Histological changes in endocrine and exocrine pancreatic tissue from patients exposed to incretin-based therapies.

AIMS: Incretin-based therapies have been associated with an increased risk of pancreatitis. Recently, various histological abnormalities have been reported in human pancreatic tissue from brain-dead organ donors who had been exposed to incretin-based drugs. In the present study we examined pancreatic tissue collected at surgery. METHODS: Human pancreatic tissue from 7 type 2-diabetic patients treated with incretin-based drugs (type 2-I), 6 diabetic patients without incretin treatment (type 2-NI), 11 patients without diabetes (no diabetes group) and 9 brain-dead organ donors (BDOD group) was examined. RESULTS: Fractional beta-cell area was reduced in the type 2-NI group compared to the group without diabetes (P < .05), but there was no difference compared to the type 2-I patients. Alpha-cell area (P = .30), beta-cell replication (P = .17) and alpha-cell replication (P = .91) were not different. There were also no differences in acinar cell (P = .13) and duct cell replication (P = .099). Insulin-positive duct cells were more frequent in the type 2-I and the BDOD groups (P = .034). No co-expression of insulin and glucagon was detected. Pancreatic intraepithelial neoplasia (PanIN) lesions were very rare, all low-grade (PanIN 1a and 1b) and tended to occur more frequently in the type 2-I group (P = .084). CONCLUSIONS: The present results did not reveal marked histological abnormalities in the pancreas of incretin-treated patients with type 2 diabetes. Low numbers of specimens available and a large inter-individual variability of the findings warrant caution regarding the interpretation of histological data concerning drug effects on the human pancreas.

Diminished glucagon suppression after ß-cell reduction is due to impaired α-cell function rather than an expansion of α-cell mass.

Impaired suppression of glucagon levels after oral glucose or meal ingestion is a hallmark of type 2 diabetes. Whether hyperglucagonemia after a ß-cell loss results from a functional upregulation of glucagon secretion or an increase in α-cell mass is yet unclear. CD-1 mice were treated with streptozotocin (STZ) or saline. Pancreatic tissue was collected after 14, 21, and 28 days and examined for α- and ß-cell mass and turnover. Intraperitoneal (ip) glucose tolerance tests were performed at day 28 as well as after 12 days of subcutaneous insulin treatment, and glucose, insulin, and glucagon levels were determined. STZ treatment led to fasting and post-challenge hyperglycemia (P < 0.001 vs. controls). Insulin levels increased after glucose injection in controls (P < 0.001) but were unchanged in STZ mice (P = 0.36). Intraperitoneal glucose elicited a 63.1 ± 4.1% glucagon suppression in control mice (P < 0.001), whereas the glucagon suppression was absent in STZ mice (P = 0.47). Insulin treatment failed to normalize glucagon levels. There was a significant inverse association between insulin and glucagon levels after ip glucose ingestion (r(2) = 0.99). ß-Cell mass was reduced by ∼75% in STZ mice compared with controls (P < 0.001), whereas α-cell mass remained unchanged (P > 0.05). α-Cell apoptosis (TUNEL) and replication (Ki67) were rather infrequently noticed, with no significant differences between the groups. These studies underline the importance of endogenous insulin for the glucose-induced suppression of glucagon secretion and suggest that the insufficient decline in glucagon levels after glucose administration in diabetes is primarily due to a functional loss of intraislet inhibition of α-cell function rather than an expansion of α-cell mass.

Protection from diabetes development by single-chain antibody-mediated delivery of a NF-κB inhibitor specifically to ß-cells in vivo.

Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human ß-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in ß-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect ß-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat ß-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1ß + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved ß-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects ß-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.

Islet Amyloid in Patients With Diabetes Due to Exocrine Pancreatic Disorders, Type 2 Diabetes, and Nondiabetic Patients.

BACKGROUND: Amyloid deposits are a typical finding in pancreatic islets from patients with type 2 diabetes. Whether this is linked to the pathogenesis of type 2 diabetes is currently unknown. Therefore, we compared the occurrence of islet amyloid in patients with type 2 diabetes, diabetes secondary to pancreatic disorders, and nondiabetic individuals. PATIENTS AND METHODS: Pancreatic tissue from 15 nondiabetic patients, 22 patients with type 2 diabetes, and 11 patients with diabetes due to exocrine pancreatic disorders (chronic pancreatitis, pancreatic carcinoma) were stained for insulin, amyloid, and apoptosis. ß-cell area, amyloid deposits, and ß-cell apoptosis were quantified by morphometric analysis. RESULTS: The proportion of islets containing amyloid deposits was significantly higher in both type 2 diabetes and diabetes due to exocrine pancreatic disorders than in healthy subjects. Islets with both amyloid and apoptosis were observed more frequently in type 2 diabetes and significantly more so in diabetes due to exocrine pancreatic disorders. In both diabetic groups, apoptotic ß-cells were found significantly more frequently in islets with more prominent amyloid deposits. CONCLUSIONS: The occurrence of amyloid deposits in both type 2 diabetes and diabetes secondary to exocrine pancreatic disorders suggests that islet amyloid formation is a common feature of diabetes mellitus of different etiologies and may be associated with a loss of pancreatic ß-cells.

Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

INTRODUCTION: The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. METHODS: To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. RESULTS: The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. CONCLUSIONS: The expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration.

Abundance and turnover of GLP-1 producing L-cells in ileal mucosa are not different in patients with and without type 2 diabetes.

INTRODUCTION: The gastrointestinal hormone GLP-1 is released from enteroendocrine L-cells and augments postprandial insulin secretion. In patients with type 2 diabetes, the incretin effect is markedly diminished. It is unclear, whether this is due to a reduction in the abundance of L-cells in the intestine. METHODS: Ileal tissue samples from 10 patients with and 10 patients without diabetes that underwent surgery for the removal of colon tumors were included. Tissue sections were stained for GLP-1, Ki67, TUNEL and chromogranin A. RESULTS: The number of L-cells was not different between patients with and without diabetes in either crypts (1.81±0.21% vs. 1.49±0.24%, respectively; p=0.31) or villi (1.07±0.16% vs. 0.83±0.10%, respectively; p=0.23). L-cell number was higher in crypts than in villi (p<0.0001). L-cell replication was detected rarely and not different between the groups. L-cell apoptosis was similar in patients with and without diabetes in both crypts (7.84±2.77% vs. 8.65±3.77%, p=0.85) and villi (4.48±2.89% vs. 8.62±4.64%, p=0.42). Chromogranin A staining was found in a subset of L-cells only. CONCLUSIONS: Intestinal L-cell density is higher in crypts than in villi. Chromogranin A is not a prerequisite for GLP-1 production. L-cell density and turnover are not different between patients with and without diabetes. Thus, alterations in the number of GLP-1 producing cells do not explain the reduced incretin effect in patients with type 2 diabetes.

In vivo visualization of single native pancreatic islets in the mouse.

The purpose of this study was to investigate the potential of a novel targeted contrast agent (CA) for the in vivo visualization of single native pancreatic islets, the sites of insulin production, in the pancreas of mice using magnetic resonance imaging (MRI). The CA for intravenous administration was composed of the ß-cell-specific single-chain antibody fragment, SCA B1, and ferromagnetic carbon-coated cobalt nanoparticles. MRI experiments were performed at 7, 9.4 and 16.4 T in excised organs (pancreas, liver, kidney, spleen), at 7 T in mice fixed in formalin and at 9.4 and 16.4 T in living mice. Image contrast in untreated control animals was compared with images from mice treated with unspecific and specific CA. For the validation of MRI results, selected pancreases were subjected to immunohistochemical staining and numerical contrast simulations were performed. Ex vivo results and the outcome of immunohistochemistry suggest that islets are marked only by the CA containing SCA B1. Strong accumulation of particles was found also in other investigated organs owing to the uptake by the reticuloendothelial system, but the contrast in the MR images is clearly distinguishable from the islet specific contrast in pancreases and numerical predictions. In vivo experiments based on averaged dynamic sampling with 66 × 66 × 100 µm³ and triggered acquisition with 90 × 90 × 200 µm³ nominal resolution resulted in similar particle contrast to in in vitro measurements. The newly developed CA and MRI strategies have the potential to be used for studying mouse diabetes models by visualizing single native pancreatic islets.

Generation of novel single-chain antibodies by phage-display technology to direct imaging agents highly selective to pancreatic beta- or alpha-cells in vivo.

OBJECTIVE: Noninvasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell-specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS: To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS: We report the generation of SCAs that bind highly selective to either beta- or alpha-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to beta- or alpha-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [(125)I]-labeled SCAs after intravenous administration in rats strongly predicted the beta-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS: Our data provide strong evidence that the presented SCAs are highly specific for pancreatic beta-cells and enable imaging and quantification in vivo.