Daniel Fraga

Osteocalcin effect on human β-cells mass and function

The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes.

Characterization of a nanogland for the autotransplantation of human pancreatic islets

Despite the clinical success of pancreatic islet transplantation, graft function is frequently lost over time due to islet dispersion, lack of neovascularization, and loss of physiological architecture. To address these problems, islet encapsulation strategies including scaffolds and devices have been developed, which produced encouraging results in preclinical models. However, islet loss from such architectures could represent a significant limitation to clinical use. Here, we developed and characterized a novel islet encapsulation silicon device, the NanoGland, to overcome islet loss, while providing a physiological-like environment for long-term islet viability and revascularization. NanoGlands, microfabricated with a channel size ranging from 3.6 nm to 60 μm, were mathematically modeled to predict the kinetics of the response of encapsulated islets to glucose stimuli, based on different channel sizes, and to rationally select membranes for further testing. The model was validated in vitro using static and perifusion testing, during which insulin secretion and functionality were demonstrated for over 30-days. In vitro testing also showed 70-83% enhanced islet retention as compared to porous scaffolds, here simulated through a 200 μm channel membrane. Finally, evidence of in vivo viability of human islets subcutaneously transplanted within NanoGlands was shown in mice for over 120 days. In this context, mouse endothelial cell infiltration suggesting neovascularization from the host were identified in the retrieved grafts. The NanoGland represents a novel, promising approach for the autotransplantation of human islets.

The effect of isolation methods and the use of different enzymes on islet yield and in vivo function

The ability to isolate high-yield pure and viable islets from human cadaver pancreas donors is dependent on donor factor as well as isolation factors. The aim of this study was to examine factors influencing islets recovery and in vivo function with an emphasis on donor and isolation methods as well as to compare the effectiveness of Liberase, widely used in clinical islet isolation, with Serva for the isolation of pure functional islets. The results of 123 islet isolations using Liberase for digestion were compared with those of 113 isolations with Serva. Islet equivalents per gram of tissue were similar between Liberase and Serva (3620 ± 1858 vs. 4132 ± 2104, p < 0.2) as well as the percent purity (75 ± 16 vs. 74 ± 15, p < 0.9). In vivo function of islets from 71 isolations (Liberase = 45, Serva = 26) were further tested by transplantation into NOD-SCID mice following short-term culture (<6 days, n = 71). Our data show that both Liberase- and Serva-isolated islets showed similar function results following short-term culture. These data demonstrate that there is no difference in islet yield, purity, and function between the two enzymes. However, when these 71 isolations were analyzed for in vivo function with emphasis on donor factors, cold ischemia time (12.0 ± 5.3 vs. 15.0 ± 5.7, p < 0.04), islet integrity (1.6 ± 0.7 vs. 1.3 ± 0.5, p < 0.05), and female gender were the only factors that correlated with in vivo function. We also compared the mechanical-shaking method for islets isolation with hand-shaking methods. Our results show that although there is no different in islet yield, purity, and integrity between different enzymes using the same method, hand-shaking method yields more islets with better integrity than mechanical-shaking method.

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow–derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates’ survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland—islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy.

Osteocalcin protects against nonalcoholic steatohepatitis in a mouse model of metabolic syndrome.

Nonalcoholic fatty liver disease, particularly its more aggressive form, nonalcoholic steatohepatitis (NASH), is associated with hepatic insulin resistance. Osteocalcin, a protein secreted by osteoblast cells in bone, has recently emerged as an important metabolic regulator with insulin-sensitizing properties. In humans, osteocalcin levels are inversely associated with liver disease. We thus hypothesized that osteocalcin may attenuate NASH and examined the effects of osteocalcin treatment in middle-aged (12-mo-old) male Ldlr(-/-) mice, which were fed a Western-style high-fat, high-cholesterol diet for 12 weeks to induce metabolic syndrome and NASH. Mice were treated with osteocalcin (4.5 ng/h) or vehicle for the diet duration. Osteocalcin treatment not only protected against Western-style high-fat, high-cholesterol diet-induced insulin resistance but substantially reduced multiple NASH components, including steatosis, ballooning degeneration, and fibrosis, with an overall reduction in nonalcoholic fatty liver disease activity scores. Further, osteocalcin robustly reduced expression of proinflammatory and profibrotic genes (Cd68, Mcp1, Spp1, and Col1a2) in liver and suppressed inflammatory gene expression in white adipose tissue. In conclusion, these results suggest osteocalcin inhibits NASH development by targeting inflammatory and fibrotic processes.

Induction of IAPP amyloid deposition and associated diabetic abnormalities by a prion-like mechanism

Although a large proportion of patients with type 2 diabetes (T2D) accumulate misfolded aggregates composed of the islet amyloid polypeptide (IAPP), its role in the disease is unknown. Here, we show that pancreatic IAPP aggregates can promote the misfolding and aggregation of endogenous IAPP in islet cultures obtained from transgenic mouse or healthy human pancreas. Islet homogenates immunodepleted with anti-IAPP–specific antibodies were not able to induce IAPP aggregation. Importantly, intraperitoneal inoculation of pancreatic homogenates containing IAPP aggregates into transgenic mice expressing human IAPP dramatically accelerates IAPP amyloid deposition, which was accompanied by clinical abnormalities typical of T2D, including hyperglycemia, impaired glucose tolerance, and a substantial reduction on &bgr; cell number and mass. Finally, induction of IAPP deposition and diabetic abnormalities were also induced in vivo by administration of IAPP aggregates prepared in vitro using pure, synthetic IAPP. Our findings suggest that some of the pathologic and clinical alterations of T2D might be transmissible through a similar mechanism by which prions propagate in prion diseases.

3D Printed Vascularized Device for Subcutaneous Transplantation of Human Islets

Transplantation of pancreatic islets or stem cell derived insulin secreting cells is an attractive treatment strategy for diabetes. However, islet transplantation is associated with several challenges including function-loss associated with dispersion and limited vascularization as well as the need for continuous immunosuppression. To overcome these limitations, here we present a novel 3D printed and functionalized encapsulation system for subcutaneous engraftment of islets or islet like cells. The devices were 3D printed with polylactic acid and the surfaces treated and patterned to increase the hydrophilicity, cell attachment, and proliferation. Surface treated encapsulation systems were implanted with growth factor enriched platelet gel, which helped to create a vascularized environment before loading human islets. The device protected the encapsulated islets from acute hypoxia and kept them functional. The adaptability of the encapsulation system was demonstrated by refilling some of the experimental groups transcutaneously with additional islets.

Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome

Background/Objectives:Our objective was to assess the sustained, low-dose and constant administration of the thyroid receptor-β (TRβ)-selective agonist GC-1 (sobetirome) from a novel nanochannel membrane device (NMD) for drug delivery. As it known to speed up metabolism, accomplish weight loss, improve cholesterol levels and possess anti-diabetic effects, GC-1 was steadily administered by our NMD, consisting of an implantable nanochannel membrane, as an alternative to conventional daily administration, which is subject to compliance issues in clinical settings.Subjects/Methods:Diet-induced obese C57BL/J6 male mice were fed a very high-fat diet (VHFD) and received NMD implants subcutaneously. Ten mice per group received capsules containing GC-1 or phosphate-buffered saline (control). Weight, lean and fat mass, as well as cholesterol, triglycerides, insulin and glucose, were monitored for 24 days. After treatment, plasma levels of thyroid-stimulating hormone (TSH) and thyroxine were compared. mRNA levels of a panel of thermogenic markers were examined using real-time PCR in white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue, liver and local inflammatory response to the implant were examined histologically. Pancreatic islet number and β-cell area were assessed.Results:GC-1 released from the NMD reversed VHFD-induced obesity and normalized serum cholesterol and glycemia. Significant reductions in body weight and fat mass were observed within 10 days, whereas reductions in serum cholesterol and glucose levels were seen within 7 days. The significant decrease in TSH was consistent with TRβ selectivity for GC-1. Levels of transcript for Ucp1 and thermogenic genes PGC1a, Cidea, Dio2 and Cox5a showed significant upregulation in WAT in NMD–GC-1-treated mice, but decreased in BAT. Although mice treated by NMD–GC-1 showed a similar number of pancreatic islets, they exhibited significant increase in β-cell area.Conclusions:Our data demonstrate that the NMD implant achieves steady administration of GC-1, offering an effective and tightly controlled molecular delivery system for treatment of obesity and metabolic disease, thereby addressing compliance.

Serum undercarboxylated osteocalcin correlates with hemoglobin A1c in children with recently diagnosed pediatric diabetes

Osteocalcin (OC), a hormone secreted by osteoblasts, improves beta‐cell function in vitro and in vivo. We aimed to understand the relationship between OC and hemoglobin A1c (HbA1c) in pediatric diabetes.

Developing a Stable Diabetic Model for Human Islet Assessment in the NOD-scid Mouse

Islet transplantation as a treatment for diabetes offers several potential advantages. These include normalization of glycemic control, a relatively non-traumatic transplant procedure and the potential for pretransplant procedures to eliminate the need for permanent immunosuppression (Ricordi et al. Diabetes 37:43, 1988). When successful, islet transplantation can keep patients insulin independent and normoglycemic for many years (Carroll et al. Transplantation 59:875–879, 1995; Kendall et al. Diabetes 45:257A, 1995; Robertson et al. Diabetes 50:47–50, 2001; Shapiro et al. Am J Transplant 3:296, 2003). However, islet grafts often suffer from immunological rejection and/or graft primary non-function (Bretzel et al. Exp Clin Endocrinol Diab 103:143–159, 1995). The non-function occurs immediately and is not affected by immunosuppressive drugs. Indeed, patients receiving islet autotransplants often experience reduced islet function below a level that allows them to remain insulin independent (Fontana et al. Transplant Proc 26:581, 1994), indicating that this problem is distinct from immunological rejection mediated by adaptive immunity. Primary non-function from nonspecific inflammatory response appears to be a problem that has impaired the success of islet transplantation in humans (Stevens et al. Transplant Proc 26:692, 1994).