Immune attack on pancreatic islet transplants in the spontaneously diabetic BioBreeding/Worcester (BB/W) rat is not MHC restricted

Spontaneous diabetes mellitus in the BB/W rat is preceded by lymphocytic insulitis that destroys pancreatic beta cells. Cultured pancreatic islets and adrenal cortex from inbred rats of variable MHC were transplanted to RT1/u BB/W rats without allograft rejection. Islet grafts from RT1/u and non-RT1/u rats evidenced lymphocytic insulitis in BB/W recipients that became diabetic or evidenced lymphocytic insulitis within endogenous islets. These findings suggest that BB immune insulitis is not MHC restricted and may be directed against islet transplants from non-RT1/u animals.     

Endothelial dysfunction in the early- and late-stage type-2 diabetic Goto-Kakizaki rat aorta

Hodgkin and Reed-Sternberg (H-RS) cells of classical Hodgkin lymphoma (cHL) present an impaired expression of immunoglobulin genes, but escape apoptotic death. We investigated whether nitric oxide synthases (NOS) are expressed by H-RS cells, studied their association with EBV status and the expression of apoptotic proteins, and investigated their relationship to the clinical outcome of 171 patients. NOS1 and NOS2 were expressed in a large number of cases, whereas NOS3 expression was not detected. Positive associations were found between NOS1 and p53, bax and NOS2, bcl-2 and NOS2, bax and p53, and between bax and fasL. Inverse correlations were established between EBV and NOS2 and between EBV and bcl-2. A shorter overall survival (OS) was associated with strong expression of NOS2. In conclusion, NOS are expressed by H-RS cells of cHL.     

Activated protein C inhibits pancreatic islet inflammation, stimulates T regulatory cells, and prevents diabetes in non-obese diabetic (NOD) mice

Activated protein C (aPC) is a natural anticoagulant with strong cyto-protective and anti-inflammatory properties. aPC inhibits pancreatic inflammation and preserves functional islets after intraportal transplantation in mice. Whether aPC prevents the onset or development of type 1 diabetes (T1D) is unknown. In this study, when human recombinant aPC was delivered intraperitoneally, twice weekly for 10 weeks (from week 6 to 15) to non-obese diabetic (NOD) mice, a model for T1D, the incidence of diabetes was reduced from 70% (saline control) to 7.6% by 26 weeks of age. Islets of aPC-treated mice exhibited markedly increased expression of insulin, aPC/protein C, endothelial protein C receptor, and matrix metalloproteinase (MMP)-2 when examined by immunostaining. The insulitis score in aPC-treated mice was 50% less than that in control mice. T regulatory cells (Tregs) in the spleen, pancreatic islets, and pancreatic lymph nodes were increased 37, 53, and 59%, respectively, in NOD mice following aPC treatment. These Tregs had potent suppressor function and, after adoptive transfer, delayed diabetes onset in NOD.severe combined immunodeficiency mice. The culture of NOD mouse spleen cells with aPC reduced the secretion of inflammatory cytokines interleukin (IL)-1β and interferon-γ but increased IL-2 and transforming growth factor-β1, two cytokines required for Treg differentiation. In summary, our results indicate that aPC prevents T1D in the NOD mouse. The aPC mechanism of action is complex, involving induction of Treg differentiation, inhibition of inflammation, and possibly direct cyto-protective effects on β cells.     

Effects of magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) on diabetic nephropathy in type 2 diabetic Goto-Kakizaki rats

We investigated the effect of magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl), a marker compound isolated from the cortex of Magnolia officinalis, in non-obese type 2 diabetic Goto-Kakizaki (GK) rats. The rats were treated orally with magnolol (100 mg/kg body weight) once a day for 13 weeks. In magnolol-treated GK rats, fasting blood glucose and plasma insulin were significantly decreased, and the pancreatic islets also showed strong insulin antigen positivity. Urinary protein and creatinine clearance (Ccr) were significantly decreased. Pathological examination revealed the prevention of the glomeruli enlargement in magnolol-treated GK rats. The overproduction of renal sorbitol, advanced glycation endproducts (AGEs), type IV collagen, and TGF-beta1 mRNA were significantly reduced in magnolol-treated GK rats. Thus based on our findings, the use of magnolol could result in good blood glucose control and prevent or retard development of diabetic complications such as diabetic nephropathy.     

A new prostaglandin E1 analogue (TFC-612) improves the reduction in motor nerve conduction velocity in spontaneously diabetic GK (Goto-Kakizaki) rats

A new prostaglandin E1 analogue, TFC-612, was given orally to 2 month-old spontaneously diabetic GK (Goto-Kakizaki) rats for 3 months to ascertain its effects on reduced motor nerve conduction velocity (MCV). A high dose of this compound (0.3 mg/kg body weight) significantly restored MCV after 2 and 3 months of administration, although the low dose (0.03 mg/kg body weight) did not. In addition, 1 month administration of TFC-612 significantly improved the reduced MCV in aged (5 month-old) GK rats only in the high dose group (0.3 mg/kg body weight), but not in the low dose group (0.03 mg/kg body weight). Although TFC-612 significantly suppressed sorbitol accumulation in the sciatic nerves of GK rats in a dose dependent manner after 3 months administration, this suppression was not observed after either 2 months administration to 2 month-old GK rats or after 1 month administration to 5 month-old GK rats. Fasting blood glucose levels of all GK rats remained high throughout the experiments, regardless of TFC-612 administration. TFC-612's improvement on reduced motor nerve conduction velocity was related partly to suppression of sorbitol accumulation, but other factors, including microcirculation, may contribute significantly to this effect. These results suggest that TFC-612 may be beneficial in the treatment of diabetic nerve impairment.     

Plasma insulin and glucagon and their release from pancreatic islet in hyperosmolar diabetic rat.

In order to investigate the metabolic abnormalities in hyperosmolar diabetes from the viewpoint of insulin or glucagon, experimental hyperosmolar diabetes was produced by a combination of cortisol injection and water deprivation or only by the latter in streptozotocin-induced moderately hyperglycemic rat. They had a high blood glucose level and high plasma osmotic pressure. Fasting plasma insulin tended to decrease in the dehydrated state whether diabetic or not. Fasting plasma glucagon was increased to 0.047 +/- 0.009 nmol/l (P less than 0.05) in the non-diabetic dehydrated state (normal 0.026 +/- 0.004 nmol/l), and a similar high level of plasma glucagon was observed in the dehydrated diabetic rat (0.052 +/- 0.020 nmol/l), especially after cortisol treatment. In isolated rat islet, insulin released from the dehydrated diabetic rat at a high concentration of glucose was to some extent lower than that of diabetic rat, and released IRG vice versa. The insulin:glucagon ratio in the presence of high glucose was significantly lower in the dehydrated diabetic rat than in the normal rat (P less than 0.01). In the diabetic rat this ratio was not significantly different. This finding was also consistent with the results of in vivo experiments. Thus more catabolic hormonal changes were found in in vivo and in vitro studies in the hyperosmolar diabetic rat.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

Differential glucose-regulation of microRNAs in pancreatic islets of non-obese type 2 diabetes model Goto-Kakizaki rat

Background

The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.

Methodology/Principal Findings

Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3′UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.

Conclusions/Significance

The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.     

Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice

Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.     

Luteinizing hormone-releasing factor (LRF)-like immunoreactivity in rat pancreatic islet cells.

Luteinizing hormone-releasing factor (LRF)-like immunoreactive material was demonstrated by the three-layer immunoperoxidase method in formalin-fixed tissue sections of the rat pancreas. Anti-LRF antiserum was prepared in rabbits by immunizing with synthetic LRF coupled to bovine serum albumin (BSA). The immunoreactive site of LRF reacting with antiserum resided between residues Tyr⁵ and Gly¹⁰-NH₂. A positive staining reaction was observed in the islet cells with the use of anti-LRF antiserum after solid phase immunoadsorption with BSA, whereas no staining was observed when adjacent control sections were prepared with anti-LRF antiserum after immunoadsorption with an LRF-BSA conjugate, or with rabbit anti-oxytocin antiserum. LRF-like immunoreactive material was isolated from the rat pancreata by methanol extraction. This material coeluted with synthetic and hypothalamic LRF in cation exchange chromatography on carboxymethyl cellulose, and dilutions of it gave an inhibition curve parallel to that of synthetic LRF in radioimmunoassay. The concentration of LRF-like material in the rat pancreas is 1.1 pg/mg wet weight. These results suggest that LRF or a closely LRF-related peptide is shared by the central nervous system and the gastrointestinal tract.     

Expression of multiple plasma membrane Ca(2+)-ATPases in rat pancreatic islet cells

When stimulated by glucose, the pancreatic beta-cell displays large oscillations of intracellular free Ca2+ concentration ([Ca2+]i). To control [Ca2+]i, the beta-cell must be equipped with potent mechanisms for Ca2+ extrusion. We studied the expression of the plasma membrane Ca(2+)-ATPases (PMCA) in three insulin secreting preparations (a pure beta-cell preparation, RINm5F cells and pancreatic islet cells), using reverse-transcribed PCR, RNase protection assay and Western blotting. The four main isoforms, PMCA1, PMCA2, PMCA3 and PMCA4 were expressed in the three preparations. Six alternative splice mRNA variants, characterized at splice sites A, B and C were detected in the three preparations (rPMCA1xb, 2yb, 2wb, 3za, 3zc, 4xb), plus two additional variants in pancreatic islet cells (PMCA4za, 1xkb). The latter variant corresponded to a novel variant of rat PMCA1 gene lacking the exon coding for the 10th transmembrane segment, at splice site B. At the mRNA and protein level, five variants predominated (1xb, 2wb, 3za, 3zc, 4xb), whilst one additional isoform (4za), predominated at the protein level only. This provides the first evidence for the presence of PMCA2 and PMCA3 isoforms at the protein level in non-neuronal tissue. Hence, the pancreatic beta-cell is equipped with multiple PMCA isoforms with possible differential regulation, providing a full range of PMCAs for [Ca2+]i regulation.     

Higher efficiency of the liver phosphorylative system in diabetic Goto-Kakizaki (GK) rats.

Liver mitochondrial bioenergetics of Goto-Kakizaki (GK) rats (a model of non-insulin dependent diabetes mellitus) reveals a Delta Psi upon energization with succinate significantly increased relatively to control animals. The repolarization rate following ADP phosphorylation is also significantly increased in GK mitochondria in parallel with increased ATPase activity. The increase in the repolarization rate and ATPase activity is presumably related to an improved efficiency of F(0)F(1)-ATPase, either from a better phosphorylative energy coupling or as a consequence of an enlarged number of catalytic units. Titrations with oligomycin indicate that diabetic GK liver mitochondria require excess oligomycin pulses to completely abolish phosphorylation, relative to control mitochondria. Therefore, accepting that the number of operational ATP synthase units is inversely proportional to the amount of added oligomycin, it is concluded that liver mitochondria of diabetic GK rats are provided with extra catalytic units relative to control mitochondria of normal rats. Other tissues (kidney, brain and skeletal muscle) were evaluated for the same bioenergetic parameters, confirming that this feature is exclusive to liver from diabetic GK rats.     

Intracranial pancreatic islet transplantation increases islet hormone expression in the rat brain and attenuates behavioral dysfunctions induced by MK-801 (dizocilpine)

The treatment of rodents with non-competitive antagonist of the N-Methyl-d-aspartate (NMDA) receptor, MK-801 (dizocilpine), induces symptoms of psychosis, deficits in spatial memory and impairment of synaptic plasticity. Recent studies have suggested that insulin administration might attenuate the cognitive dysfunctions through the modulatory effect on the expression of NMDA receptors and on the brain insulin signaling. Intrahepatic pancreatic islet transplantation is known as an efficient tool for correcting impaired insulin signaling. We examined the capacity of syngeneic islets grafted into the cranial subarachnoid cavity to attenuate behavioral dysfunctions in rats exposed to MK-801. Animals were examined in the open field (OF) and the Morris Water Maze (MWM) tests following acute or subchronic administration of MK-801. We found well-vascularized grafted islets expressing insulin, glucagon and somatostatin onto the olfactory bulb and prefrontal cortex. Significantly higher levels of insulin were detected in the hippocampus and prefrontal cortex of transplanted animals compared to the non-transplanted rats. All animals expressed normal peripheral glucose homeostasis for two months after transplantation. OF tests revealed that rats exposed to MK-801 treatment, showed hyper-responsiveness in motility parameters and augmented center field exploration compared to intact controls and these effects were attenuated by the grafted islets. Moreover, in the MWM, the rats treated with MK-801 showed impairment of spatial memory that were partially corrected by the grafted islets. In conclusion, intracranial islet transplantation leads to the expression of islet hormones in the brain and attenuates behavioral and cognitive dysfunctions in rats exposed to MK-801 administration without altering the peripheral glucose homeostasis.     

A morphological study of the primary cilia in the rat pancreatic ductal system: ultrastructural features and variability

Primary cilia in the pancreas of the rat were studied by transmission electron microscopy. Their presence is very common, and each ductal cell seems to be provided with a single cilium. The basal body showed anchoring apparatus such as transitional fibers and basal feet. The shaft can show a number of different patterns according to the level of the sections. Proceeding towards the tip, the microtubules decrease in number, although not always in the same way. Near the tip, it is possible to detect patterns, with only 1 microtubule. Three kinds of tips are described. The function of the cilia is discussed.     

Adenylyl cyclase isoform expression in non-diabetic and diabetic Goto-Kakizaki (GK) rat pancreas. Evidence for distinct overexpression of type-8 adenylyl cyclase in diabetic GK rat islets

Glucose-induced insulin release is markedly decreased in the spontaneously diabetic Goto-Kakizaki (GK) rat pancreas. This defect was recently shown to be reversed by forskolin which markedly enhances cAMP generation in GK islets. These effects of forskolin were associated with overexpression of type-3 adenylyl cyclase (AC) mRNA due to the presence of two functional point mutations in the promoter region of AC3 gene in GK rat. Nine AC isoforms have been described, but their expression pattern in relation to the main pancreatic islet cell types, as well as their involvement in the diabetic state, is still unknown. Using antibodies raised against AC1–8, we have studied by double immunofluorescence the localisation of these AC isoforms in different endocrine cell types in both normal and diabetic GK rat pancreas. Our results demonstrated a clear immunoreaction (IR) to AC1–4 and 6 in normal and GK islet β-cells, while a smaller number of ACs were expressed in α- and δ-cells. No AC-IR was observed in pancreatic polypeptide cells. Moreover, we have found an increased IR of the Ca²⁺-stimulated AC1, AC3 and AC8 in diabetic β- and α-cells, compared with the corresponding IR in control pancreas. Most noticeable was the eliciting of a markedly enhanced AC8-IR in GK rat β- and α-cells, in contrast to a barely discernible AC8-IR in corresponding normal cells. In conclusion, AC expression exhibits a complex pattern in the endocrine pancreas, with specific differences between the normal and diabetic state. Accepted: 25 November 1999     

Chromosomal mapping and developmental study of tattered-hokkaido (Td ho)

We found a new X-linked dominant mouse mutation. This mouse has the same phenotype as Td, which exhibits hyperkeratotic skin, reduced viability in affected females, a tendency to be smaller, lighter weight than the normal sibs during weaning age, and prenatal lethality in affected males. To map the locus, we tested 267 progeny from an intraspecific backcross between affected females and wild-origin strain males. Polymerase chain reaction (PCR) was performed with microsatellite markers of the proximal region of the mouse X Chromosome (Chr). This mutant showed no recombination with DXMit 123, DXMit 55, or DXMit 26. The gene position and phenotype of this mutant were very similar to those of Td. Therefore, it is speculated that the new mutant gene is a multiple allele of Td, and we designated it Tattered-Hokkaido (Td ^ho ). Linkage analysis of these animals suggested a possible gene order of cen-(Td ^ho , DXMit123, DXMit55, DXMit26)–DXMit161–DXMit54–DXMit103–DXMit52–DXMit190–DXMit138) in the X Chr. Prenatal lethality of male mutants was also investigated, with 12.5 to 16.5 embryonic day (E) backcrossed embryos from affected F₁ females. It was found that the male mutants died between E12.5 and E14.5. The cause of death of male mutants is discussed in relation with the other proximal genes of the X Chr. Received: 15 December 1997 / Accepted: 1 April 1997