Differentiation of mouse nuclear transfer embryonic stem cells into functional pancreatic beta cells

AIMS/HYPOTHESIS: Therapeutic cloning has been reported to have potential in the treatment of several degenerative diseases. However, it has yet to be determined whether mouse nuclear transfer-embryonic stem cells (NT-ESCs) can be differentiated into pancreatic beta cells and used to reverse diabetes in an animal model. METHODS: We first used the somatic nuclear transfer technique to generate mouse NT-ESCs and then developed a chemically defined stepwise protocol to direct the NT-ESCs into functional pancreatic beta cells. We examined the gene expression pattern of the differentiated NT-ESCs and transplanted the NT-ESC-derived insulin-producing cells into recipient diabetic mice. RESULTS: Four mouse NT-ESC lines were first established using an improved nuclear transfer technique and insulin-producing cells were efficiently generated from NT-ESCs by mimicking pancreatic in vivo development. Most of the insulin-producing cells that we generated co-produced pancreatic and duodenal homeobox 1, but not glucagon at the final stage of this differentiation method, which differed from the insulin and glucagon co-production reported by other groups. The differentiated NT-ESCs were able to release insulin in response to glucose stimuli and normalise the blood glucose level of diabetic mice for at least 2 months. CONCLUSIONS/INTERPRETATION: These results demonstrate the potential of therapeutic cloning for cell therapy of type 1 diabetes in a mouse model.     

Evidence for the presence of type I insulin-like growth factor receptors on rat pancreatic A and B cells

Purified rat pancreatic islet cells express somatomedin receptors which are identified by their affinity for insulin-like growth factor (IGF)-I, IGF-II, and insulin. Binding of [125I]IGF-I to islet A cells was half-maximally inhibited by 7.10(-10) M IGF-I, while IGF-II, insulin, and proinsulin were respectively 10-, 500-, and 10,000-fold less potent displacers of IGF-I binding. Unrelated hormones such as glucagon or GH did not compete with [125I]IGF-I binding to A cells. The concentration of IGF-I receptors on A cells was estimated at 5000 IGF-I binding sites per cell with affinity constant (Ka) of 2 X 10(9) M-1. Islet B cells were found to exhibit a reversible time- and temperature-dependent binding with [125I]IGF-I. Specificity and affinity of IGF-I binding sites were identical for islet A and B cells. Linear Scatchard plots of competitive binding data on B cells suggest 1 single class of IGF-I receptors in a concentration of 12,000 sites per cell. The presence of high affinity receptors for IGF-I on adult islet A and B cells provides a molecular basis for this growth factor to influence growth, survival, and/or function of these endocrine cell types. Their low affinity for insulin should be considered as a potential mechanism for this hormone to influence, at high concentration, the function of islet A and B cells.     

Lysosomes in normal pancreatic beta cells

Summary Lysosomes and their relationships with surrounding organelles were studied in pancreatic B-cells of normal Wistar rats by electron microscopy and cytochemical localization of acid phosphatase (AcPase) and arylsulphatase. Several forms of lysosomes were distinguished and shown to interact frequently with the secretory granules. Two different digestive processes were recognized. During crinophagy, lysosomes directly fuse with secretory granules while, during autophagy, a cytoplasmic area containing secretory granules and/or other organelles is circumscribed before the enzymatic digestion. In addition, lysosomes may transport arylsulphatase into secretory granules apparently not involved in a destructive process. Such a process could also account, at least in part, for the presence of AcPase in a certain number of mature and immature secretory granules.     

Incretin-based therapy and pancreatic beta cells

Type 2 diabetes (T2D) is a complex, progressive disease with life-threatening complications and one of the most serious public-health problems worldwide. The two main mechanisms of T2D pathogenesis are pancreatic beta cell dysfunction and insulin resistance. It is now recognized that pancreatic beta cell dysfunction is a necessary factor for T2D development. Traditional therapies for controlling blood glucose are suboptimal as they fail to meet target goals for many patients. Glucagon-like peptide-1 receptor agonists (GLP1RA) and dipeptidyl peptidase-4 inhibitors (DPP4I) are an attractive class of therapy because they reduce blood glucose by targeting the incretin hormone system and, in particular, have the potential to positively affect pancreatic beta cell biology. This review outlines our current understanding of pancreatic beta cell incretin system dysfunction in T2D and summarizes recent evidence of the effect of incretin-based therapies on beta cell function and mass. Incretin-based therapies have shown strong evidence for beneficial effects on beta cell function and mass in animal studies. In humans, incretin-based therapies are effective glucose-lowering agents, but further study is still required to evaluate their long-term effects on beta cell function and safety as well as beta cell mass expansion.     

Evidence for thymopoietin and thymopoietin/alpha-bungarotoxin/nicotinic receptors within the brain.

Thymopoietin, a polypeptide hormone of the thymus that has pleiotropic actions on the immune, endocrine, and nervous systems, potently interacts with the neuromuscular nicotinic acetylcholine receptor. Thymopoietin binds to the nicotinic alpha-bungarotoxin (alpha-BGT) receptor in muscle and, like alpha-BGT, inhibits cholinergic transmission at this site. Evidence is given that radiolabeled thymopoietin similarly binds to a nicotinic alpha-BGT-binding site within the brain and does so with the characteristics of a specific receptor ligand. Thus specific binding to neuronal membranes was saturable, of high affinity (Kd = 8 nM), linear with increased tissue concentration, and readily reversible; half-time was approximately 5 min for association and 10 min for dissociation. Binding of 125I-labeled thymopoietin was displaced not only by unlabeled thymopoietin but also by alpha-BGT and the nicotinic receptor ligands d-tubocurarine and nicotine; various other receptor ligands (muscarinic, adrenergic, and dopaminergic) did not affect binding of 125I-labeled thymopoietin. Thymopoietin was shown by ELISA to be present in brain extracts, displacement curves of thymus and brain extracts being parallel to the standard thymopoietin curve, and Western (immuno) blot identified in brain and thymus extracts a thymopoietin-immunoreactive polypeptide of the same molecular mass as purified thymopoietin polypeptide. We conclude that thymopoietin and thymopoietin-binding sites are present within the brain and that the receptor for thymopoietin is the previously identified nicotinic alpha-BGT-binding site of neuronal tissue.     

beta -Amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of beta-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the alpha7 gene product (alpha7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch-clamp recording we show here that nanomolar concentrations of beta-amyloid peptides specifically and reversibly block alpha7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. beta-Amyloid blockade is likely to be a common feature of alpha7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because alpha7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.     

Insulin granule dynamics in pancreatic beta cells

Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter 0.3 µm) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.Abbreviations [Ca²⁺]_i Free cytoplasmic Ca²⁺-concentration - EGFP enhanced GFP - fF femtofarad (10^-15F) - GFP green fluorescent protein - K_ATP-channel ATP-regulated K⁺-channel - LDCV large dense-core vesicle - ms millisecond - mV millivolt - RRP readily releasable pool of granules - SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptors     

Insulin receptors on pancreatic acinar cells in guinea pigs

The characteristics of interaction of insulin with specific receptors on exocrine pancreatic cells of the guinea pig have been studied. Insulins from different species as well as certain insulin analogs were found to have affinities to receptors on pancreatic acinar cells which are similar to what have been described for insulin receptors in other organs of different mammalian species. Binding was rapid and reversible at 37 C but dissociation was markedly slower at 12 C. Clear indications of negative cooperativity between binding sites were not seen. Bovine and chicken insulin bound with approximately a 100-fold higher affinity to guinea pig insulin receptors than guinea pig insulin itself. The number of insulin receptors per acinar cell were comparable with what has been described for other mammalian cells. Part of cell-associated insulin was internalized. After 60 min of incubation the major part of radioactivity in the incubation medium as well as in cells appeared as intact [125I] iodoinsulin on a Sephadex G-50 column and less than 12% of radioactivity was eluted as breakdown products together with Na 125I.