Erwin Ilegems

T1R3 and gustducin in gut sense sugars to regulate expression of Na-glucose cotransporter 1

Dietary sugars are transported from the intestinal lumen into absorptive enterocytes by the sodium-dependent glucose transporter isoform 1 (SGLT1). Regulation of this protein is important for the provision of glucose to the body and avoidance of intestinal malabsorption. Although expression of SGLT1 is regulated by luminal monosaccharides, the luminal glucose sensor mediating this process was unknown. Here, we show that the sweet taste receptor subunit T1R3 and the taste G protein gustducin, expressed in enteroendocrine cells, underlie intestinal sugar sensing and regulation of SGLT1 mRNA and protein. Dietary sugar and artificial sweeteners increased SGLT1 mRNA and protein expression, and glucose absorptive capacity in wild-type mice, but not in knockout mice lacking T1R3 or α-gustducin. Artificial sweeteners, acting on sweet taste receptors expressed on enteroendocrine GLUTag cells, stimulated secretion of gut hormones implicated in SGLT1 up-regulation. Gut-expressed taste signaling elements involved in regulating SGLT1 expression could provide novel therapeutic targets for modulating the guts capacity to absorb sugars, with implications for the prevention and/or treatment of malabsorption syndromes and diet-related disorders including diabetes and obesity.

Donor Islet Endothelial Cells in Pancreatic Islet Revascularization

OBJECTIVE Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets. RESEARCH DESIGN AND METHODS Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts’ metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy. RESULTS DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets. CONCLUSIONS In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.

REEP2 enhances sweet receptor function by recruitment to lipid rafts.

Heterologously expressed sensory receptors generally do not achieve the ligand sensitivity observed in vivo, and may require specific accessory proteins to ensure optimal function. We searched for taste cell-expressed receptor transporting protein (RTP) and receptor expression enhancing protein (REEP) family members that might serve as accessory molecules to enhance gustatory receptor function. We determined that REEP2 is an integral membrane protein expressed in taste cells, physically associates with both subunits of the type 1 taste receptor 2 and type 1 taste receptor 3 sweet receptor and specifically enhances responses to tastants of heterologously expressed sweet and bitter taste receptors. Downregulation of endogenously expressed REEP2 in the chemosensory enteroendocrine GLUTag cell line dramatically reduced sensitivity of endogenous sweet receptors. In contrast to the observation that RTP1, RTP2, and REEP1 enhance function of olfactory receptors by promoting their transit to the cell surface, we found that REEP2 does not increase cell surface expression of sweet receptors but instead alters their spatial organization. REEP2 recruits sweet receptors into lipid raft microdomains localized near the taste cells apical region, thereby improving G-protein-coupled receptor signaling and promoting receptor access to tastants arriving through the apical taste pore.

Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes

Significance Insulin resistance and β-cell failure are the major defects in type 2 diabetes. We now demonstrate that local insulin resistance-induced increase in apolipoprotein CIII (apoCIII) within pancreatic islets causes promotion of an intraislet inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i), and apoptosis. Decreasing apoCIII in vivo in animals with insulin resistance improves glucose tolerance, and apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of apoCIII, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes. Insulin resistance and β-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

Imaging dynamics of CD11c(+) cells and Foxp3(+) cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes

Aims/hypothesisThe aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes.MethodsWe adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets.ResultsWe demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour.Conclusions/interpretationTogether, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.

Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion.

The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

Nucleobindin 1 (NUCB1) is a Golgi-resident marker of neurons.

Nucleobindin 1 (NUCB1; also known as CALNUC or NUC) is a putative DNA- and calcium-binding protein and exhibits significant structural homology with the protein nucleobindin 2 (NUCB2; also known as nesfatin). While NUCB2 has been mapped in detail in the brain and implicated in the hypothalamic control of energy metabolism, no study has to date addressed the presence of NUCB1 in the central nervous system. Here we have explored the expression and distribution of NUCB1 in the rat brain and spinal cord, using RT-PCR, immunofluorescence and in situ hybridization. NUCB1 mRNA and protein was found to be present in all brain regions, extending to the spinal cord and dorsal root ganglia. Double-staining for NUCB1 and NeuN, glial fibrillary acidic protein and myelin basic protein revealed that NUCB1 is exclusively found in neurons, and not in glial or ependymal cells. Notably, NUCB1-immunoreactivity was observed in all neurons examined, making no distinction between previously identified glutamatergic and GABAergic populations, including those that are known not to stain for NeuN. This included the markedly more restricted population of NUCB2-expressing neurons in the brain. The protein was detected in cell somata and proximal dendrites, but not in axons or terminal structures. Further examination of the subcellular distribution of NUCB1 using organelle-specific markers revealed its consistent presence in the Golgi apparatus. These findings identify NUCB1 as a novel pan-neuronal marker. Along with the recent demonstration of broad expression of the protein in endocrine cells, the present results suggest that NUCB1 may play a role in spatiotemporal calcium handling in signaling cells.

Expression of nucleobindin 1 (NUCB1) in pancreatic islets and other endocrine tissues

The protein nucleobindin 1 (NUCB1; also known as CALNUC or Nuc) contains an intriguing combination of DNA- and calcium-binding motifs, a trait that it shares with the protein nucleobindin 2 (NUCB2; also known as nesfatin). NUCB2 has been implicated in several aspects of metabolic control and has been identified in a number of endocrine organs. No such comprehensive mapping of NUCB1 has been presented. We have explored the expression and distribution of NUCB1 in tissues and cells of the mouse endocrine system, with particular focus on the endocrine pancreas. Using reverse transcription plus the polymerase chain reaction (RT-PCR) and Western blot, we demonstrate that NUCB1 is present in the endocrine islets of Langerhans but absent from the exocrine acinar cells. Immunofluorescence studies have revealed that all islet cell types contain NUCB1, including the NUCB2-expressing beta cells. RT-PCR, Western blot and immunofluorescence have shown that NUCB1 is expressed in the pituitary, thyroid, parathyroid, gastrointestinal tract, adrenals and gonads. However, within these tissues, NUCB1 expression is not ubiquitous. For example, in the testis, NUCB1 occurs in the seminiferous tubules but not in the Leydig-cell-containing interstitial tissue. Similarly, the lamina propria of the duodenum lacks NUCB1, despite its presence in enterocytes. Where present, NUCB1 consistently appears to be associated with the Golgi apparatus. Thus, NUCB1 is broadly, but not ubiquitously, expressed in cells of the mouse endocrine system. Together with its location in the Golgi apparatus and its putative Ca2+-binding ability, this distribution suggests a role for NUCB1 in Ca2+ handling/sensing in secretory cells.

Biochemical profiling of diabetes disease progression by multivariate vibrational microspectroscopy of the pancreas

Despite the dramatic increase in the prevalence of diabetes, techniques for in situ studies of the underlying pancreatic biochemistry are lacking. Such methods would facilitate obtaining mechanistic understanding of diabetes pathophysiology and aid in prognostic and/or diagnostic assessments. In this report we demonstrate how a multivariate imaging approach (orthogonal projections to latent structures - discriminant analysis) can be applied to generate full vibrational microspectroscopic profiles of pancreatic tissues. These profiles enable extraction of known and previously unrecorded biochemical alterations in models of diabetes, and allow for classification of the investigated tissue with regards to tissue type, strain and stage of disease progression. Most significantly, the approach provided evidence for dramatic alterations of the pancreatic biochemistry at the initial onset of immune-infiltration in the Non Obese Diabetic model for type 1 diabetes. Further, it enabled detection of a previously undocumented accumulation of collagen fibrils in the leptin deficient ob/ob mouse islets. By generating high quality spectral profiles through the tissue capsule of hydrated human pancreata and by in vivo Raman imaging of pancreatic islets transplanted to the anterior chamber of the eye, we provide critical feasibility studies for the translation of this technique to diagnostic assessments of pancreatic biochemistry in vivo.

You are what you eat—Do not blame your mother

The incidence of diabetes mellitus worldwide is increasing and especially in case of type 2 diabetes (T2DM) reaching epidemic proportions. According to the Diabetes Atlas recently published by the International Diabetes Federation, in 2011 more than 360 million people suffered from the disease and this number will increase to more than 550 million by 2030 [1]. This represents a huge personal burden for the people suffering from the disease but also a financial challenge for society to combat all resulting consequences. Genome-wide association studies revealed a considerable number of candidate genes having a role potentially associated with the development and function of the pancreatic islet of Langerhans [2], thus placing this micro-organ back into the center of attention. However, the outcome of these studies made also clear that only a small proportion of T2DM can be solely explained by genetic factors, and underlines the contribution of a changing lifestyle in the development of obesity and insulin resistance. Indeed a decrease in calorie expenditure, due to reduced physical activity in combination with an increased calorie intake, resulted over the last three decades in an increase in obesity especially in the younger generation, including young parents as well as their children. Obesity is one of the biggest risk factors for developing T2DM, however the underlying mechanism(s) remain unclear. One of the key questions is how maternal high-calorie consumption during pregnancy and by the child after birth influences the development of the pancreatic islets of Langerhans in the offspring. In this issue, Comstock and colleagues [3] address this important question by studying the impact of early programming (maternal diet) versus postnatal programming (post-weaning diet) on pancreatic islet development in non-human primates (NHP). To accomplish this, the authors compared pancreata from offspring in four different groups up to the age of 13 months. The first group, named HFD/HFD, was offspring to mothers that were fed a high-fat diet (HFD) during pregnancy and where the offspring continued HFD feeding after the weaning period. The second group, CTR/HFD, was offspring to mothers that were fed a control diet (CTR) during pregnancy and where the offspring continued HFD feeding after the weaning period. The third group, HFD/CTR, was offspring to mothers that were fed HFD during pregnancy and where the offspring was fed a control diet post-weaning. These three groups were compared to a control group, CTR/CTR, i.e. offspring to mothers that were fed a control diet during pregnancy and where the offspring was fed a control diet post-weaning. While pancreata of the HFD/HFD and CTR/HFD groups showed an increase in islet mass, islet mass in the HFD/CTR group normalized to control levels indicating the high plasticity during islet development. Interestingly, while the islet mass of both HFD/HFD and CTR/HFD groups were increased, this was accomplished by different mechanisms (Fig. 1A). In the HFD/HFD group this increase was gained by an increase in islet diameter while in the CTR/HFD group the authors observed an increase in islet number and islet density. Another striking difference concerned the ratio of insulin-producing β-cells to glucagon-producing α-cells. Islets of the CTR/HFD group showed an increased number of both β-cells and α-cells. Islets of the HFD/HFD group, however, showed no increase in α-cell number, which resulted in an almost doubling of the β-cell/α-cell ratio. The close to normal serum glucagon levels in the HFD/HFD group were very likely gained by a hyperactivity of these α-cells. These novel observations are highly interesting and intensify our desire to get more insights into the molecular mechanisms underlying the striking differences in the development of pancreatic islets in the HFD/HFD versus CTR/HFD groups. Earlier data published by the same group showed that HFD during pregnancy in NHP leads to placental insufficiency and placental inflammation, the latter resulting in increased circulating concentration of cytokines in the fetus which may affect organ development [4]. Other observations by these authors presented in this issue [3] and earlier [5] demonstrate that maternal HFD leads to the development of early hepatic insulin resistance in the fetus, which is reflected by decreased clearing of circulating insulin, increased serum triglyceride levels and increased hepatic expression of genes involved in gluconeogenesis. Figure 1 (A) The endocrine cells within the islets of Langerhans in non-human primates, similarly to those in humans, have specific paracrine interactions due a majority of heterotypic cellular contacts. This is in contrast to endocrine cells in the mouse, which ... A major strength of the present study is the experimental model, namely the use of non-human primates. Although there is a wealth of data on pancreatic islet development and function in health and disease gained from studies on various rodent models, their relevance to human pancreatic islet function and dysfunction has to be taken with caution in the light of novel findings demonstrating that differences in the islet architecture between rodents and NHP/human have functional consequences (see [6] and references therein). With regard to the study by Comstock and colleagues in this issue [3], a change in the islet composition as seen in the HFD/HFD group will certainly have consequences in islet function in general and in α-cell and β-cell function in particular. Data by Rodriguez-Diaz et al. demonstrated that human α-cells in addition to secrete glucagon also secrete the neurotransmitter acetylcholine, which primes the secretory response of human β-cell to glucose by paracrine action [7]. A reduction in α-cell number thus may also lead to reduced paracrine priming of NHP/human β-cells leading to reduced insulin secretion, as reflected by lower circulating insulin C-peptide levels in the HFD/HFD group in the present study [3]. Another highly interesting point concerns the plasticity of NHP/human islets. Pancreatic islets develop until post-puberty in both humans and NHPs. The present study on NHP macaques, which was carried out until age 13 months after birth, showed that islet composition in the HFD/CTR group, despite maternal HFD diet and potential associated complications in organ development during pregnancy, normalized to control levels. It is of great interest to learn what time-frame is critical for islet development during infancy/puberty and what is the ‘point-of-no-return’ in islet programming by HFD during development. Such a longitudinal study would ideally be non-invasive and allow monitoring of pancreatic islet morphology and function in a living NHP under different dietary regimes. A potential strategy to achieve this would be by applying an in vivo imaging approach, such as published by Speier and colleagues [8,9]. Following surgical capture of a small part of the pancreas and subsequent isolation of pancreatic islets, a few islets will be used for auto-transplantation into the anterior chamber of the eye (Figure 1B). Following engraftment, these islets can be readily monitored by fluorescence microscopy through the cornea and will serve as representatives of the in situ pancreatic islets. Such an approach would not only allow to non-invasively monitor islet morphology and function longitudinally at single-cell resolution, but would also allow evaluating the consequences of changing dietary regimes in the same NHP.