Therapy Of Insulin-dependent Diabetes Mellitus Research Articles

Simultaneous Pancreas and Kidney Transplantation: An Initial Experience in Southern India

Abstract Introduction: Simultaneous pancreas-kidney transplantation (SPKT) is a well-documented therapy for insulin-dependent diabetes mellitus (IDDM) with end-stage renal disease (ESRD). Here, we share the initial reports of SPKT conducted at our institution. Patients and Methods: We performed five SPKT procedures in patients with IDDM and ESRD between April 2021 and April 2023. All grafts were procured from healthy, in-house, brain-dead donors. Results: The recipients included three males and two females, with a mean age of 35 ± 11.2 years. The mean duration of diabetes mellitus and time on dialysis were 17 ± 9.7 years and 21 ± 8.7 months, respectively. Donors had a mean age and body mass index of 27 ± 5.8 years and 23 ± 2.7 kg/m2. The mean glycated hemoglobin (HbA1c) and creatinine levels of donors at the time of donation were 5.6% ±0.27% and 0.98 ± 0.4 mg/dL. One recipient underwent pancreas removal due to a duodenal leak, and one developed kidney acute tubular necrosis. All recipients were on proper follow-up for a median period of 12 (4–18) months, with 100% patient survival, 80% pancreas, and 100% kidney survival. Average HbA1c, fasting blood sugar, and serum creatinine on follow-up were 5.5% ±0.7%, 84 ± 9.9 mg/dL, and 1 ± 0.16 mg/dL, respectively. Conclusion: In our initial experience with SPKT, recipient outcomes were comparable to results published in the literature. With proper graft selection, SPKT can be established in small-volume centers. Meticulous surgical technique and postoperative management remain key to the success of the transplant program.

Efficacy and safety of allogenic canine adipose tissue-derived mesenchymal stem cell therapy for insulin-dependent diabetes mellitus in four dogs: A pilot study

Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and can control the immune dysregulation that leads to β-cell destruction. Stem-cell transplantation could thus manage insulin-dependent diabetes mellitus (IDDM) in dogs. In this pilot study, we aimed to assess canine adipose tissue-derived MSCs (cAT-MSCs) transplantation as a treatment for canine diabetes mellitus. This study included four dogs with over a year of insulin treatment for IDDM, following diagnosis at the Veterinary Medicine Teaching Hospital of Seoul National University. Allogenic cAT-MSCs were infused intravenously three or five times monthly to dogs with IDDM. Blood and urine samples were obtained monthly. General clinical symptoms, including changes in body weight, vitality, appetite, and water intake were assessed. Three of the four owners observed improvement of vitality after stem cell treatment. Two of the four dogs showed improvement in appetite and body weight, polyuria, and polydipsia. C-peptide has increased by about 5–15% in three of the cases, and fructosamine and HbA1c levels have improved in two of the cases. Hyperlipidemia was resolved in two of the dogs, and there was no concurrent bacterial cystitis in any of the dogs. C-peptide secretion and lipid metabolism are associated with diabetic complications. Improvement in these parameters following the treatment suggests that cAT-MSC transplantation in dogs with IDDM might help to improve their insulin secretory capacity and prevent diabetic complications.

Interim report on the effective intraperitoneal therapy of insulin-dependent diabetes mellitus in pet dogs using "Neo-Islets," aggregates of adipose stem and pancreatic islet cells (INAD 012-776).

We previously reported that allogeneic, intraperitoneally administered “Neo-Islets,” composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012–776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively.

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

Microencapsulation of Islets with Living Cells Using PolyDNA-PEG-Lipid Conjugate

Microencapsulation of islets with a semipermeable membrane, i.e., bioartificial pancreas, is a promising way to transplant islets without the need for immunosuppressive therapy for insulin-dependent diabetes mellitus (type I diabetes). However, materials composing a bioartificial pancreas are not ideal and might activate defense reactions against foreign materials. In this study, we propose an original method for microencapsulation of islets with living cells using an amphiphilic poly(ethylene glyocol)-conjugated phospholipid derivative (PEG-lipid) and DNA hybridization. PolyA and polyT were introduced onto the surfaces of the islets and HEK 293 cells, respectively, using amphiphilic PEG-lipid derivatives. PolyA20 modified HEK cells were immobilized onto the islet surface where polyT20-PEG-lipid was incorporated. The cells spread and proliferated on the islet surface, and the islet surface was completely encapsulated with a cell layer after culture. The encapsulated islets retained the ability to control insulin release in response to glucose concentration changes.

Phases I–III Clinical Trials Using Adult Stem Cells

Mesenchymal progenitor/stromal/stem cell (MSC) research has made substantial progress during the past decades. Investigators have published a wealth of basic science and preclinical data documenting the potential utility, efficacy, and safety of MSCs. The majority of this work has focused on the bone marrow- or adipose-derived MSCs. Since this body of evidence has set the stage for clinicians to advance from the bench to the bedside, Stem Cells International set out to publish a special issue devoted to the topic of Phases I–III Clinical Trials Using Adult Stem Cells. The result is a collection of ten outstanding articles submitted by investigators representing ten countries across Asia, Europe, and North America. In all cases, the methods section of each manuscript included a statement documenting that the clinical investigations were performed following institutional review board approval and/or that patient informed consent had been provided prior to the conduct of the study. Bieback et al. in Germany (Translating research into clinical scale manufacturing of mesenchymal stromal cells) and B. Philippe et al. in France (in “Culture and Use of Mesenchymal Stromal Cells in Phase I and II Clinical Trials”) have focused on the process of MSC isolation and expansion with respect to current Good Manufacturing Practices (cGMP). These authors highlight the state of the art as well as the challenges facing the development of clinical grade cells for therapeutic applications. From a regulatory perspective, the simplest application of MSC is to use them in the context of their tissue of origin. S. Akita et al. in Japan (in “Noncultured Autologous Adipose-Derived Stem Cell Therapy for Chronic Radiation Injury”) have exploited adipose-derived stem cells for soft tissue regeneration. They describe their clinical experience using autologous adipose-derived stem cells in combination with growth factors and scaffold to treat ten patients suffering from dermal radiation lesions. A series of three review papers from Belgium (“Phase 1–3 Clinical Trials Using Adult Stem Cells in Osteonecrosis and Nonunion Fractures” by J. –P. Hauzeur and V. Gangji), the USA and China (“Mesenchymal Progenitor Cells and Their Orthopedic Applications: Forging a Path towards Clinical Trials” by D. S. Shenaq et al.), and the Netherlands (“Clinical Applications of Human Mesenchymal Stromal Cells for Bone Tissue Engineering” by Chatterjea et al.) have examined the use of bone marrow-derived MSC for orthopedic applications. Each work provides a unique perspective on the published clinical trials using MSC to treat cartilage and tendon repair, critical sized defects, metabolic bone diseases, nonunion fractures, and/or osteonecrosis. From a public health perspective, MSC applications for common cardiac and central nervous system disorders could have substantial health and societal benefits. R. Sanz-Ruiz et al. in Spain (in “Phases I–III Clinical Trials Using Adult Stem Cells”) have emphasized the critical importance of randomized clinical trials to assess the utility of MSCs as they focus on the clinical evidence supporting MSC-based treatment of cardiac disease. Furthermore, they review the guidance documents on future clinical trials provided by a task force convened by the European Society for Cardiology. P. A. Walker et al. in the USA (in “Progenitor Cell Therapy for The Treatment of Central Nervous System Injury: A Review of the State of Current Clinical Trials) provide a similar perspective relating to central nervous system disorders. They evaluate the use of bone marrow-derived MSCs for ischemic stroke, spinal cord injury, and traumatic brain injury and conclude that there remains a need for further evidence to support these clinical applications. There are potential MSC applications for metabolic disorders as well. A. C. Piscaglia et al. in Italy (in “Stem Cell Therapies for Liver Disease: State of the Art and New Perspectives) have reviewed the limited number of clinical trials using hematopoietic stem cell (HSC), MSC infusion, or cytokine (G-CSF) therapy to reverse hepatic injury due to cirrhosis, drug exposure, or other causes of end stage liver disease. Likewise, A. V. Vanikar et al. from India (in “Cotransplantation of Adipose Tissue-Derived Insulin Secreting Mesenchymal Stem Cells A Novel Therapy for Insulin-Dependent Diabetes Mellitus”) describe their experience combining adipose- and bone marrow-derived MSC to treat a cohort of 11 diabetic subjects for a period of up to 23 months. They report improved hemoglobin A1C levels as well as decreased daily requirements for exogenous insulin. Uniformly, these authors highlight both the promise and the challenges faced by this emerging field of medicine. Their manuscripts identify the critical need for additional prospective, randomized controlled clinical trials evaluating all cell-based therapies, regardless of the underlying disorder. In summary, this special issue provides a snapshot of the current status of MSC-based clinical trials across the globe. Hopefully, this publication will provide a benchmark for future meta-analyses evaluating a far greater body of clinical evidence regarding the safety and efficacy of MSC therapies. Jeffrey M. Gimble Bruce A. Bunnell Louis Casteilla Jin Sup Jung Kotaro Yoshimura

Acceleration of functional maturation and differentiation of neonatal porcine islet cell monolayers shortly in vitro cocultured with microencapsulated sertoli cells.

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

Further evidence for a preventive therapy of insulin-dependent diabetes mellitus in the offspring by avoiding maternal hyperglycaemia during pregnancy.

A highly significantly decreased prevalence of insulin-dependent childhood-onset diabetes (less than 1/3 of the initial prevalence rate) could be achieved in Berlin/GDR since 1973 by improving systematically diagnostic and therapeutic measures for pregnant diabetics, particularly for non-insulin-dependent gestational diabetics. In addition, a highly significantly increased incidence rate of diagnosed, diet-treated and delivered non-insulin-dependent pregnant diabetics was found between 1979 and 1983 in Berlin/GDR, Halle and Leipzig as compared to the other districts of the GDR. Simultaneously, a highly significantly decreased prevalence rate of diabetic children (less than 1/3), who were born during this period, was found in 1983 for Berlin, Halle and Leipzig as compared to the other districts of the GDR. Finally, a highly significant inverse correlation could be demonstrated for the 15 districts of the GDR between the incidence rates of diagnosed, diet-treated and delivered non-insulin-dependent pregnant diabetics and the prevalence rates of diabetic children who were born during this period (1979-1983). In view of these findings, an interruption and even a reversal of the continued dramatic increase of idiopathic insulin-dependent diabetes mellitus appears to be possible in the developed countries by preventing maternal hyperglycaemia during pregnancy and hence hyperinsulinism in the foetuses and newborns.

Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus

To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy.

Islet transplant model in nonhuman primates: use of streptozotocin

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease resulting from progressive destruction of the insulin-producing pancreatic β cells. Insulin-dependent diabetes mellitus is a common disease, the second most prevalent chronic illness in children. The clinical results reported by the Edmonton group in recipients of allogeneic islet grafts, who all achieved at least temporary insulin independence, have rekindled interest in islet transplantation as a therapy for IDDM. However, for islet transplantation to become the therapy of choice, it is essential to (1) overcome the shortage of donor tissue; (2) develop islet-friendly immunosuppressive protocols; (3) develop donor-specific tolerance; and (4) improve the rate of long-term engraftment as to rival outcomes with whole pancreas organ transplantation. As improvements are needed, a reliable diabetic animal model to develop strategies to improve clinical outcomes is important. The nonhuman primates are well suited for this purpose, although spontaneous diabetes in large animals is rare ( J Clin Invest 1996;98:1417–22). Thus, induction of diabetes is necessary in this model. A nonhuman primate diabetic model using streptozotocin with stable insulin dependence has been achieved in several laboratories and will be discussed here.

Heat-regulated production and secretion of insulin from a human artificial chromosome vector

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-β-cells as a host for the vector. To facilitate the production of mature insulin in non-β-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21ΔpqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-β-cells, which might provide a direction for future treatment for diabetes.

Hepatic expression of the human insulin gene reduces glucose levels in vivo in diabetic mice model

The objective of these studies was to evaluate human insulin gene expression following intraliver plasmid injection in diabetic mice as a potential approach to gene therapy for insulin-dependent diabetes mellitus. The fragment containing human proinsulin gene lacking its own promoter, was cloned into plasmids containing promoter and enhancer of cytomegalovirus or human hepatitis B virus. The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo applying streptozotocin-induced diabetic mice. We found significant reduction in glucose levels in both experimental systems, giving evidence that prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cell line in vitro and in mice following intra-liver plasmid injection. Our data demonstrate the reduction of glucose levels in vitro in 3T3 fibroblast cells and in vivo in diabetic mice after treatment with plasmids expressing proinsulin, giving evidence that those constructs may have certain usage also in human gene therapy of diabetes mellitus type 1.

Pancreas transplantation in Asan Medical Center, Korea

PANCREAS transplantation has become an accepted form of therapy for insulin-dependent diabetes mellitus (IDDM). In particular, simultaneous pancreas-kidney transplantation (SPK) has become a widely accepted treatment modality for uremic diabetic patients. From 1980 through December 2000, according to the International Pancreas Transplant Registry (IPTR), there were more than 13,000 pancreas transplantations performed worldwide. The number of pancreas transplantations has been increasing continuously over time with improvement of graft survival. However, a couple of problems still need to be solved following pancreas transplantation, namely the surgical complications and immunologic rejection. We analyzed these problem in our pancreas transplant recipients by studying patient and graft survival rates according to the chronological stages.

Genes induced by growth arrest in a pancreatic β cell line: identification by analysis of cDNA arrays

Pancreatic β cell lines are a potentially attractive source of material for cell therapy of insulin-dependent diabetes mellitus. However, induction of proliferation in post-mitotic, differentiated β cells is likely to affect the expression of multiple genes associated with cell function, resulting in dedifferentiation. We have developed a murine β cell line by conditional transformation with the SV40 T antigen oncoprotein. These cells can undergo reversible induction of proliferation and growth arrest. Here we utilized this model to identify differences in gene expression between proliferating and quiescent β cells, by analyzing known β cell genes and differentially secreted proteins, as well as by a systematic survey of a mouse cDNA array. Our findings demonstrate that growth arrest stimulates expression of the insulin gene and genes encoding components of the insulin secretory vesicles. Screening of the cDNA array revealed the activation of multiple genes following growth arrest, many of them novel genes which may be related to β cell function. Characterization of these genes is likely to contribute to our understanding of β cell function and the ability to employ β cell lines in cell therapy of diabetes.

Human proinsulin production in primary rat hepatocytes after retroviral vector gene transfer.

The development of autologous somatic cells, engineered for the synthesis and release of human insulin under physiological stimuli, would certainly represent a major breakthrough in the therapy of insulin-dependent diabetes mellitus. We generated a retroviral vector containing the human proinsulin cDNA and the gene coding for the human nerve growth factor receptor for quantitative analysis of transduced cells. Primary rat hepatocytes were selected as target cells because of the constitutive expression of the pancreatic beta-cell glucose transporter GLUT-2 and the glycolitic enzyme glucokinase. Appropriate conditions for culture and retroviral transduction are described. The highest transduction efficiency, evaluated as percentage of LNGFr expressing cells was obtained by repeated infection cycles (40+/-10%). Human proinsulin accumulated in the culture medium of transduced rat hepatocytes (mean+/-SD): 18.1+/-7.9 (range 8.7-36.4) ng/24h/10(6) cells. Primary rat hepatocytes can be efficiently transduced by a retroviral vector and the de novo synthesis of human proinsulin can be induced. Primary cultured hepatocytes represent an useful model to test retroviral constructs engineered for the glucose-inducible expression of insulin under the control of liver-specific promoters.

Transplantation of Allogeneic/Xenogeneic Pancreatic Islets Containing Coherent Microcapsules in Adult Pigs

WE HAVE PREVIOUSLY described a method for fabrication of alginate/polyaminoacidic CM. Thereafter, we completed assessment of either morphological properties of structural as well as ultrastructural level, or in vitro immunoselectivity, or in vivo post-transplant (Tx) functional performance in diabetic rodents of these new biomembranes for islet Tx immunoprotection in full absence of the recipient’s pharmacological immunosuppression. Because CM, unlike conventional-size microcapsules (CSM), measuring an average 600 to 800 mm in equatorial diameter, hold the unique advantage of occupying a volume which is almost coincident with that of naked islets, these new microimmunobarriers could address a number of unsolved problems with regard to encapsulated islet cell Tx in diabetic large mammalians. One of the most formidable hurdles to this approach for the therapy of insulin-dependent diabetes mellitus has consisted of inadequacy of implant sites for encapsulated islets. Because of the final excessive CSM’s Tx size, these capsules had been only implanted in the peritoneal cavity. Unfortunately, even if highly biocompatible, intraperitoneally grafted, islet-containing microcapsules had often provoked severe, possibly mass-related (60 to 80 mL/diabetic dog) inflammatory cell reaction. Dense connective tissue infiltration invariably resulted in impairment of biochemical exchange, ultimately leading to Tx failure. After testing the new CM in either in vitro functional and immunological or in vivo rodent diabetes-correction studies, we had embarked on assessment of CM biocompatibility, after multiple-site transplantation into adult pigs. Preliminarily, we have shown that empty CM, per se, indeed were biocompatible in this large-size mammal animal model, with a very low degree of sensitization being induced by repeated CM-Tx booster. In an attempt to determine whether CM, because of their minimal size, would permit access to Tx sites so far interdicted to CSM, including parenchymatous organs, with full retention of the encapsulated allogeneic/xenogeneic islet cell viability, we have transplanted porcine or canine islet containing CM into multiple Tx sites, and examined them histologically, at 30 days post-Tx, in normal, adult pigs. MATERIALS AND METHODS Animals

Cell-based therapy for insulin-dependent diabetes mellitus.

Insulin-secreting pancreatic beta-cell lines represent a promising approach for treatment of insulin-dependent diabetes mellitus. Such cell lines can provide an abundant and reproducible source of beta-cell material for transplantation. A number of highly differentiated beta-cell lines have been developed using transgenic mice. These cells produce insulin amounts comparable to normal pancreatic islets and release it in response to physiological insulin secretagogues. Our laboratory has employed a reversible transformation approach to tightly regulate cell replication in these beta-cell lines, both in culture and in vivo. Beta-cell lines can be modulated by gene transfer to improve their function and survival. We have utilized adenovirus genes, which downregulate antigen presentation and increase cell resistance to cytokines, to facilitate transplantation of mouse beta cells across allogeneic barriers. These approaches could be applied to the development of human beta-cell lines by genetic engineering of isolated human islets.

Regulated expression of mature human insulin in the liver of transgenic mice

Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.

Genetic engineering of insulin expression in nonislet cells: implications for beta-cell replacement therapy for insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) occurs as a consequence of autoimmune destruction of the insulin-producing pancreatic beta-cells. Although progress has been made in the field of islet transplantation, an appealing alternative strategy for beta-cell replacement therapy for IDDM is to target insulin expression to non-islet cells. We have recently generated transgenic nonobese diabetic (NOD) mice in which insulin gene expression was targeted to proopiomelanocortin (POMC)-expressing cells of the intermediate lobe (IL) of the pituitary. We have shown that POMC-expressing IL pituitary cells secreted large amounts of mature insulin, similar to islet beta-cells. However, in contrast to the insulin-producing islet beta-cells, the insulin-producing IL pituitary cells were not attacked by the immune system. Remarkably, transplantation of small amounts of the transgenic IL tissues into diabetic NOD mice resulted in the restoration of near-normogylcemia and the complete reversal of diabetic symptoms. In separate experiments, IL allografts showed enhanced viability and were highly vascularized, compared with similarly transplanted islet allografts. These features are highly advantageous in the transplantation setting and demonstrate the considerable potential of these non-islet cell types for insulin-gene delivery in IDDM.

Long-Term Acting and Orally Active Vanadyl-Methylpicolinate Complex with Hypoglycemic Activity in Streptozotocin-Induced Diabetic Rats.

The therapy of insulin-dependent diabetes mellitus (IDDM) is achieved only by daily subcutaneous injections of insulin, and compounds that can replace insulin or insulin-mimetics for oral administration need to be developed. Vanadate ion, vanadyl ion and their complexes have been reported to possess insulin-mimetic activities in both in vitro and in vivo experiments. On the basis of our recent preliminary finding that a bis(picolinato)oxovanadium (VO-PA) complex has a possible hypoglycemic activity when given by oral administration to streptozotocin-induced diabetic rats (STZ-rats), we synthesized several analogs of the VO-PA complex and examined the relationship between their structures and insulin-mimetic activity. Bis(methylpicolinato)oxovanadium (VO-MPA) complex, which has a relatively high partition coefficient among the prepared complexes, was found to be effective to inhibit in vitro release of free fatty acid from isolated rat adipocytes, similar to VO-PA. VO-MPA complex was thus given to STZ-rats by intraperitoneal injection or oral administration, and was found to normalize the serum glucose levels without the body weight loss. Especially, on oral administration of the complex, the normal serum glucose level was maintained for 80 days after the cessation of the complex administration. The long-acting character of the complex was suggested by the fact that vanadium is incorporated in bone as well as in kidney or other organs. Based on these observations, VO-MPA was proposed to be an useful agent not only to treat IDDM in experimental animals but to analyze the mechanism for the insulin mimetic activity of vanadium compounds.