Molecular Mechanisms of Amylin Turnover, Misfolding and Toxicity in the Pancreas.

Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet ß-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of ß-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.

Therapeutic Ultrasound-Induced Insulin Release in Vivo.

The tolerability and efficacy of low-frequency, low-intensity therapeutic ultrasound-induced insulin release was investigated in a pre-clinical in vivo murine model. The treatment groups received a single 5-min continuous sonication at 1 MHz and 1.0 W/cm2. Insulin and glucagon levels in the serum were determined using enzyme-linked immunosorbent assay. The pancreas was excised and sectioned for histologic analysis. In terminal studies, we observed a moderate (∼50 pM) but significant increase in blood insulin concentration in vivo immediately after sonication compared with a decrease of approximately 60 pM in sham animals (n < 6, p < 0.005). No difference was observed in the change in glucose or glucagon concentrations between groups. Comparisons of hematoxylin and eosin-stained terminal and survival pancreatic tissue revealed no visible differences or evidence of damage. This study is the first step in assessing the translational potential of therapeutic ultrasound as a treatment for early stages of type 2 diabetes.

An inducible model of human amylin overexpression reveals diverse transcriptional changes.

Human Islet Amyloid Polypeptide or amylin is a neuroendocrine peptide with important endocrine and paracrine functions. Excessive production and accumulation of human amylin in the pancreas can lead to its aggregation and apoptosis of islet ß-cells. Amylin has been shown to function within the central nervous system to decrease food intake, and more recently, it has been revealed that amylin is directly transcribed from neurons of the central nervous system, including the hypothalamus, arcuate nucleus, medial preoptic area, and nucleus accumbens. These findings alter the current model of how amylin targets the nervous system, and as a result may lead to obesity and type II diabetes mellitus. Here we set out to use Caenorhabditis elegans as an inducible in vivo model system to study the effects of amylin overexpression in tissues that include the nervous system. We profiled the transcriptional changes in transgenic animals expressing human amylin through RNA-seq. Using this genome-wide approach our results revealed for the first time that expression of human amylin in tissues including the nervous system induce diverse physiological responses in various signaling pathways. From our characterization of transgenic C. elegans animals expressing human amylin, we also observed specific defects in neural developmental programs as well as sensory behavior. Taken together, our data demonstrate the utility of using C. elegans as a valuable in vivo model to study human amylin toxicity.

Ultrasound-Induced Insulin Release as a Potential Novel Treatmentfor Type 2 Diabetes Mellitus.

Therapeutic ultrasound presents a potential novel treatment for type 2 diabetes mellitus that utilizes the non-invasive application of ultrasound energy to treat secretory defects in the earlier stages of the disease. Our previous studies have shown that ultrasound is capable of stimulating insulin release from pancreatic beta cells, safely and effectively. This study aims to both examine the calcium-dependent mechanisms of ultrasound-mediated insulin release from pancreatic beta cells using three complementary modalities - carbon fiber amperometry, ELISA studies, and Ca2+ fluorescence imaging - and to study the translational potential of therapeutic ultrasound using transgenic hyperglycemic mice for safety and efficacy studies.

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells.

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet ß-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to ß-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic ß-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. ß-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in ß-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic ß-cells.

The Molecular Physiopathogenesis of Islet Amyloidosis.

Human islet amyloid polypeptide or amylin (hA) is a 37-amino acid peptide hormone produced and co-secreted with insulin by pancreatic ß-cells. Under physiological conditions, hA regulates a broad range of biological processes including insulin release and slowing of gastric emptying, thereby maintaining glucose homeostasis. However, under the pathological conditions associated with type 2 diabetes mellitus (T2DM), hA undergoes a conformational transition from soluble random coil monomers to alpha-helical oligomers and insoluble ß-sheet amyloid fibrils or amyloid plaques. There is a positive correlation between hA oligomerization/aggregation, hA toxicity, and diabetes progression. Because the homeostatic balance between hA synthesis, release, and uptake is lost in diabetics and hA aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies investigating molecular mechanisms of hA uptake, trafficking, and degradation in pancreatic cells and its relevance to h's toxicity. We will also discuss the regulatory role of endocytosis and proteolytic pathways in clearance of toxic hA species. Finally, we will discuss potential pharmacological approaches for specific targeting of hA trafficking pathways and toxicity in islet ß-cells as potential new avenues toward treatments of T2DM patients.

Calcium-dependent ultrasound stimulation of secretory events from pancreatic beta cells.

BACKGROUND: Our previous studies have indicated that ultrasound can stimulate the release of insulin from pancreatic beta cells, providing a potential novel treatment for type 2 diabetes. The purpose of this study was to explore the temporal dynamics and Ca2+-dependency of ultrasound-stimulated secretory events from dopamine-loaded pancreatic beta cells in an in vitro setup. METHODS: Carbon fiber amperometry was used to detect secretion from INS-1832/13 beta cells in real time. The levels of released insulin were also measured in response to ultrasound treatment using insulin-specific ELISA kit. Beta cells were exposed to continuous wave 800 kHz ultrasound at intensities of 0.1 W/cm2, 0.5 W/cm2 and 1 W/cm2 for several seconds. Cell viability tests were done with trypan blue dye exclusion test and MTT analysis. RESULTS: Carbon fiber amperometry experiments showed that application of 800 kHz ultrasound at intensities of 0.5 and 1 W/cm2 was capable of stimulating secretory events for durations lasting as long as the duration of the stimulus. Furthermore, the amplitude of the detected peaks was reduced by 64% (p < 0.01) when extracellular Ca2+ was chelated with 10 mM EGTA in cells exposed to ultrasound intensity of 0.5 W/cm2. Measurements of released insulin in response to ultrasound stimulation showed complete inhibition of insulin secretion by chelating extracellular Ca2+ with 10 mM EGTA (p < 0.01). Viability studies showed that 800 kHz, 0.5 W/cm2 ultrasound did not cause any significant effects on viability and metabolic activity in cells exposed to ultrasound as compared to sham-treated cells. CONCLUSIONS: Our results demonstrated that application of ultrasound was capable of stimulating the release of insulin from pancreatic beta cells in a safe, controlled and Ca2+-dependent manner.

Role of Cholesterol and Phospholipids in Amylin Misfolding, Aggregation and Etiology of Islet Amyloidosis.

Amyloidosis is a biological event in which proteins undergo structural transitions from soluble monomers and oligomers to insoluble fibrillar aggregates that are often toxic to cells. Exactly how amyloid proteins, such as the pancreatic hormone amylin, aggregate and kill cells is still unclear. Islet amyloid polypeptide, or amylin, is a recently discovered hormone that is stored and co-released with insulin from pancreatic islet ß-cells. The pathology of type 2 diabetes mellitus (T2DM) is characterized by an excessive extracellular and intracellular accumulation of toxic amylin species, soluble oligomers and insoluble fibrils, in islets, eventually leading to ß-cell loss. Obesity and elevated serum cholesterol levels are additional risk factors implicated in the development of T2DM. Because the homeostatic balance between cholesterol synthesis and uptake is lost in diabetics, and amylin aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies exploring molecular mechanisms by which cholesterol and phospholipids modulate secondary structure, folding and aggregation of human amylin and other amyloid proteins on membranes and in cells. Amylin turnover and toxicity in pancreatic cells and the regulatory role of cholesterol in these processes are also discussed.

Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells.

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.