Pharmacological induction of pancreatic islet cell transdifferentiation: relevance to type I diabetes

Type I diabetes (T1D) is an autoimmune disease in which an immune response to pancreatic β-cells results in their loss over time. Although the conventional view is that this loss is due to autoimmune destruction, we present evidence of an additional phenomenon in which autoimmunity promotes islet endocrine cell transdifferentiation. The end result is a large excess of δ-cells, resulting from α- to β- to δ-cell transdifferentiation. Intermediates in the process of transdifferentiation were present in murine and human T1D. Here, we report that the peptide caerulein was sufficient in the context of severe β-cell deficiency to induce efficient induction of α- to β- to δ-cell transdifferentiation in a manner very similar to what occurred in T1D. This was demonstrated by genetic lineage tracing and time course analysis. Islet transdifferentiation proceeded in an islet autonomous manner, indicating the existence of a sensing mechanism that controls the transdifferentiation process within each islet. The finding of evidence for islet cell transdifferentiation in rodent and human T1D and its induction by a single peptide in a model of T1D has important implications for the development of β-cell regeneration therapies for diabetes.The response of a tissue to stress/injury can involve cell death and proliferation. However, it has become increasingly recognized that changes in cellular differentiation state can have an important role.¹ In type I diabetes (T1D), the established view has been that the primary pathophysiological event is β-cell apoptosis due to a β-cell specific autoimmune response,² leading to profound β-cell deficiency. Thus, there has been great interest in inducing β-cell neogenesis, but there has been controversy over how and even whether β-cell regeneration occurs.³Activation of dedicated stem/progenitor cells within the pancreas and transdifferentiation of other differentiated cell types to β-cells are two potential mechanisms. In the past, the prevailing paradigm has been that neogenesis proceeds by the activation of facultative β-cell stem/progenitors within pancreatic ducts.^{4, 5, 6, 7} However, more recent studies have not found evidence of robust β-cell neogenesis from ducts.^{8, 9, 10, 11} β-cell neogenesis from other cell types within the pancreas, including acinar¹² and centroacinar¹³ cells has also been reported.Recently, we demonstrated robust β-cell neogenesis by transdifferentiation from preexisting α-cells in a model of T1D where severe β-cell deficiency was induced by high-dose alloxan.^{14, 15} In this model, β-cell neogenesis from α-cells was stimulated by pancreatic duct ligation (PDL).^{9, 15} Surgical reversal of PDL led to the recovery of β-cell mass and function by a combination of β-cell replication and β-cell neogenesis, demonstrating that β-cell regeneration by α- to β-cell neogenesis could be a robust approach to diabetes therapy,¹⁶ but PDL, which involves major surgery, is not a practical approach to therapy. Importantly, the relevance of α- to β-cell transdifferentiation to human biology remained unclear, as previous studies were performed in rodents.Here, we report the occurrence of efficient islet cell transdifferentiation using an entirely pharmacologically based approach where the peptide caerulein,^{17, 18} substituting for PDL, stimulated β-cell transdifferentiation from α-cells in mice rendered severely β-cell deficient by alloxan. Following caerulein plus alloxan, many of the neogenic β-cells went on to form δ-cells. In murine and human T1D, a similar process appeared to occur, where α-cells transdifferentiated into β-cells, which went on to form δ-cells. This led to a marked δ-cell excess in both murine and human T1D.The finding of endocrine cell transdifferentiation in T1D supports a new paradigm where β-cells, in addition to undergoing destruction by inflammatory mediators, undergo a dynamic process of neogenesis from α-cells and transdifferentiation to δ-cells. Controlling the neogenic process could lead to a new approach to diabetes therapy.     

Cellular hypersensitivity to human pancreatic B-cell clone in diabetes mellitus and its relationship to the presence of islet cell antibodies

A leukocyte migration inhibition test on the human pancreatic B-cell clone (JHPI-1) was performed in 13 IDDM patients with islet cell cytoplasmic antibody (ICCA) and/or islet cell surface antibody (ICSA), 15 IDDM patients without ICCA or ICSA, 34 NIDDM patients and 17 healthy controls. The mean values for the migration index (M.I. %) in each group were 85.4 +/- 6.9, 89.1 +/- 10.9, 98.3 +/- 7.9 and 100.0 +/- 8.5. The M.I. values were significantly decreased in IDDM patients than in NIDDM patients and controls irrespective of whether or not there were islet cell antibodies in the patients' sera. When M.I. values less than 0.83 (Mean-2 S.D.) were taken as indicative of inhibition, the percentage of IDDM and NIDDM patients with migration inhibition were 32% and 0% respectively. And the decreased M.I. values in IDDM patients proved not to be due to non-specific migration inhibition by normal M.I. values, with the human fetal lung fibroblast cells (W 138) as antigen. Our data suggested that the lymphocytes of IDDM patients might be sensitized by pancreatic B-cell antigen(s) present in the JHPI-1 cells, which promoted leukocyte migration inhibition. No correlation between the migration indices and duration of diabetes mellitus in IDDM patients was observed (r = 0.254, Y = 84.9 + 0.49 X). LMT to JHPI-1 seems to be useful in detecting the abnormal cell-mediated immunity even in patients with longstanding IDDM.     

Role of islet amyloid in type 2 diabetes mellitus

Diabetes mellitus is one of the most common metabolic diseases worldwide and its prevalence is rapidly increasing. Due to its chronic nature (diabetes mellitus can be treated but as yet not cured) and its serious complications, it is one of the most expensive diseases with regard to total health care costs per patient. The elevated blood glucose levels in diabetes mellitus are caused by a defect in production and/or secretion of the polypeptide hormone insulin, which normally promotes glucose-uptake in cells. Insulin is produced by the pancreatic 'beta-cells' in the 'islets of Langerhans', which lie distributed within the exocrine pancreatic tissue. In type 2 diabetes mellitus, the initial defect in the pathogenesis of the disease in most of the patients is believed to be 'insulin resistance'. Hyperglycemia (clinically overt diabetes mellitus) will not develop as long as the body is able to produce enough insulin to compensate for the reduced insulin action. When this compensation fails ('beta-cell failure') blood glucose levels will become too high. In this review, we discuss one of the mechanisms that have been implicated in the development of beta-cell failure, i.e. amyloid formation in the pancreatic islets. This islet amyloid is a characteristic histopathological feature of type 2 diabetes mellitus and both in vitro and in vivo studies have revealed that its formation causes death of islet beta-cells. Being a common pathogenic factor in an otherwise heterogeneous disease, islet amyloidosis is an attractive novel target for therapeutic intervention in type 2 diabetes mellitus.     

Antibodies specific for the pancreatic islet amyloid polypeptide associated with type 2 diabetes mellitus

Antibodies raised to a lysine solubilized peptide composed of residues 20-29 of the pancreatic islet amyloid polypeptide react selectively and specifically with this polypeptide and with islet amyloid deposits in Type 2 diabetes mellitus. These antibodies may prove useful in studies employing radioimmunoassay of body fluids and islet cell cultures in order to define if a pathogenic relationship exists between the islet amyloid polypeptide and Type 2 diabetes mellitus.     

Intrasplenic xenotransplantation of human fetal pancreatic islet cell cultures in rats with experimental diabetes mellitus

Islet cell cultures obtained from the pancreas of human embryos were transplanted to the spleen pulp of rats with alloxan diabetes mellitus. During 1-2 weeks after transplantation, 6 of the 8 recipients manifested a decrease in glycemia to normal or almost normal. The antidiabetic effect of xenotransplantation of islet cell cultures was well preserved throughout the entire observation period (up to 4 months). Two recipient rats with stable normoglycemia were subjected to splenectomy. One week after operation the animals manifested the recurrent grave diabetic status. Histological study of the removed spleen has shown the red pulp to contain the accumulations of implanted islet cells.     

Increased frequency of islet cell antibodies in unaffected brothers of children with type 1 diabetes

AIM: To assess the relation between islet cell antibody (ICA) positivity and demographic characteristics in an extensive series of first-degree relatives of children with type 1 diabetes (T1D). METHODS: Family members of children diagnosed with T1D before the age of 16 years and attending one of 27 participating paediatric units in Finland taking care of children with diabetes were invited to volunteer for an ICA screening program aimed at identifying individuals eligible for inclusion in the European Nicotinamide Diabetes Intervention Trial (ENDIT). The final series comprised 2,522 first-degree relatives (1,107 males) with a mean age of 20.4 (range 0.1-51.9) years, out of whom 390 were fathers, 622 mothers, 717 brothers, and 793 sisters of affected cases. RESULTS: Two hundred and four family members (8.1%) tested positive for ICA with levels ranging from 3 to 564 (median 18) Juvenile Diabetes Foundation (JDF) units. One hundred and five relatives (4.2%) had an ICA level of 18 JDF units or more. Males had detectable ICA more often than females (9.6 vs. 6.9%; p = 0.02). Antibody-positive family members under the age of 20 years had higher ICA levels than the older ones [median 18 (range 3-514) JDF units vs. 10 (range 3-564) JDF units; p = 0.008]. Among the adult relatives (>or=20 years of age) antibody-positive females had higher ICA levels than the males [median 10 (range 5-564) JDF units vs. 9 (range 3-130) JDF units; p = 0.04]. Siblings had an increased frequency of high-titre ICA (>or=18 JDF units) when compared to the parents (4.8 vs. 3.2%; p = 0.05). Among siblings, we found a higher frequency of ICA positivity in brothers than in sisters (10.8 vs. 6.9%; p = 0.01), and this was also true for high-titre ICA (6.0 vs. 3.8 %; p = 0.04). Geographically, the highest ICA prevalence was seen among relatives living in the middle of Finland (10.4 vs. 7.2% in the other parts of Finland; p = 0.01). CONCLUSIONS: These results imply that male gender and young age favour positive ICA reactivity among family members of children with T1D. Siblings test positive for high ICA titres (>or=18 JDF units) more frequently than parents. Accordingly, judged from demographic characteristics, the yield of ICA screening in first-degree relatives would be maximized by targeting young brothers of affected cases.     

Islet transplantation in type I diabetes mellitus

For most patients with type I diabetes, insulin therapy and glucose monitoring are sufficient to maintain glycemic control. However, hypoglycemia is a potentially lethal side effect of insulin treatment in patients who are glycemically labile or have hypoglycemia-associated autonomic failure [1]. For those patients, an alternative therapy is beta cell replacement via pancreas or islet transplantation. Pancreas transplants using cadaveric donor organs reduce insulin dependence but carry risks involved in major surgery and chronic immunosuppression. Islet transplantation, in which islets are isolated from donor pancreases and intravenously infused, require no surgery and can utilize islets isolated from pancreases unsuitable for whole organ transplantation. However, islet transplantation also requires immunosuppression, and standard steroid regimens may be toxic to beta cells [2]. The 2000 Edmonton Trial demonstrated the first long-term successful islet transplantation by using a glucocorticoid-free immunosuppressive regimen (sirolimus and tacrolimus). The Clinical Islet Transplantation (CIT) Consortium seeks to improve upon the Edmonton Protocol by using anti-thymocyte globulin (ATG) and TNFα antagonist (etanercept). The trials currently in progress, in addition to research efforts to find new sources of islet cells, reflect enormous potential for islet transplantation in treatment of type I diabetes.     

The prevalence of type II diabetes mellitus is haptoglobin phenotype-independent

Haptoglobin (Hp) phenotype distribution and the association between Hp polymorphism and type II diabetes mellitus was investigated in a Jordanian sample population consisting of 618 nondiabetics and 265 diabetics. In nondiabetics, Hp 2-2 was the most predominant type occurring at a frequency of 0.529 followed by Hp 2-1 occurring at a frequency of 0.387. In diabetics, the Hp 2-2 frequency was 0.540 while that of Hp 2-1 was 0.381. No statistically significant variation was detected in Hp type distribution between the two groups. The Hp2 allele occurred at a frequency of 0.722 in nondiabetics and 0.730 in diabetics. In both groups, the Hp type distribution was in agreement with the Hardy-Weinberg equilibrium calculations. These results suggest that type II diabetes mellitus is Hp phenotype-independent.     

Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures

Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line. ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index. Cells on ECM were heterogeneous in size and insulin content. They showed extended areas of confluence. Cultures on plastic demonstrated an organisation in clusters and low mitotic activity. However, ECM did not allow for reconstitution of an islet-like structure. When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture. Glucagon and somatostatin secretion were similar on both substrates. These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.     

Islet cell antibodies, circulating immune complexes and antinuclear antibodies in diabetes mellitus

Serum samples of patients suffering from diabetes mellitus were tested for complement-fixing and non complement-fixing islet cell antibodies, antinuclear antibodies and circulating immune complexes. There was no correlation between circulating immune complexes or antinuclear antibodies and secondary diabetic complications. A close relationship was found between the ICA titer and complement fixation of ICA. The incidence of ICA at the onset of the disease was higher in the patients under the age of 10 (85%) and decreased with increasing age up to 45% in patients with onset above age 20. In five patients being positive and four patients being negative for ICA at onset of disease, changes and fluctuations in antibody titers were observed over 38 months. Since manifestation of diabetes mellitus is believed to be an endpoint of a long lasting autoimmune process, our observations indicate that the autoimmune phenomena are merely indicators of ongoing autoimmune reactions not necessarily reflecting the state of autoaggression or islet cell destruction.     

Autoantibodies predicting diabetes mellitus type I in celiac disease

Celiac disease (CD) and diabetes mellitus type I (DM-I) are both autoimmune diseases. Abnormal first-phase insulin response (FPIR) is associated with the prediabetic phase. Glutamic acid decarboxylase (GAD) and islet cell antibodies (ICAs) - especially the tyrosine phosphatase-like protein IA-2 antibodies - are considered to be serological markers of DM-I future development. The aim of this study is to investigate the presence of autoantibodies (GAD, IA-2) in individuals with CD, on a gluten-free diet, who have normal intestinal morphology. Thirty patients with CD (4-22, mean 15 years), 30 newly diagnosed diabetic children (2.5-16, mean 10 years) and 30 healthy subjects (7-35, mean 18 years) were investigated. Serum GAD and IA-2 autoantibodies were assessed by a quantitative enzyme-linked immunosorbent assay (ELISA) method in all patients and controls. Seven CD patients (23%), 28 diabetic children (93%) and none in the control group had positive GAD and IA-2 antibodies. The FPIR was normal in CD patients (>/=46 mU/l). Conclusions: GAD and IA-2 antibodies are detected in 23% of patients with CD. These patients may be at risk to develop DM-I. Regular follow-up and determination of FPIR for the early diagnosis of the prediabetic phase in patients with CD having circulating autoantibodies is recommended.     

Population based study of prevalence of islet cell autoantibodies in monozygotic and dizygotic Danish twin pairs with insulin dependent diabetes mellitus.

OBJECTIVE: To study the comparative importance of environment and genes in the development of islet cell autoimmunity associated with insulin dependent diabetes mellitus. DESIGN: Population based study of diabetic twins. SETTING: Danish population. SUBJECTS: 18 monozygotic and 36 dizygotic twin pairs with one or both partners having insulin dependent diabetes. MAIN OUTCOME MEASURES: Presence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase (GAD65) in serum samples from twin pairs 10 years (range 0-30 years) and 9.5 years (2-30 years) after onset of disease. RESULTS: In those with diabetes the prevalence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase in the 26 monozygotic twins was 38%, 85%, and 92%, respectively, and in the dizygotic twins was 57%, 70%, and 57%, respectively. In those without diabetes the proportions were 20%, 50%, and 40% in the 10 monozygotic twins and 26%, 49%, and 40% in the 35 dizygotic twins. CONCLUSION: There is no difference between the prevalence of islet cell autoantibodies in dizygotic and monozygotic twins without diabetes, suggesting that islet cell autoimmunity is environmentally rather than genetically determined. Furthermore, the prevalence of islet cell antibodies was higher in the non-diabetic twins than in other first degree relatives of patients with insulin dependent diabetes. This implies that the prenatal or early postnatal period during which twins are exposed to the same environment, in contrast with that experienced by first degree relatives, is of aetiological importance.     

Anti-GAD-positive patients with type 1 diabetes mellitus have higher prevalence of autoimmune thyroiditis than anti-GAD-negative patients with type 1 and type 2 diabetes mellitus

The aim of our study was to evaluate antibodies against thyroglobulin (anti-TG) and thyroid peroxidase (anti-TPO) - markers of autoimmune thyroiditis - in several groups of adult patients with type 1 and type 2 diabetes mellitus (DM). We were particularly interested whether the presence of thyroid antibodies is related to the positivity of glutamic acid decarboxylase antibodies (anti-GAD). We found elevated anti-GAD in 46 % (97/210) patients with type 1 DM. All patients with type 2 diabetes were anti-GAD-negative. At least one thyroid antibody (anti-TG and/or anti-TPO) was found in 30 % (62/210) patients with type 1 DM and 27 % (22/83) type 2 diabetes patients. The patients with type 1 DM were further grouped according to their anti-GAD status. The anti-GAD-positive patients had a higher prevalence of anti-TG antibodies than the anti-GAD-negative patients (25 % vs. 12 %, p=0.03) as well as anti-TPO antibodies (32 % vs. 12 %, p<0.001). At least one thyroid antibody was detected in 39 % (38/97) of anti-GAD-positive but only in 21 % (24/113) of anti-GAD-negative patients with type 1 DM (p=0.006). No significant difference in the frequency of thyroid antibodies was found between anti-GAD-negative patients with type 1 and type 2 DM (21 % vs. 27 %, p=0.4). The groups with or without thyroid antibodies in both type 1 and type 2 diabetic patients did not differ in actual age, the age at diabetes onset, duration of diabetes, body mass index or HbA1c level. Patients with elevated thyroid antibodies had significantly higher levels of TSH than those without thyroid antibodies (1.86 vs. 3.22 mIU/l, p=0.04 in type 1 DM; 2.06 vs. 4.89 mIU/l, p=0.003 in type 2 DM). We conclude that there is a higher frequency of thyroid-specific antibodies in anti-GAD-positive adult patients with type 1 DM than in anti-GAD-negative patients or in patients with type 2 DM. Patients with or without thyroid antibodies do not differ in age, DM onset and duration, BMI or HbA1c. Thyroid antibodies-positive patients have higher levels of thyroid stimulating hormone (TSH).     

Genomics of type I diabetes mellitus and its late complications

In ethnic Russians, MHC (HLA) was shown to be the major locus determining the predisposition to type 1 diabetes mellitus (T1DM). To map the regions linked to T1DM, families with concordant or discordant sib pairs were selected from the Russian population of Moscow. With these families, linkage to T1DM was demonstrated for CTLA4 (IDDM12, 2q32.1-q33), which codes for a T-cell surface antigen, and PDCD2 (IDDM8, 6q25-q27), which is homologous to the mouse programmed cell death activator gene. With polymorphic microsatellites, regions 3q21-q25 (IDDM9) and 10p12.2 (IDDM10) were also linked to T1DM. Case/control and family studies of the polymorphic markers from region 11p13 revealed a new T1DM-associated locus in the vicinity of the catalase gene (CAT); linkage to this locus was not reported earlier for other populations. Diabetic polyneuropathy (DPN) proved to be associated with single-nucleotide polymorphisms Ala(-9)Val (SOD2), Arg213Gly (SOD3), and T(-262)C (CAT) and with a polymorphic microsatellite of the NOS2 promoter. Hence oxidative stress, which results from hyperglycemia, increased mitochondrial production of superoxide radicals, and insufficient activities of antioxidative enzymes, was assumed to play an important part in DPN development in T1DM. Diabetic nephropathy (DN) showed no association with the antioxidative enzyme genes. However, the association was observed for the insertion/deletion (I/D) polymorphism of ACE and the ecNOS34a/4b polymorphism of NOS3, two genes involved in controlling vascular tonicity, and for the I/D polymorphism of APOB and the epsilon 2/epsilon 3/epsilon 4 polymorphism of APOE, two genes involved in lipid transport. In addition, polymorphic microsatellites of chromosome 3q21-q25 proved to be closely associated with DN. The tightest association was established for D3S1550, carriers of allele 12 or genotype 12/14 having high risk of DN (OR = 4.85 and 6.25, respectively). Region 3q21-q25 was assumed to contain a major gene determining DN development, while the other DN-associated genes mostly affect the progression of DN.     

Polymorphonuclear cell derangements in type I diabetes

Polymorphonuclear cell function had been studied in 58 Type I diabetic subjects. Chemotaxis, phagocytosis, adherence, bactericidal activity, NBT reduction capacity were evaluated. We enumerated gamma Fc receptor bearing polymorphonuclear cells and the percentage of immune complexes containing polymorphonuclear cells. These data were studied in accordance with glycemic levels and the presence of infections. All polymorphonuclear functions were decreased compared to non-diabetic subjects with the exception of phagocytosis. The efficiency of the diabetic sera on normal subjects polymorphonuclear cells was decreased (bactericidal activity, chemotactic index and phagocytosis). These abnormalities were independent of the presence of infection. No correlation was found with glycemic level. The percentage of cells bearing an Fc gamma receptor was less in diabetics than in normal (70.1 +/- 17.4 vs 80.2 +/- 7.8%). The percentage of immune complexes containing polymorphonuclear cells was increased (n = 16, 9.06 +/- 4.7 vs 4.75 +/- 2.1% in normals). There again, these data are without correlation with the presence of infections or glycemic level.     

Mesenchymal stem cells and differentiated insulin producing cells are new horizons for pancreatic regeneration in type I diabetes mellitus

BackgroundDiabetes mellitus has become the third human killer following cancer and cardiovascular disease. Millions of patients, often children, suffer from type 1 diabetes (T1D). Stem cells created hopes to regenerate damaged body tissues and restore their function.AimThis work aimed at clarifying and comparing the therapeutic potential of differentiated and non-differentiated mesenchymal stem cells (MSCs) as a new line of therapy for T1D.Methods40 Female albino rats divided into group I (control): 10 rats and group II (diabetic), III and IV, 10 rats in each, were injected with streptozotocin (50 mg/kg body weight). Group III (MSCs) were transplanted with bone marrow derived MSCs from male rats and group IV (IPCs) with differentiated insulin producing cells. Blood and pancreatic tissue samples were taken from all rats for biochemical and histological studies.ResultsMSCs reduced hyperglycemia in diabetic rats on day 15 while IPCs normalizes blood glucose level on day 7. Histological and morphometric analysis of pancreas of experimental diabetic rats showed improvement in MSCs-treated group but in IPCs-treated group, β-cells insulin immunoreactions were obviously returned to normal, with normal distribution of β-cells in the center and other cells at the periphery. Meanwhile, most of the pathological lesions were still detected in diabetic rats.ConclusionMSCs transplantation can reduce blood glucose level in recipient diabetic rats. IPCs initiate endogenous pancreatic regeneration by neogenesis of islets. IPCs are better than MSCs in regeneration of β-cells. So, IPCs therapy can be considered clinically to offer a hope for patients suffering from T1D.