Ake Andersson

Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice

SummaryBy using a previously described culture technique for the midgestational fetal porcine pancreas, islet-like cell clusters with a Beta-cell frequency of approximately 5% have been produced in large numbers. These islet-like cell clusters were transplanted beneath the kidney capsule to either normoglycaemic or alloxan-treated nude mice. The grafts consistently failed to cure the alloxan-treated mice immediately after implantation, however, normoglycaemia was restored in a majority of the mice within 2 months after transplantation and in all animals after 4 and 6 months. Indeed, the insulin released from the transplanted fetal Beta cells was able to normalize the serum glucose concentration at porcine levels (4–5 mmol/l) rather than at the level maintained in mice (8–10 mmol/l). In the cured mice there was a normal secretory response to glucose in the grafts as evidenced by normal glucose profiles during intravenous glucose tolerance test and a biphasic insulin response to high glucose when perfusing the graft bearing kidney. On the other hand, in the normoglycaemic animals the second phase faded before the glucose stimulus had been withdrawn. Two months after transplantation the edocrine cells were arranged so that the endocrine non-Beta cells were randomly scattered among a majority of Beta cells. The cell replication of the Beta cells, measured by 3H-thymidine incorporation, was within the lower range of that seen in the native islets of adult mice. No major differences between the controls and the alloxan-treated animals were observed in this respect. Cultured islet-like cell clusters had high rates of glucose utilization, paralleled by low rates of glucose oxidation, compared with adult mouse islets. Following transplantation there was a progressive decrease in glucose utilization and an increase in glucose oxidation. It is concluded that after transplantation the epitheloid cells comprising the porcine islet-like cell clusters can develop into insulin-producing cells with the ability to cure diabetic nude mice. Provided the rejection problems can be overcome the fetal porcine pancreas may be suitable for future clinical xenogeneic transplantations.

Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt

Aims/hypothesisPro-inflammatory cytokines cause beta cell secretory dysfunction and apoptosis—a process implicated in the pathogenesis of type 1 diabetes. Cytokines induce the expression of inducible nitric oxide (NO) synthase (iNOS) leading to NO production. NO contributes to cytokine-induced apoptosis, but the underlying mechanisms are unclear. The aim of this study was to investigate whether NO modulates signalling via mitogen-activated protein kinases (MAPKs) and Akt.Materials and methodsMAPK activities in INS-1 cells and isolated islets were determined by immunoblotting and in vitro kinase assay. Apoptosis was determined by ELISA measurement of histone–DNA complexes present in cytoplasm.ResultsApoptosis in INS-1 cells induced by IL-1β plus IFNγ was dependent on NO production as demonstrated by the use of the NOS blocker NG-methyl-l-arginine. Accordingly, an NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP) dose-dependently caused apoptosis in INS-1 cells. SNAP activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but suppressed the activity of extracellular signal-regulated kinase MAPK. In rat islets, NOS inhibition decreased JNK and p38 activities induced by a 6-h exposure to IL-1β. Likewise, IL-1β-induced JNK and p38 activities were lower in iNOS(−/−) mouse islets than in wild-type islets. In human islets, SNAP potentiated IL-1β-induced JNK activation. The constitutive level of active, Ser473-phosphorylated Akt in INS-1 cells was suppressed by SNAP. IGF-I activated Akt and protected against SNAP-induced apoptosis. The anti-apoptotic effect of IGF-I was not associated with reduced JNK activation.Conclusions/interpretationWe suggest that NO contributes to cytokine-induced apoptosis via potentiation of JNK activity and suppression of Akt.

Rapid deposition of amyloid in human islets transplanted into nude mice

SummaryHuman islets of Langerhans were transplanted to the subcapsular space of the kidneys of nude mice which were either normoglycaemic or made diabetic with alloxan. After 2 weeks, the transplants were processed for light and electron microscopical analyses. In all transplants, islet amyloid polypeptide (IAPP)-positive cells were found with highest frequency in normoglycaemic animals. IAPP-positive amyloid was seen in 16 out of 22 transplants (73%), either by polarisation microscopy after Congo red staining or by immune electron microscopy. At variance with previous findings of amyloid deposits exclusively in the extracellular space of islets of non-insulin-dependent diabetic patients, the grafted islets contained intracellular amyloid deposits as well. There was no clear difference in occurrence of amyloid between diabetic and non-diabetic animals. The present study indicates that human islets transplanted into nude mice very soon present IAPP-positive amyloid deposits. This technique may provide a valuable model for studies of the pathogenesis of islet amyloid and its impact on islet cell function.

Human autoantibodies react with glutamic acid decarboxylase antigen in human and rat but not in mouse pancreatic islets.

SummaryThe presence of one of the major targets for auto-antibodies in Type 1 (insulin-dependent) diabetes mellitus, the enzyme glutamic acid decarboxylase, was studied in human, rat and mouse pancreatic tissue using immunoprecipitation and immunohistochemical techniques. Immunoprecipitation of glutamic acid decarboxylase was attempted with lysates of [35S]-methionine-labelled rat or mouse pancreatic islets using two different glutamic acid decarboxylase antisera, one mouse monoclonal antibody raised against the 65 kDa isoform of the enzyme, sera from six patients with Type 1 diabetes, one patient with stiff-man syndrome and sera from 19 non-obese diabetic mice. The same sera were used for immunoperoxidase staining of cryosections of human, rat or mouse pancreas. Using patient sera glutamic acid decarboxylase was detected by immunoprecipitations from isolated rat islets but not from islets of five different mouse strains tested, including the non-obese diabetic mouse. When using the non-obese diabetic mouse sera, glutamic acid decarboxylase could not be detected in either rat or mouse tissue. Immunoperoxidase staining demonstrated high levels of glutamic acid decarboxylase in human and rat pancreatic islets but low levels in mouse islets. Direct measurements of enzyme activity showed glutamic acid decarboxylase to be present in mouse islets at a level of about 40% of that in rat islets, and subsequent Western blot analyses indicated that mouse islets express the 67 kDa isoform, whereas in rat islets both the 67 and 65 kDa isoforms are present. The species difference at the level of one of the major islet cell autoantigens in Type 1 diabetes thus indicates that human or rat and mouse islets, respectively, express immunologically distinct forms of glutamic acid decarboxylase and differ in their way of regulating the enzyme production.

New perspectives on the structure and function of the normal and diabetic beta-cell.

The pancreatic 0-cell has the unique capability to express the insulin gene and to synthesize, store and release insulin exactly according to the needs of the body. In a broad sense the 0-cell can be regarded as a biological sensor of circulating metabolic substrates, which are controlled by a feed-back system involving insulin secretion. Insulin is synthesized on the endoplasmic reticulum in the form of preproinsulin which is rapidly cleaved to proinsulin, transported to the Golgi zone and packed into secretory granules. Proinsulin is converted into C-peptide and biologically active insulin which is stored in the granules until released as a result of one or several specific signals reaching the 0-cell. The most important of these is an elevation of the blood glucose level, which causes an insulin response directly proportional to the glucose concentration. This process is of decisive importance for the short-term regulation of the blood glucose level. Of equal importance, however, is the total 0cell mass, which determines the capacity for insulin production in the long-term and is the result of an adaptive growth process. A deficient insulin production in relation to the insulin requirement is a common denominator of all types of diabetes. This state may be induced by the action of toxins, viruses or autoimmune assaults on the 0-cell or, perhaps, exhaustion from a long-standing functional demand on the insulin production. There is still poor knowledge on the various states of insulin deficiency and a deeper insight into the underlying mechanisms would greatly enhance the attempts to find a treatment or cure for diabetes and also to introduce measures for complete prevention of the disease. The present paper will summarize some recent observations on factors regulating insulin production and mechanisms of 0-cell proliferation and degeneration.