Pancreatic beta-cell insulin signaling in genetic and dietary models of obesity and insulin resistance

Type 2 Diabetes Mellitus (T2DM) is a heterogeneous metabolic disease characterized by elevated blood glucose levels that has reached pandemic proportions. Genome-wide association studies have linked T2DM to the function of the insulin-producing pancreatic βcell residing in the micro-organ islet of Langerhans. An individual´s risk to develop T2DM depends on genetic predisposition and environmental factors, e.g. life style. Central for disease development is the interplay between insulin resistance in insulin target tissues like muscle, liver and fat and deficient β-cell insulin secretion. Since the β-cell is an insulin target itself, βcell insulin resistance can contribute to β-cell dysfunction and the development of T2DM. This was shown in several genetic (knockout) mouse models, however the dynamics of β-cell insulin resistance and its relevance in a diet-induced context has so far not been explored. Furthermore the consequences of diet-induced β-cell insulin resistance for β-cell function remain to be understood. The difficulty to study β-cell insulin resistance in vivo has partly been due to the lack of a technique to monitor β-cell insulin resistance non-invasively and longitudinally in the living organism. In my thesis I employed the anterior chamber of the eye of mice as a transplantation site for biosensor-expressing reporter islets and the cornea as a natural body window to monitor β-cell insulin resistance non-invasively and longitudinally by microscopic imaging. The β-cell insulin resistance biosensor is based on GFP-labeled FoxO1, that changes its intracellular localization from cytoplasmic (insulin responsive) to nuclear (insulin resistant). With this technique we investigated β-cell insulin resistance dynamics in ob/ob and NZO mice and demonstrated that β-cell insulin resistance dynamics vary in animal models of insulin resistance and obesity. Furthermore, we showed that β-cell insulin resistance developed in the presence of whole-body insulin resistance, impaired glucose tolerance and increased body weight, but independently from liver insulin resistance. To study the relevance of β-cell insulin resistance in diet-induced T2DM development, we treated diabetes-prone male C57BL/6J mice with different combinations of solid high fat diet and drinking water containing either sucrose or fructose. Employing our new monitoring technique we showed that only mice that were fed a High-FatHigh-Sucrose-Diet developed β-cell insulin resistance. This demonstrated the importance of βcell insulin resistance in a model of diet-induced obesity and insulin resistance and highlighted the importance of diet composition for the development of T2DM. The β-cell insulin resistance was accompanied by a decreased functional β-cell mass and impaired insulin secretion downstream of glucose-stimulated Ca2+ influx, due to a reduction of syntaxin-1A. We were also able to show that β-cell insulin resistance in one insulin signaling cascade can re-route the insulin signal, thus allowing the co-existence of reduced and increased insulin response in the same cell. In conclusion, my in vivo studies of diet-induced β-cell insulin resistance and its consequences on β-cell function and survival contribute to better understanding of the development of T2DM

Ectopic leptin production by intraocular pancreatic islet organoids ameliorates the metabolic phenotype of ob/ob mice

The pancreatic islets of Langerhans consist of endocrine cells that secrete peptide hormones into the blood circulation in response to metabolic stimuli. When transplanted into the anterior chamber of the eye (ACE), pancreatic islets engraft and maintain morphological features of native islets as well as islet-specific vascularization and innervation patterns. In sufficient amounts, intraocular islets are able to maintain glucose homeostasis in diabetic mice. Islet organoids (pseudo-islets), which are formed by self-reassembly of islet cells following disaggregation and genetic manipulation, behave similarly to native islets. Here, we tested the hypothesis that genetically engineered intraocular islet organoids can serve as production sites for leptin. To test this hypothesis, we chose the leptin-deficient ob/ob mouse as a model system, which becomes severely obese, hyperinsulinemic, hyperglycemic, and insulin resistant. We generated a Tet-OFF-based beta-cell-specific adenoviral expression construct for mouse leptin, which allowed efficient transduction of native beta-cells, optical monitoring of leptin expression by co-expressed fluorescent proteins, and the possibility to switch-off leptin expression by treatment with doxycycline. Intraocular transplantation of islet organoids formed from transduced islet cells, which lack functional leptin receptors, to ob/ob mice allowed optical monitoring of leptin expression and ameliorated their metabolic phenotype by improving bodyweight, glucose tolerance, serum insulin, and C-peptide levels

Human islet microtissues as an in vitro and an in vivo model system for diabetes

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents

Tissue‐specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes

PI3K-C2α Knockdown Results in Rerouting of Insulin Signaling and Pancreatic Beta Cell Proliferation

Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic β cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the β cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance

Diet-induced -cell insulin resistance results in reversible loss of functional -cell mass

Although convincing in genetic models, the relevance of -cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that -cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased -cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional -cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional -cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional -cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.Paschen, M., Moede, T., Valladolid-Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., Garcia-Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.-O. Diet-induced -cell insulin resistance results in reversible loss of functional -cell mass

Apolipoprotein CIII links islet insulin resistance to β-cell failure in 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.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted Versio

Surfactant proteins in pediatric interstitial lung disease

BACKGROUND: Children's interstitial lung diseases (chlLD) comprise a broad spectrum of diseases. Besides the genetically defined surfactant dysfunction disorders, most entities pathologically involve the alveolar surfactant region, possibly affecting the surfactant proteins SP-B and SP-C. Therefore, our objective was to determine the value of quantitation of SP-B and SP-C levels in bronchoalveolar lavage fluid (BALF) for the diagnosis of chlLD. METHODS: Levels of SP-B and SP-C in BALF from 302 children with chlLD and in controls were quantified using western blotting. In a subset, single-nucleotide polymorphisms (SNPs) in the SFTPC promoter were genotyped by direct sequencing. RESULTS: While a lack of dimeric SP-B was found only in the sole subject with hereditary SP-B deficiency, low or absent SP-C was observed not only in surfactant dysfunction disorders but also in patients with other diffuse parenchymal lung diseases pathogenetically related to the alveolar surfactant region. Genetic analysis of the SFTPC promoter showed association of a single SNP with SP-C level. CONCLUSION: SP-B levels may be used for screening for SP-B deficiency, while low SP-C levels may point out diseases caused by mutations in TTF1, SFTPC, ABCA3, and likely in other genes involved in surfactant metabolism that remain to be identified. We conclude that measurement of levels of SP-B and SP-C was useful for the differential diagnosis of chlLD, and for the precise molecular diagnosis, sequencing of the genes is necessary