Mild diabetes models and their maternal-fetal repercussions

The presence of diabetes in pregnancy leads to hormonal and metabolic changes making inappropriate intrauterine environment, favoring the onset of maternal and fetal complications. Human studies that explore mechanisms responsible for changes caused by diabetes are limited not only for ethical reasons but also by the many uncontrollable variables. Thus, there is a need to develop appropriate experimental models. The diabetes induced in laboratory animals can be performed by different methods depending on dose, route of administration, and the strain and age of animal used. Many of these studies are carried out in neonatal period or during pregnancy, but the results presented are controversial. So this paper, addresses the review about the different models of mild diabetes induction using streptozotocin in pregnant rats and their repercussions on the maternal and fetal organisms to propose an adequate model for each approached issue. © 2013 D. C. Damasceno et al.

THE MOLECULAR INTERACTION BETWEEN TYPE II DIABETES AND ALZHEIMER’S DISEASE THROUGH CROSS-SEEDING OF PROTEIN MISFOLDING

With the population of the world aging, the prominence of diseases such as Type II Diabetes (T2D) and Alzheimer’s disease (AD) are on the rise. In addition, patients with T2D have an increased risk of developing AD compared to age-matched individuals, and the number of AD patients with T2D is higher than among aged-matched non-AD patients. AD is a chronic and progressive dementia characterized by amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs), neuronal loss, brain inflammation, and cognitive impairment. T2D involves the dysfunctional use of pancreatic insulin by the body resulting in insulin resistance, hyperglycemia, hyperinsulinemia, pancreatic beta cell (β-cell) death, and other complications. T2D and AD are considered protein misfolding disorders (PMDs). PMDs are characterized by the presence of misfolded protein aggregates, such as in T2D pancreas (islet amyloid polypeptide - IAPP) and in AD brain (amyloid– Aβ) of affected individuals. The misfolding and accumulation of these proteins follows a seeding-nucleation model where misfolded soluble oligomers act as nuclei to propagate misfolding by recruiting other native proteins. Cross-seeding occurs when oligomers composed by one protein seed the aggregation of a different protein. Our hypothesis is that the pathological interactions between T2D and AD may in part occur through cross-seeding of protein misfolding. To test this hypothesis, we examined how each respective aggregate (Aβ or IAPP) affects the disparate disease pathology through in vitro and in vivo studies. Assaying Aβ aggregates influence on T2D pathology, IAPP+/+/APPSwe+/- double transgenic (DTg) mice exhibited exacerbated T2D-like pathology as seen in elevated hyperglycemia compared to controls; in addition, IAPP levels in the pancreas are highest compared to controls. Moreover, IAPP+/+/APPSwe+/- animals demonstrate abundant plaque formation and greater plaque density in cortical and hippocampal areas in comparison to controls. Indeed, IAPP+/+/APPSwe+/- exhibit a colocalization of both misfolded proteins in cerebral plaques suggesting IAPP may directly interact with Aβ and aggravate AD pathology. In conclusion, these studies suggest that cross-seeding between IAPP and Aβ may occur, and that these protein aggregates exacerbate and accelerate disease pathology, respectively. Further mechanistic studies are necessary to determine how these two proteins interact and aggravate both pancreatic and brain pathologies.

Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes

Background: The HNF1A, HNF1B and HNF4A genes are part of an autoregulatory network in mammalian pancreas, liver, kidney and gut. The layout of this network appears to be similar in rodents and humans, but inactivation of HNF1A, HNF1B or HNF4A genes in animal models cause divergent phenotypes to those seen in man. We hypothesised that some differences may arise from variation in the expression profile of alternatively processed isoforms between species. Methodology/Principal Findings: We measured the expression of the major isoforms of the HNF1A, HNF1B and HNF4A genes in human and rodent pancreas, islet, liver and kidney by isoform-specific quantitative real-time PCR and compared their expression by the comparative Ct (??Ct) method. We found major changes in the expression profiles of the HNF genes between humans and rodents. The principal difference lies in the expression of the HNF1A gene, which exists as three isoforms in man, but as a single isoform only in rodents. More subtle changes were to the balance of HNF1B and HNF4A isoforms between species; the repressor isoform HNF1B(C) comprised only 6% in human islets compared with 24–26% in rodents (p = 0.006) whereas HNF4A9 comprised 22% of HNF4A expression in human pancreas but only 11% in rodents (p = 0.001). Conclusions/Significance: The differences we note in the isoform-specific expression of the human and rodent HNF1A, HNF1B and HNF4A genes may impact on the absolute activity of these genes, and therefore on the activity of the pancreatic transcription factor network as a whole. We conclude that alterations to expression of HNF isoforms may underlie some of the phenotypic variation caused by mutations in these genes.

Patient-reported outcomes following islet cell or pancreas transplantation (alone or after kidney) in type 1 diabetes: a systematic review

Aims For selected individuals with complex Type 1 diabetes, pancreatic islet transplantation (IT) offers the potential of excellent glycaemic controlwithout significant hypoglycaemia, balanced by the need for ongoing systemic immunosuppression. Increasingly, patient-reported outcomes (PROs) are considered alongside biomedical outcomes as a measure of transplant success. PROs in IT have not previously been compared directlywith the closest alternate treatment option, pancreas transplant alone (PTA) or pancreas after kidney (PAK).

Methods We used a Population, Intervention, Comparisons, Outcomes (PICO) strategy to search Scopus and screened 314 references for inclusion.

Results Twelve studies [including PRO assessment of PAK, PTA, islet-after kidney (IAK) and islet transplant alone (ITA); n = 7–205] used a total of nine specified and two unspecified PRO measures. Results were mixed but identified some benefits which remained apparent up to 36 months post-transplant, including improvements in fear of hypoglycaemia, as well as some aspects of diabetes-specific quality of life (QoL) and general health status. Negative outcomes included short-term pain associated with the procedure, immunosuppressant side effects and depressed mood associated with loss of graft function.

Conclusions The mixed resultsmay be attributable to limited sample sizes. Also, some PROmeasures may lack sensitivity to detect actual changes, as they exclude issues and domains of life likely to be important forQoL post-transplantation and when patients may no longer perceive themselves to have diabetes. Thus, the full impact of islet ⁄ pancreas transplantation (alone or after kidney) on QoL is unknown. Furthermore, no studies have assessed patient satisfaction, which may highlight further advantages and disadvantages of transplantation.

Long-term effects of insulin therapy, islet transplantation, and pancreas transplantation in the prevention of glomerular changes in kidneys of alloxan-induced diabetic rats

Groups of inbred alloxan-induced diabetic rats were treated with insulin (I), islets (IT), or pancreas transplantation (PT). Nondiabetic (N) and untreated diabetic (D) control groups were concurrently included. Each group was divided into five subgroups of 10 rats and killed after follow-up of 1, 3, 6, 9, and 12 months. Clinical and laboratory parameters were recorded, and kidney ultrastructural and morphometric analyses performed in each 12-month subgroup, namely glomerular basement membrane (GM) thickening, podocyte number, and number/extension of slit diaphragms (S). Rats from the I group showed poor metabolic control of diabetes compared with N group control rats. However, successfully transplanted rats (IT and PT) had complete restoration to normal levels for all metabolic parameters. GM thickening was significantly higher in diabetic compared with control rats. In contrast, the numbers of podocytes and slits as well as slit extensions were significantly decreased. Insulin therapy did not prevent any alterations upon comparison of diabetic vs control rats. Despite good metabolic control in IT rats, the degree of kidney lesion control never compared with that achieved in PT rats. In this group all glomerular changes were similar to the age-dependent lesions observed in control rats. We conclude that either IT or PT may be a good option for diabetes treatment, although pancreas transplantation seems to be the most effective treatment to control chronic complications.

Islet of Langerhans transplantation. A comparative study of two different methods for isolating islet cells from rat pancreas

Two different methods for isolation of islet of Langerhans on control of metabolic abnormalities of alloxan-induced diabetic rat were tested. Sixty rats were randomly assigned to four experimental groups: GI included 10 non-diabetic control rats, GII included 10 diabetic control rats, without treatment, GIII included 20 diabetic rats (10 inbred and 10 outbred rats) that received islet of Langerhans transplantation (ILT) using islet cells prepared by collagenase, and GIV included 20 diabetic rats (10 inbred and 10 outbred rats) submitted to ILT using islet cells prepared by nonenzymatic method. Clinical and laboratory parameters at beginning and 4, 7, 14, 21 and 30 days of follow-up were recorded. Outbred rats were immunosuppressed with cyclosporin A, diabetes was induced by e.v. alloxan administration, and islet cells were isolated from normal donor Lewis rats and injected into the portal vein. ILT corrected the body weight gain, polyuria, polydipsia, polyphagia, and the high levels of blood and urine glucose in 73.7% of rats treated by enzymatic method and in 64.7% of those ones treated by nonenzymatic method. However, there was no significantly difference between the two methods (P > 0.50). We did not also observe significantly difference between the two methods when ILT was performed either in inbred or outbred rats. We concluded that ILT performed by nonenzymatic method may be an alternative treatment for diabetes due to be less expensive and to have possible advantages in the isolation process.

The pancreatic islet system of the rock bass /|nby Edward Francis Squires. -- 260 St. Catharines, Ont. : [s. n.],

The endocrine pancreas of the rock bass (Ambloplites rupestris) was examined by light and electron microscopy. Two cell types with staining properties similar to mammalian A and B cells, and a third, non-staining cell type were found in the spherical pancreatic islets that were surrounded by a connective tissue capsule and embedded in two small masses of exocrine tissue. From an analysis of the ultrastructure of the A and B cells, a secretory cycle for each of these cell types was proposed. The secretory cycle of the A cell consisted of three well defined stages: (1) A cell production stage: during which A granule formation occurred in the sacs of the Golgi apparatus and the cell was characterized by the presence of numerous secretory granules, some elements of lamellar endoplasmic reticulum, and a homogeneously granular nucleus. The cytoplasm contained few distended cisternae, variable numbers of free ribosomes, microtubules and small vesicles. (2) A cell release stage: during which the release of A granules occurred and the cell usually contained several large distended cisternae and variable numbers of secretory granules. Granule release mechanisms included exocytosis, by which individual granules were released into the extracellular space after their membranes fused with the plasmalemma, and emiocytosis, by which one or more granules were released into a large cisterna whose membrane fused with the plasmalemma and formed a pore through which the cisternal contents passed out of the cell. (3) A cell reorganization stage: during which the changeover from the release stage to the production stage occurred and the reorganization of organelles and membrane structures took place. The cell contained few secretory granules and numerous small endoplasmic reticular cisternae. The cytoplasm exhibited less electron density than either of the other two stages. The A granule after formation underwent a series of morphological changes which were described in four numerically identified phases. The secretory cycle of the B cell consisred of two stages: (1) B cell production stage: during which the B granule formation occurred in the sacs of the Go1gi apparatus. The cell was characterized by an irregular outline, the presence of numerous secretory granules, and an irregularly shaped nucleus which contained variable amounts of clumped chromatin. The cytoplasm contained moderate amounts of lamellar endoplasmic reticulum studded with ribosomes, several small vesicles, and an active Go1gi apparatus. (2) B cell release stage: during which the release of B granules occurred. The cell contained a rounded nucleus with dispersed chromatin, several distended endoplasmic reticular cisternae and a variable number of secretory granules. Granule release occu~ by emiocytosis and exocytosis similar to that found for the A cell.

Anatomic and molecular characterization of the 1 endocrine pancreas of a teleostean fish: Atlantic wolffish (Anarhichas lupus)

Background: The biologic attributes of the endocrine pancreas and the comparative endocrinology of islet amyloid polypeptide (IAPP) of fish are not well described in the literature. This study describes the endocrine pancreas of one teleostean fish. Ten captive Atlantic wolffish (Anarhichas lupus) from the Montreal Biodome were submitted for necropsy and their pancreata were collected. Results: Grossly, all the fish pancreata examined contained 1-3 nodules of variable diameter (1-8 mm). Microscopically, the nodules were uniform, highly cellular, and composed of polygonal to elongated cells. Immunofluorescence for pancreatic hormones was performed. The nodules were immunoreactive for insulin most prominent centrally, but with IAPP and glucagon only in the periphery of the nodules. Exocrine pancreas was positive for chromogranin A. Not previously recognized in fish, IAPP immunoreactivity occurred in α, glucagon-containing, cells and did not co-localize with insulin in β cells. The islet tissues were devoid of amyloid deposits. IAPP DNA sequencing was performed to compare the sequence among teleost fish and the potency to form amyloid fibrils. In silico analysis of the amino acid sequences 19-34 revealed that it was not amyloidogenic. Conclusions: Amyloidosis of pancreatic islets would not be expected as a spontaneous disease in the Atlantic wolffish. Our study underlines that this teleost fish is a potential candidate for pancreatic xenograft research.

Enhanced /3-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an inyortant role in the pancreatic It-cell proliferation and insulin secretion. In the present study. we have investigaled how sympathetic system mgulales the panrrealic n I rnerui nr ht an:ilyiing I'pinephi inn 1111 ), Norepinephrinc (NE) and /1-adrenergic receptor changes in the brain as (%eli is in the I swirls. Fill and NII showed a significant decrease in the brain regions, pancreas and plasma :rt 72Ius iller partial prurcrealectonty. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using I CHI propranolol showed a significant increase in the number of loth the low affinity and high affinity t-adrenergic receplors in cerebral cortex and hypothalamus of partially pancreatectornised rats during peak DNA synthesis. The affinity of the receptors decrea,ed significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In file brain stein, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic ff-adrenergic receptors were also up regulated at 72 firs after partial panerealectony. In vitro studies showed that /i-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the t-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic ft-cell proliferation an(hilisulin secretion in wean hug rats.

Inflammation of the Hypothalamus Leads to Defective Pancreatic Islet Function

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic beta-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic beta-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor a leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-gamma coactivator Delta a and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1 alpha expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.

B-cell loss and B-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition

Amyloid deposition and reduced β-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased β-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased β-cell area quantified on histological sections is correlated with increased β-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and β cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic β cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased -cell area, and increased β-cell βapoptosis, as expected. There was a strong inverse correlation between β-cell area and amyloid deposition (r=0.42, P < 0.001). β-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had β-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with β-cell apoptosis (r= 0.56, P < 0.01). Thus, islet amyloid is associated with decreased β-cell area and increased β-cell apoptosis, suggesting that islet myloid deposition contributes to the decreased β-cell mass that characterizes type 2 diabetes.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress

Aims/hypothesis Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP.

Methods Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1).

Results Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers.

Conclusions/interpretation Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

Neprilysin impedes islet amyloid formation by inhibition of fibril formation rather than peptide degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.