ins2f/f PdxCreER and Ins1-/-Ins2f/f mice administered AAV Ins1-Cre)]. Although AAV Ins1-INS could successfully infect and confer insulin expression to ß-cells, insulin null ß-cells had a prohormone processing defect. Secretion of abundant proinsulin transiently reversed diabetes. We reattempted therapy with AAV Ins1-Ins1, but Ins1-/-Ins2-/- ß-cells still had a processing defect of both replaced Ins1 and pro-islet amyloid polypeptide (proIAPP). In adult inducible models, ß-cells that lost insulin expression developed a processing defect that resulted in impaired proIAPP processing and elevated circulating proIAPP, and cells infected with AAV Ins1-Ins1 to rescue insulin expression secreted proinsulin. We assessed the subcellular localization of prohormone convertase 1/3 (PC1/3) and detected defective sorting of PC1/3 to glycogen-containing vacuoles and retention in the endoplasmic reticulum as a potential mechanism underlying defective processing. We provide evidence that persistent production of endogenous proinsulin within ß-cells is necessary for ß-cells to be able to properly store and process proinsulin.

Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice.

miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, ß-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of ß-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-ß signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how ß-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.

Role of myeloid cell leptin signaling in the regulation of glucose metabolism.

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.

AAV8 Ins1-Cre can produce efficient ß-cell recombination but requires consideration of off-target effects.

In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on ß-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 ß-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 ß-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 ß-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 ß-cells provides an effective alternative to transgenic approaches for inducible knockout studies.

SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes.

SNAP23 is the ubiquitous SNAP25 isoform that mediates secretion in non-neuronal cells, similar to SNAP25 in neurons. However, some secretory cells like pancreatic islet ß cells contain an abundance of both SNAP25 and SNAP23, where SNAP23 is believed to play a redundant role to SNAP25. We show that SNAP23, when depleted in mouse ß cells in vivo and human ß cells (normal and type 2 diabetes [T2D] patients) in vitro, paradoxically increased biphasic glucose-stimulated insulin secretion corresponding to increased exocytosis of predocked and newcomer insulin granules. Such effects on T2D Goto-Kakizaki rats improved glucose homeostasis that was superior to conventional treatment with sulfonylurea glybenclamide. SNAP23, although fusion competent in slower secretory cells, in the context of ß cells acts as a weak partial fusion agonist or inhibitory SNARE. Here, SNAP23 depletion promotes SNAP25 to bind calcium channels more quickly and longer where granule fusion occurs to increase exocytosis efficiency. ß Cell SNAP23 antagonism is a strategy to treat diabetes.

AAV GCG-EGFP, a new tool to identify glucagon-secreting α-cells.

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.

Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues.

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.

Leptin contributes to the beneficial effects of insulin treatment in streptozotocin-diabetic male mice.

It was long thought that the only hormone capable of reversing the catabolic consequences of diabetes was insulin. However, various studies have demonstrated that the adipocyte-derived hormone leptin can robustly lower blood glucose levels in rodent models of insulin-deficient diabetes. In addition, it has been suggested that some of the metabolic manifestations of insulin-deficient diabetes are due to hypoleptinemia as opposed to hypoinsulinemia. Because insulin therapy increases leptin levels, we sought to investigate the contribution of leptin to the beneficial effects of insulin therapy. To do this, we tested insulin therapy in streptozotocin (STZ) diabetic mice that were either on an ob/ ob background or that were given a leptin antagonist to determine if blocking leptin action would blunt the glucose-lowering effects of insulin therapy. We found that STZ diabetic ob/ ob mice have a diminished blood glucose-lowering effect in response to insulin therapy compared with STZ diabetic controls and exhibited more severe weight loss post-STZ injection. In addition, STZ diabetic mice administered a leptin antagonist through daily injection or plasmid expression respond less robustly to insulin therapy as assessed by both fasting blood glucose levels and blood glucose levels during an oral glucose tolerance test. However, leptin antagonism did not prevent the insulin-induced reduction in ß-hydroxybutyrate and triglyceride levels. Therefore, we conclude that elevated leptin levels can contribute to the glucose-lowering effect of insulin therapy in insulin-deficient diabetes.

The Role of ARX in Human Pancreatic Endocrine Specification.

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

FGF21-Mediated Improvements in Glucose Clearance Require Uncoupling Protein 1.

Fibroblast growth factor 21 (FGF21)-mediated weight loss and improvements in glucose metabolism correlate with increased uncoupling protein 1 (Ucp1) levels in adipose tissues, suggesting that UCP1-dependent thermogenesis may drive FGF21 action. It was reported that FGF21 is equally effective at reducing body weight and improving glucose homeostasis without UCP1. We find while FGF21 can lower body weight in both wild-type and Ucp1 knockout mice, rapid clearance of glucose by FGF21 is defective in the absence of UCP1. Furthermore, in obese wild-type mice there is a fall in brown adipose tissue (BAT) temperature during glucose excursion, and FGF21 improves glucose clearance while preventing the fall in BAT temperature. In Ucp1 knockout mice, the fall in BAT temperature during glucose excursion and FGF21-mediated changes in BAT temperature are lost. We conclude FGF21-mediated improvements in clearance of a glucose challenge require UCP1 and evoke UCP1-dependent thermogenesis as a method to increase glucose disposal.

Overexpression of PAX4 reduces glucagon expression in differentiating hESCs.

Human embryonic stem cells (hESCs) are pluripotent and capable of generating new ß-cells, but current in vitro differentiation protocols generally fail to produce mature, glucose-responsive, unihormonal ß-cells. Instead, these methods tend to produce immature polyhormonal endocrine cells which mature in vivo into glucagon-positive α-cells. PAX4 is an established transcription factor in ß-cell development and function, and is capable of converting glucagon-positive cells to insulin-positive cells in mice. Work in human and mouse ESCs has shown that constitutive PAX4 expression promotes the development of insulin-positive cells, but whether acute PAX4 expression is sufficient to guide specific endocrine cell fates has not been addressed in hESCs. In this study, we applied recombinant adenovirus to ectopically express human PAX4 in hESC-derived pancreatic progenitors, with the aim of influencing the endocrine developmental cascade away from polyhormonal cells toward unihormonal insulin-positive cells. Gene delivery to pancreatic progenitors was efficient and dose-dependent. By the end of in vitro differentiation, PAX4 reduced ARX expression, but only the high dose tested significantly reduced glucagon release. Single cell analysis revealed that while PAX4 did not alter the proportion of endocrine cells, it did reduce the number of glucagon-positive cells and increased the number of unihormonal insulin-positive cells. These data suggest that acute PAX4 overexpression can reduce expression of ARX and glucagon resulting in improved numbers of unihormonal insulin-positive cells.

Overexpression of PPARγ specifically in pancreatic ß-cells exacerbates obesity-induced glucose intolerance, reduces ß-cell mass, and alters islet lipid metabolism in male mice.

The contribution of peroxisomal proliferator-activated receptor (PPAR)-γ agonism in pancreatic ß-cells to the antidiabetic actions of thiazolidinediones has not been clearly elucidated. Genetic models of pancreatic ß-cell PPARγ ablation have revealed a potential role for PPARγ in ß-cell expansion in obesity but a limited role in normal ß-cell physiology. Here we overexpressed PPARγ1 or PPARγ2 specifically in pancreatic ß-cells of mice subjected to high-fat feeding using an associated adenovirus (ß-PPARγ1-HFD and ß-PPARγ2-HFD mice). We show ß-cell-specific PPARγ1 or PPARγ2 overexpression in diet-induced obese mice exacerbated obesity-induced glucose intolerance with decreased ß-cell mass, increased islet cell apoptosis, and decreased plasma insulin compared with obese control mice (ß-eGFP-HFD mice). Analysis of islet lipid composition in ß-PPARγ2-HFD mice revealed no significant changes in islet triglyceride content and an increase in only one of eight ceramide species measured. Interestingly ß-PPARγ2-HFD islets had significantly lower levels of lysophosphatidylcholines, lipid species shown to enhance insulin secretion in ß-cells. Gene expression profiling revealed increased expression of uncoupling protein 2 and genes involved in fatty acid transport and ß-oxidation. In summary, transgenic overexpression of PPARγ in ß-cells in diet-induced obesity negatively impacts whole-animal carbohydrate metabolism associated with altered islet lipid content, increased expression of ß-oxidative genes, and reduced ß-cell mass.

IGFBP2 is neither sufficient nor necessary for the physiological actions of leptin on glucose homeostasis in male ob/ob mice.

The ability of leptin to improve metabolic abnormalities in models of leptin deficiency, lipodystrophy, and even type 1 diabetes is of significant interest. However, the mechanism by which leptin mediates these effects remains ill-defined. Leptin was recently reported to regulate insulin-like growth factor-binding protein-2 (IGFBP2), and adenoviral overexpression of pharmacological levels of IGFBP2 ameliorates diabetic symptoms in many models of diabetes. We sought to determine the role of physiological levels of IGFBP2 in the glucoregulatory action of leptin. To investigate whether physiological levels of IGFBP2 are sufficient to mimic the action of leptin, we treated male ob/ob mice with low-dose IGFBP2 adenovirus (Ad-IGFBP2) or low-dose leptin. Despite similar levels of circulating IGFBP2, leptin but not Ad-IGFBP2 lowered body weight and plasma insulin and improved glucose and insulin tolerance. To elucidate the role of IGFBP2 in normal glucose homeostasis, we knocked down IGFBP2 in male C57BL/6 mice using small interfering RNA to determine whether this would recapitulate any aspect of the ob/ob phenotype. Despite successful IGFBP2 knockdown, body weight, blood glucose, and plasma insulin were unchanged. Finally, to determine whether IGFBP2 is required for the glucoregulatory actions of leptin, we prevented leptin-mediated increases in IGFBP2 in male ob/ob mice using RNA interference. Even though increases in IGFBP2 were blocked, the ability of leptin to decrease body weight, blood glucose, and plasma insulin levels were unaltered. In conclusion, physiological levels of IGFBP2 are neither sufficient to mimic nor required for the physiological action of leptin.

Overexpression of peroxisome proliferator-activated receptor α in pancreatic ß-cells improves glucose tolerance in diet-induced obese mice.

Lipotoxicity is implicated in pancreatic ß-cell dysfunction in obesity-induced type 2 diabetes. In vitro, activation of peroxisome proliferator-activated receptor α (PPARα) has been shown to protect pancreatic ß-cells from the lipotoxic effects of palmitate, thereby preserving insulin secretion. Utilizing an adeno-associated virus (dsAAV8), overexpression of PPARα was induced specifically in pancreatic ß-cells of adult, C57Bl/6 mice fed a high-fat diet for 20 weeks and carbohydrate metabolism and ß-cell mass assessed. We show that overexpression of PPARα in pancreatic ß-cells in vivo preserves ß-cell function in obesity, and this improves glucose tolerance by preserving insulin secretion in comparison to control mice with diet-induced obesity. No changes in ß-cell mass were observed in PPARα-overexpressing mice compared with diet-induced obese control animals. This model of ß-cell-specific PPARα overexpression provides a useful in vivo model for elucidating the mechanisms underlying ß-cell lipotoxicity in obesity-induced type 2 diabetes.