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.

Thyroid Hormone Levels Correlate With the Maturation of Implanted Pancreatic Endoderm Cells in Patients With Type 1 Diabetes.

BACKGROUND: Macroencapsulated pancreatic endoderm cells (PECs) can reverse diabetes in rodents and preclinical studies revealed that thyroid hormones in vitro and in vivo bias PECs to differentiate into insulin-producing cells. In an ongoing clinical trial, PECs implanted in macroencapsulation devices into patients with type 1 diabetes were safe but yielded heterogeneous outcomes. Though most patients developed meal responsive C-peptide, levels were heterogeneous and explanted grafts had variable numbers of surviving cells with variable distribution of endocrine cells. METHODS: We measured circulating triiodothyronine and thyroxine levels in all patients treated at 1 of the 7 sites of the ongoing clinical trial and determined if thyroid hormone levels were associated with the C-peptide or glucagon levels and cell fate of implanted PECs. RESULTS: Both triiodothyronine and thyroxine levels were significantly associated with the proportion of cells that adopted an insulin-producing fate with a mature phenotype. Thyroid hormone levels were inversely correlated to circulating glucagon levels after implantation, suggesting that thyroid hormones lead PECs to favor an insulin-producing fate over a glucagon-producing fate. In mice, hyperthyroidism led to more rapid maturation of PECs into insulin-producing cells similar in phenotype to PECs in euthyroid mice. CONCLUSION: These data highlight the relevance of thyroid hormones in the context of PEC therapy in patients with type 1 diabetes and suggest that a thyroid hormone adjuvant therapy may optimize cell outcomes in some PEC recipients.

Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

BACKGROUND: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). METHODS: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. RESULTS: The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. CONCLUSIONS: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD). Methods: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. Results: The speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. Conclusions: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

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.

A Century-long Journey From the Discovery of Insulin to the Implantation of Stem Cell-derived Islets.

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing ß cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing ß cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature ß-like cells in vitro that produce insulin but do not yet fully mimic healthy mature ß cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell-derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.

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.

Altered islet prohormone processing: a cause or consequence of diabetes?

Peptide hormones are first produced as larger precursor prohormones that require endoproteolytic cleavage to liberate the mature hormones. A structurally conserved but functionally distinct family of nine prohormone convertase enzymes (PCs) are responsible for cleavage of protein precursors, of which PC1/3 and PC2 are known to be exclusive to neuroendocrine cells and responsible for prohormone cleavage. Differential expression of PCs within tissues defines prohormone processing; whereas glucagon is the major product liberated from proglucagon via PC2 in pancreatic α-cells, proglucagon is preferentially processed by PC1/3 in intestinal L cells to produce glucagon-like peptides 1 and 2 (GLP-1, GLP-2). Beyond our understanding of processing of islet prohormones in healthy islets, there is convincing evidence that proinsulin, pro-islet amyloid polypeptide (proIAPP), and proglucagon processing is altered during prediabetes and diabetes. There is predictive value of elevated circulating proinsulin or proinsulin-to-C-peptide ratio for progression to type 2 diabetes, and elevated proinsulin or proinsulin-to-C-peptide ratio is predictive for development of type 1 diabetes in at-risk groups. After onset of diabetes, patients have elevated circulating proinsulin and proIAPP, and proinsulin may be an autoantigen in type 1 diabetes. Furthermore, preclinical studies reveal that α-cells have altered proglucagon processing during diabetes, leading to increased GLP-1 production. We conclude that despite strong associative data, current evidence is inconclusive on the potential causal role of impaired prohormone processing in diabetes and suggest that future work should focus on resolving the question of whether altered prohormone processing is a causal driver or merely a consequence of diabetes pathology.

Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes.

An open-label, first-in-human phase 1/2 study is being conducted to evaluate the safety and efficacy of pancreatic endoderm cells (PECs) implanted in non-immunoprotective macroencapsulation devices for the treatment of type 1 diabetes. We report an analysis on 1 year of data from the first cohort of 15 patients from a single trial site that received subcutaneous implantation of cell products combined with an immunosuppressive regimen. Implants were well tolerated with no teratoma formation or severe graft-related adverse events. After implantation, patients had increased fasting C-peptide levels and increased glucose-responsive C-peptide levels and developed mixed meal-stimulated C-peptide secretion. There were immunosuppression-related transient increases in circulating regulatory T cells, PD1high T cells, and IL17A+CD4+ T cells. Explanted grafts contained cells with a mature ß cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. These data, and associated findings (Shapiro et al., 2021), are the first reported evidence of meal-regulated insulin secretion by differentiated stem cells in patients.

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.

Pancreatic islets in bed with microvasculature-companions for life.

In Cell Stem Cell, Aghazadeh et al.1 show that human embryonic stem cell-derived pancreatic progenitors can reverse hyperglycemia for several weeks in streptozotocin-induced diabetic mice when co-transplanted with microvessel fragments into the subcutaneous space.

Process parameter development for the scaled generation of stem cell-derived pancreatic endocrine cells.

Diabetes is a debilitating disease characterized by high blood glucose levels. The global prevalence of this disease has been projected to reach 700 million adults by the year 2045. Type 1 diabetes represents about 10% of the reported cases of diabetes. Although islet transplantation can be a highly effective method to treat type 1 diabetes, its widespread application is limited by the paucity of cadaveric donor islets. The use of pluripotent stem cells as an unlimited cell source to generate insulin-producing cells for implant is a promising alternative for treating diabetes. However, to be clinically relevant, it is necessary to manufacture these stem cell-derived cells at sufficient scales. Significant advances have been made in differentiation protocols used to generate stem cell-derived cells capable of reversing diabetes in animal models and for testing in clinical trials. We discuss the potential of both stem cell-derived pancreatic progenitors and more matured insulin-producing cells to treat diabetes. We discuss the need for rigorous bioprocess parameter optimization and identify some critical process parameters and strategies that may influence the critical quality attributes of the cells with the goal of facilitating scalable manufacturing of human pluripotent stem cell-derived pancreatic endocrine cells.

I. Progress Towards a Stem Cell Based Therapy for Diabetes.

Diabetes results from insufficient production of the hormone insulin from beta cells in pancreatic islets. Islet transplantation can replace the lost beta cells in patients but is limited by the scarcity of available donor organs. Our aim is to differentiate pluripotent stem cells into functional islets that can serve as an unlimited source for transplantation to treat diabetes. We have investigated the therapeutic potential of pancreatic endoderm cells derived from human embryonic stem cells. Several weeks following transplant into diabetic rodents, these cells mature and secrete sufficient human insulin, in a regulated manner, to reverse diabetes. In rats, we observed inconsistent survival of pancreatic endoderm cells implanted subcutaneously in macroencapsulation devices designed to be immunoprotective via use of a cell impermeable layer, but this was rectified by the addition of portals designed to enable direct capillary vascular permeation into the device interior. In contrast both device types supported cell survival, differentiation and function in mice, with more rapid C-peptide release and better glucose tolerance observed using the devices containing portals. Kidney capsule grafts often contained ductal cells and cysts, whereas cells implanted subcutaneously within macroencapsulation devices differentiated predominantly to endocrine cells. In collaboration with Aspect Biosystems, we are also exploring the feasibility of using 3D bioprinting as an approach to both contain implanted islet cells and protect them from immune attack. As part of a ViaCyte clinical trial (clinicaltrial.gov identifier: NCT03163511), we investigated the safety and efficacy of pancreatic endoderm cells implanted in nonimmunoprotective macroencapsulation devices for the treatment of patients with type 1 diabetes and hypoglycemic unawareness. Patients underwent subcutaneous implant of cell products combined with an immunosuppressive regimen. After implant, patients had increased fasting Cpeptide levels, increased glucose-responsive C-peptide levels, and developed mixed meal-stimulated C-peptide secretion. Patients had reduced insulin requirements, increased time in target blood glucose range, and improved hypoglycemic awareness. Explanted grafts contained cells with a mature beta cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. Collectively, these findings support future investigation into optimizing cell therapies for diabetes.

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.

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.

Specific loss of adipocyte CD248 improves metabolic health via reduced white adipose tissue hypoxia, fibrosis and inflammation.

BACKGROUND: A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored. METHODS: The role of CD248 in adipocyte function and glucose metabolism was evaluated by omics analyses in human WAT, gene knockdowns in human in vitro differentiated adipocytes and by adipocyte-specific and inducible Cd248 gene knockout studies in mice. FINDINGS: CD248 is upregulated in white but not brown adipose tissue of obese and insulin-resistant individuals. Gene ontology analyses showed that CD248 expression associated positively with pro-inflammatory/pro-fibrotic pathways. By combining data from several human cohorts with gene knockdown experiments in human adipocytes, our results indicate that CD248 acts as a microenvironmental sensor which mediates part of the adipose tissue response to hypoxia and is specifically perturbed in white adipocytes in the obese state. Adipocyte-specific and inducible Cd248 knockouts in mice, both before and after diet-induced obesity and insulin resistance/glucose intolerance, resulted in increased microvascular density as well as attenuated hypoxia, inflammation and fibrosis without affecting fat cell volume. This was accompanied by significant improvements in insulin sensitivity and glucose tolerance. INTERPRETATION: CD248 exerts detrimental effects on WAT phenotype and systemic glucose homeostasis which may be reversed by suppression of adipocyte CD248. Therefore, CD248 may constitute a target to treat obesity-associated co-morbidities.

Navigating Two Roads to Glucose Normalization in Diabetes: Automated Insulin Delivery Devices and Cell Therapy.

Incredible strides have been made since the discovery of insulin almost 100 years ago. Insulin formulations have improved dramatically, glucose levels can be measured continuously, and recently first-generation biomechanical "artificial pancreas" systems have been approved by regulators around the globe. However, still only a small fraction of patients with diabetes achieve glycemic goals. Replacement of insulin-producing cells via transplantation shows significant promise, but is limited in application due to supply constraints (cadaver-based) and the need for chronic immunosuppression. Over the past decade, significant progress has been made to address these barriers to widespread implementation of a cell therapy. Can glucose levels in people with diabetes be normalized with artificial pancreas systems or via cell replacement approaches? Here we review the road ahead, including the challenges and opportunities of both approaches.

Vegfa/vegfr2 signaling is necessary for zebrafish islet vessel development, but is dispensable for beta-cell and alpha-cell formation.

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.

Glucose-dependent insulinotropic polypeptide deficiency reduced fat accumulation and insulin resistance, but deteriorated bone loss in ovariectomized mice.

Given the established roles of glucose-dependent insulinotropic polypeptide (GIP) in promoting fat storage and bone formation, we assessed the contribution of GIP to obesity and osteopenia in ovariectomized mice with a gene encoding green fluorescent protein (GFP) inserted into the GIP locus, in which GIP was either reduced (GIPgfp/+ ) or absent (GIPgfp/gfp ). In GIPgfp/gfp mice, weight gain, subcutaneous and visceral fat mass were reduced, and glucose intolerance was improved compared with wild-type mice with the same magnitude of insulin responses. Cancellous bone mineral density and bone cortical thickness were reduced in GIPgfp/gfp mice compared with wild-type mice. In GIPgfp/+ mice, weight gain, glucose intolerance and cancellous bone mineral density were not different from that of wild-type mice. These results indicate that the total elimination of GIP ameliorates weight gain and adiposity in ovariectomized mice, but it enhances osteopenia, particularly in cancellous bone by partly suppressing bone formation.

Insulin-Deficient Mouse ß-Cells Do Not Fully Mature but Can Be Remedied Through Insulin Replacement by Islet Transplantation.

Insulin receptor (IR) insufficiency in ß-cells leads to impaired insulin secretion and reduced ß-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in ß-cells in later embryological development may lead to compensatory ß-cell hyperplasia. Although these findings suggest insulin signaling on the ß-cell is important for ß-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for ß-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1-/-Ins2-/-). We immunostained neonatal Ins1-/-Ins2-/- and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing ß-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1-/-Ins2-/-ß-cells expressed progenitor factors associated with stem cells or dedifferentiated ß-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient ß-cells. Short-term insulin glargine (Lantus®) injections partially rescued the ß-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature ß-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for ß-cell maturation.