Exploring optimal settings for safe and effective thulium fibre laser lithotripsy in a kidney model.

OBJECTIVES: To explore the optimal laser settings and treatment strategies for thulium fibre laser (TFL) lithotripsy, namely, those with the highest treatment efficiency, lowest thermal injury risk, and shortest procedure time. MATERIALS AND METHODS: An in vitro kidney model was used to assess the efficacy of TFL lithotripsy in the upper calyx. Stone ablation experiments were performed on BegoStone phantoms at different combinations of pulse energy (EP ) and frequency (F) to determine the optimal settings. Temperature changes and thermal injury risks were monitored using embedded thermocouples. Experiments were also performed on calcium oxalate monohydrate (COM) stones to validate the optimal settings. RESULTS: High EP /low F settings demonstrated superior treatment efficiency compared to low EP /high F settings using the same power. Specifically, 0.8 J/12 Hz was the optimal setting, resulting in a twofold increase in treatment efficiency, a 39% reduction in energy expenditure per unit of ablated stone mass, a 35% reduction in residual fragments, and a 36% reduction in total procedure time compared to the 0.2 J/50 Hz setting for COM stones. Thermal injury risk assessment indicated that 10 W power settings with high EP /low F combinations remained below the threshold for tissue injury, while higher power settings (>10 W) consistently exceeded the safety threshold. CONCLUSIONS: Our findings suggest that high EP /low F settings, such as 0.8 J/12 Hz, are optimal for TFL lithotripsy in the treatment of COM stones. These settings demonstrated significantly improved treatment efficiency with reduced residual fragments compared to conventional settings while keeping the thermal dose below the injury threshold. This study highlights the importance of using the high EP /low F combination with low power settings, which maximizes treatment efficiency and minimizes potential thermal injury. Further studies are warranted to determine the optimal settings for TFL for treating kidney stones with different compositions.

In vitro investigation of stone ablation efficiency, char formation, spark generation, and damage mechanism produced by thulium fiber laser.

To investigate stone ablation characteristics of thulium fiber laser (TFL), BegoStone phantoms were spot-treated in water at various fiber tip-to-stone standoff distances (SDs, 0.5 ~ 2 mm) over a broad range of pulse energy (Ep, 0.2 ~ 2 J), frequency (F, 5 ~ 150 Hz), and power (P, 10 ~ 30 W) settings. In general, the ablation speed (mm3/s) in BegoStone decreased with SD and increased with Ep, reaching a peak around 0.8 ~ 1.0 J. Additional experiments with calcium phosphate (CaP), uric acid (UA), and calcium oxalate monohydrate (COM) stones were conducted under two distinctly different settings: 0.2 J/100 Hz and 0.8 J/12 Hz. The concomitant bubble dynamics, spark generation and pressure transients were analyzed. Higher ablation speeds were consistently produced at 0.8 J/12 Hz than at 0.2 J/100 Hz, with CaP stones most difficult yet COM and UA stones easier to ablate. Charring was mostly observed in CaP stones at 0.2 J/100 Hz, accompanied by strong spark-generation, explosive combustion, and diminished pressure transients, but not at 0.8 J/12 Hz. By treating stones in parallel fiber orientation and leveraging the proximity effect of a ureteroscope, the contribution of bubble collapse to stone ablation was found to be substantial (16% ~ 59%) at 0.8 J/12 Hz, but not at 0.2 J/100 Hz. Overall, TFL ablation efficiency is significantly better at high Ep/low F setting, attributable to increased cavitation damage with less char formation.

Shock waves generated by toroidal bubble collapse are imperative for kidney stone dusting during Holmium:YAG laser lithotripsy.

Holmium:yttrium-aluminum-garnet (Ho:YAG) laser lithotripsy (LL) has been the treatment of choice for kidney stone disease for more than two decades, yet the mechanisms of action are not completely clear. Besides photothermal ablation, recent evidence suggests that cavitation bubble collapse is pivotal in kidney stone dusting when the Ho:YAG laser operates at low pulse energy (Ep) and high frequency (F). In this work, we perform a comprehensive series of experiments and model-based simulations to dissect the complex physical processes in LL. Under clinically relevant dusting settings (Ep = 0.2 J, F = 20 Hz), our results suggest that majority of the irradiated laser energy (>90 %) is dissipated by heat generation in the fluid surrounding the fiber tip and the irradiated stone surface, while only about 1 % may be consumed for photothermal ablation, and less than 0.7 % is converted into the potential energy at the maximum bubble expansion. We reveal that photothermal ablation is confined locally to the laser irradiation spot, whereas cavitation erosion is most pronounced at a fiber tip-stone surface distance about 0.5 mm where multi foci ring-like damage outside the thermal ablation zone is observed. The cavitation erosion is caused by the progressively intensified collapse of jet-induced toroidal bubble near the stone surface (<100 µm), as a result of Raleigh-Taylor and Richtmyer-Meshkov instabilities. The ensuing shock wave-stone interaction and resultant leaky Rayleigh waves on the stone surface may lead to dynamic fatigue and superficial material removal under repeated bombardments of toroidal bubble collapses during dusting procedures in LL.

Verapamil Prevents Decline of IGF-I in Subjects With Type 1 Diabetes and Promotes ß-Cell IGF-I Signaling.

Verapamil promotes functional ß-cell mass and improves glucose homeostasis in diabetic mice and humans with type 1 diabetes (T1D). Now, our global proteomics analysis of serum from subjects with T1D at baseline and after 1 year of receiving verapamil or placebo revealed IGF-I as a protein with significantly changed abundance over time. IGF-I, which promotes ß-cell survival and insulin secretion, decreased during disease progression, and this decline was blunted by verapamil. In addition, we found that verapamil reduces ß-cell expression of IGF-binding protein 3 (IGFBP3), whereas IGFBP3 was increased in human islets exposed to T1D-associated cytokines and in diabetic NOD mouse islets. IGFBP3 binds IGF-I and blocks its downstream signaling, which has been associated with increased ß-cell apoptosis and impaired glucose homeostasis. Consistent with the downregulation of IGFBP3, we have now discovered that verapamil increases ß-cell IGF-I signaling and phosphorylation/activation of the IGF-I receptor (IGF1R). Moreover, we found that thioredoxin-interacting protein (TXNIP), a proapoptotic factor downregulated by verapamil, promotes IGFBP3 expression and inhibits the phosphorylation/activation of IGF1R. Thus, our results reveal IGF-I signaling as yet another previously unappreciated pathway affected by verapamil and TXNIP that may contribute to the beneficial verapamil effects in the context of T1D. ARTICLE HIGHLIGHTS: Verapamil prevents the decline of IGF-I in subjects with type 1 diabetes (T1D). Verapamil decreases the expression of ß-cell IGF-binding protein 3 (IGFBP3), whereas IGFBP3 is increased in human and mouse islets under T1D conditions. Verapamil promotes ß-cell IGF-I signaling by increasing phosphorylation of IGF-I receptor and its downstream effector AKT. Thioredoxin-interacting protein (TXNIP) increases IGFBP3 expression and inhibits the phosphorylation/activation of IGF1R in ß-cells. Regulation of IGFBP3 and IGF-I signaling by verapamil and TXNIP may contribute to the beneficial verapamil effects in the context of T1D.

In Pursuit of the Optimal Dusting Settings with the Thulium Fiber Laser: An In Vitro Assessment.

Objective: Low energy and high frequency settings are used in stone dusting for holmium lasers. Such settings may not be optimal for thulium fiber laser (TFL). With the seemingly endless combination of settings, we aim to provide guidance to the practicing urologists and assess the efficiency of the TFL platform in an automated in vitro "dusting model." Materials/Methods: Three experimental setups were designed to investigate stone dusting produced by an IPG Photonics TLR-50 W TFL system using 200 µm fiber and soft BegoStone phantoms. The most popular 10 and 20 W dusting settings among endourologist familiar with TFL were evaluated. We directly compared short pulse (SP) vs long pulse (LP) mode using various combinations of pulse energy (Ep) and pulse frequency (F). Thereafter, we tested the 10 and 20 W settings and compared them among each other to elucidate the most efficient settings at each power. Treatments were performed under the same total laser energy delivered to the stone at four different standoff distances (SDs) with a clinically relevant scanning speed of either 1 or 2 mm/sec. Ablation volumes were quantified by optical coherence tomography to assess stone dusting efficiency. Fragment size after ablation at different pulse energies was evaluated by sieving and evaluating under a microscope after treatment. Results: Overall, SP provided greater ablation volume when compared with LP. Our dusting efficiency model demonstrated that the maximum stone ablation was achieved at the combination of high energy/low frequency settings (p < 0.005) and at a SD of 0.2 mm. At all tested pulse energies, no stone phantoms were broken into fragments >1 mm. Conclusions: During stone dusting with TFL, SP offers superior ablation to LP settings. Optimal dusting at clinically relevant scanning speeds of 1 and 2 mm/sec occurs at high energy/low frequency settings. Thulium lithotripsy with high Ep does not result in increased fragment size.

Dissimilar cavitation dynamics and damage patterns produced by parallel fiber alignment to the stone surface in holmium:yttrium aluminum garnet laser lithotripsy.

Recent studies indicate that cavitation may play a vital role in laser lithotripsy. However, the underlying bubble dynamics and associated damage mechanisms are largely unknown. In this study, we use ultra-high-speed shadowgraph imaging, hydrophone measurements, three-dimensional passive cavitation mapping (3D-PCM), and phantom test to investigate the transient dynamics of vapor bubbles induced by a holmium:yttrium aluminum garnet laser and their correlation with solid damage. We vary the standoff distance (SD) between the fiber tip and solid boundary under parallel fiber alignment and observe several distinctive features in bubble dynamics. First, long pulsed laser irradiation and solid boundary interaction create an elongated "pear-shaped" bubble that collapses asymmetrically and forms multiple jets in sequence. Second, unlike nanosecond laser-induced cavitation bubbles, jet impact on solid boundary generates negligible pressure transients and causes no direct damage. A non-circular toroidal bubble forms, particularly following the primary and secondary bubble collapses at SD = 1.0 and 3.0 mm, respectively. We observe three intensified bubble collapses with strong shock wave emissions: the intensified bubble collapse by shock wave, the ensuing reflected shock wave from the solid boundary, and self-intensified collapse of an inverted "triangle-shaped" or "horseshoe-shaped" bubble. Third, high-speed shadowgraph imaging and 3D-PCM confirm that the shock origins from the distinctive bubble collapse form either two discrete spots or a "smiling-face" shape. The spatial collapse pattern is consistent with the similar BegoStone surface damage, suggesting that the shockwave emissions during the intensified asymmetric collapse of the pear-shaped bubble are decisive for the solid damage.

Model-based simulations of pulsed laser ablation using an embedded finite element method.

A model of thermal ablation with application to multi-pulsed laser lithotripsy is presented. The approach is based on a one-sided Stefan-Signorini model for thermal ablation, and relies on a level-set function to represent the moving interface between the solid phase and a fictitious gas phase (representing the ablated material). The model is discretized with an embedded finite element method, wherein the interface geometry can be arbitrarily located relative to the background mesh. Nitsche's method is adopted to impose the Signorini condition on the moving interface. A bound constraint is also imposed to deal with thermal shocks that can arise during representative simulations of pulsed ablation with high-power lasers. We report simulation results based on experiments for pulsed laser ablation of wet BegoStone samples treated in air, where Begostone has been used as a phantom material for kidney stone. The model is calibrated against experimental measurements by adjusting the percentage of incoming laser energy absorbed at the surface of the stone sample. Simulation results are then validated against experimental observations for the crater area, volume, and geometry as a function of laser pulse energy and duration. Our studies illustrate how the spreading of the laser beam from the laser fiber tip with concomitantly reduced incident laser irradiance on the damaged crater surface explains trends in both the experimental observations and the model-based simulation results.

The Effects of Scanning Speed and Standoff Distance of the Fiber on Dusting Efficiency during Short Pulse Holmium: YAG Laser Lithotripsy.

To investigate the effects of fiber lateral scanning speed across the stone surface (vfiber) and fiber standoff distance (SD) on dusting efficiency during short pulse holmium (Ho): YAG laser lithotripsy (LL), pre-soaked BegoStone samples were treated in water using 0.2 J/20 Hz at SD of 0.10~0.50 mm with vfiber in the range of 0~10 mm/s. Bubble dynamics, pressure transients, and stone damage were analyzed. To differentiate photothermal ablation vs. cavitation damage, experiments were repeated in air, or in water with the fiber tip at 0.25 mm proximity from the ureteroscope end to mitigate cavitation damage. At SD = 0.10 mm, the maximum dusting efficiency was produced at vfiber = 3.5 mm/s, resulting in long (17.5 mm), shallow (0.15 mm), and narrow (0.4 mm) troughs. In contrast, at SD = 0.50 mm, the maximum efficiency was produced at vfiber = 0.5 mm/s, with much shorter (2.5 mm), yet deeper (0.35 mm) and wider (1.4 mm), troughs. With the ureteroscope end near the fiber tip, stone damage was significantly reduced in water compared to those produced without the ureteroscope. Under clinically relevant vfiber (1~3 mm/s), dusting at SD = 0.5 mm that promotes cavitation damage may leverage the higher frequency of the laser (e.g., 40 to 120 Hz) and, thus, significantly reduces the procedure time, compared to at SD = 0.1 mm that promotes photothermal ablation. Dusting efficiency during short pulse Ho: YAG LL may be substantially improved by utilizing an optimal combination of vfiber, SD, and frequency.

Alpha Cell Thioredoxin-interacting Protein Deletion Improves Diabetes-associated Hyperglycemia and Hyperglucagonemia.

Thioredoxin-interacting protein (TXNIP) has emerged as a key factor in pancreatic beta cell biology, and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of TXNIP protects against diabetes in different mouse models. However, while TXNIP is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We generated an alpha cell TXNIP knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-induced diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed, and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus together the current studies suggest that downregulation of alpha cell TXNIP is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

Deletion of Gdf15 Reduces ER Stress-induced Beta-cell Apoptosis and Diabetes.

Endoplasmic reticulum (ER) stress contributes to pancreatic beta-cell apoptosis in diabetes, but the factors involved are still not fully elucidated. Growth differentiation factor 15 (GDF15) is a stress response gene and has been reported to be increased and play an important role in various diseases. However, the role of GDF15 in beta cells in the context of ER stress and diabetes is still unclear. In this study, we have discovered that GDF15 promotes ER stress-induced beta-cell apoptosis and that downregulation of GDF15 has beneficial effects on beta-cell survival in diabetes. Specifically, we found that GDF15 is induced by ER stress in beta cells and human islets, and that the transcription factor C/EBPß is involved in this process. Interestingly, ER stress-induced apoptosis was significantly reduced in INS-1 cells with Gdf15 knockdown and in isolated Gdf15 knockout mouse islets. In vivo, we found that Gdf15 deletion attenuates streptozotocin-induced diabetes by preserving beta cells and insulin levels. Moreover, deletion of Gdf15 significantly delayed diabetes development in spontaneous ER stress-prone Akita mice. Thus, our findings suggest that GDF15 contributes to ER stress-induced beta-cell apoptosis and that inhibition of GDF15 may represent a novel strategy to promote beta-cell survival and treat diabetes.

Exploratory study reveals far reaching systemic and cellular effects of verapamil treatment in subjects with type 1 diabetes.

Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.

Cavitation Plays a Vital Role in Stone Dusting During Short Pulse Holmium:YAG Laser Lithotripsy.

Objective: To investigate the mechanism of stone dusting in Holmium (Ho): YAG laser lithotripsy (LL). Materials and Methods: Cylindrical BegoStone samples (6 × 6 mm, H × D) were treated in water using a clinical Ho:YAG laser lithotripter in dusting mode (0.2-0.4 J with 70-78 µs in pulse duration, 20 Hz) at various fiber tip to stone standoff distances (SD = 0, 0.5, and 1 mm). Stone damage craters were quantified by optical coherence tomography and bubble dynamics were captured by high-speed video imaging. To differentiate the contribution of cavitation vs thermal ablation to stone damage, three additional experiments were performed. First, presoaked wet stones were treated in air to assess stone damage without cavitation. Second, the laser fiber was advanced at various offset distances (OSD = 0.25, 1, 2, 3, and 10 mm) from the tip of a flexible ureteroscope to alter the dynamics of bubble collapse. Third, stones were treated with parallel fiber to minimize photothermal damage while isolating the contribution of cavitation to stone damage. Results: Treatment in water resulted in 2.5- to 90-fold increase in stone damage compared with those produced in air where thermal ablation dominates. With the fiber tip placed at OSD = 0.25 mm, the collapse of the bubble was distracted away from the stone surface by the ureteroscope tip, leading to significantly reduced stone damage compared with treatment without the scope or with scope at large OSD of 3-10 mm. The average crater volume produced by parallel fiber orientation at 0.2 J after 100 pulses, where cavitation is the dominant mechanism of stone damage, was comparable with those produced by using perpendicular fiber orientation within SD = 0.25-1 mm. Conclusion: Cavitation plays a dominant role over photothermal ablation in stone dusting during short pulse Ho:YAG LL when 10 or more pulses are delivered to the same location.

The Role of Cavitation in Energy Delivery and Stone Damage During Laser Lithotripsy.

Purpose: Although cavitation during laser lithotripsy (LL) contributes to the Moses effect, the impact of cavitation on stone damage is less clear. Using different laser settings, we investigate the role of cavitation bubbles in energy delivery and stone damage. Materials and Methods: The role of cavitation in laser energy delivery was characterized by using photodetector measurements synced with high-speed imaging for laser pulses of varying durations. BegoStone samples were treated with the laser fiber oriented perpendicularly in contact with the stone in water or in air to assess the impact of cavitation on crater formation. Crater volume and geometry were quantified by using optical coherence tomography. Further, the role of cavitation in stone damage was elucidated by treatment in water with the fiber oriented parallel to the stone surface and by photoelastic imaging. Results: Longer pulse durations resulted in higher energy delivery but smaller craters. Stones treated in water resulted in greater volume, wider yet shallower craters compared with those treated in air. Stones treated with the parallel fiber showed crater formation after 15 pulses, confirmed by high-speed imaging of the bubble collapse with the resultant stress field captured by photoelastic imaging. Conclusions: Despite improved energy delivery, the longer pulse mode produced smaller crater volume, suggesting additional processes secondary to photothermal ablation are involved in stone damage. Our critical observations of the difference in stone damage treated in water vs in air, combined with the crater formation by parallel fiber, suggest that cavitation is a contributor to stone damage during LL.

Human Glucagon Expression Is under the Control of miR-320a.

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes.

Identification of an Anti-diabetic, Orally Available Small Molecule that Regulates TXNIP Expression and Glucagon Action.

Diabetes is characterized by hyperglycemia, loss of functional islet beta cell mass, deficiency of glucose-lowering insulin, and persistent alpha cell secretion of gluconeogenic glucagon. Still, no therapies that target these underlying processes are available. We therefore performed high-throughput screening of 300,000 compounds and extensive medicinal chemistry optimization and here report the discovery of SRI-37330, an orally bioavailable, non-toxic small molecule, which effectively rescued mice from streptozotocin- and obesity-induced (db/db) diabetes. Interestingly, in rat cells and in mouse and human islets, SRI-37330 inhibited expression and signaling of thioredoxin-interacting protein, which we have previously found to be elevated in diabetes and to have detrimental effects on islet function. In addition, SRI-37330 treatment inhibited glucagon secretion and function, reduced hepatic glucose production, and reversed hepatic steatosis. Thus, these studies describe a newly designed chemical compound that, compared to currently available therapies, may provide a distinct and effective approach to treating diabetes.

Serum miR-204 is an early biomarker of type 1 diabetes-associated pancreatic beta-cell loss.

Pancreatic beta-cell death is a major factor in the pathogenesis of type 1 diabetes (T1D), but straightforward methods to measure beta-cell loss in humans are lacking, underlining the need for novel biomarkers. Using studies in INS-1 cells, human islets, diabetic mice, and serum samples of subjects with T1D at different stages, we have identified serum miR-204 as an early biomarker of T1D-associated beta-cell loss in humans. MiR-204 is a highly enriched microRNA in human beta-cells, and we found that it is released from dying beta-cells and detectable in human serum. We further discovered that serum miR-204 was elevated in children and adults with T1D and in autoantibody-positive at-risk subjects but not in type 2 diabetes or other autoimmune diseases and was inversely correlated with remaining beta-cell function in recent-onset T1D. Thus, serum miR-204 may provide a much needed novel approach to assess early T1D-associated human beta-cell loss even before onset of overt disease.

miR-204 Controls Glucagon-Like Peptide 1 Receptor Expression and Agonist Function.

Glucagon-like peptide 1 receptor (GLP1R) agonists are widely used to treat diabetes. However, their function is dependent on adequate GLP1R expression, which is downregulated in diabetes. GLP1R is highly expressed on pancreatic ß-cells, and activation by endogenous incretin or GLP1R agonists increases cAMP generation, which stimulates glucose-induced ß-cell insulin secretion and helps maintain glucose homeostasis. We now have discovered that the highly ß-cell-enriched microRNA, miR-204, directly targets the 3' UTR of GLP1R and thereby downregulates its expression in the ß-cell-derived rat INS-1 cell line and primary mouse and human islets. Furthermore, in vivo deletion of miR-204 promoted islet GLP1R expression and enhanced responsiveness to GLP1R agonists, resulting in improved glucose tolerance, cAMP production, and insulin secretion as well as protection against diabetes. Since we recently identified thioredoxin-interacting protein (TXNIP) as an upstream regulator of miR-204, we also assessed whether in vivo deletion of TXNIP could mimic that of miR-204. Indeed, it also enhanced islet GLP1R expression and GLP1R agonist-induced insulin secretion and glucose tolerance. Thus, the present studies show for the first time that GLP1R is under the control of a microRNA, miR-204, and uncover a previously unappreciated link between TXNIP and incretin action.

Islet ChREBP-ß is increased in diabetes and controls ChREBP-α and glucose-induced gene expression via a negative feedback loop.

OBJECTIVE: Carbohydrate-response element-binding protein (ChREBP) is the major transcription factor conferring glucose-induced gene expression in pancreatic islets, liver and adipose tissue. Recently, a novel ChREBP isoform, ChREBP-ß, was identified in adipose tissue and found to be also expressed in islets and involved in glucose-induced beta cell proliferation. However, the physiological function of this less abundant ß-isoform in the islet, and in diabetes, is largely unknown. The aims of the present study, therefore, were to determine how diabetes affects ChREBP-ß and elucidate its physiological role in pancreatic beta cells. METHODS: Non-obese diabetic and obese, diabetic ob/ob mice were used as models of T1D and T2D and human islets and the rat INS-1 beta cell line were exposed to low/high glucose and used for ChREBP isoform-specific gain-and-loss-of-function experiments. Changes in ChREBP-ß and ChREBP-α were assessed by qRT-PCR, immunoblotting, promoter luciferase, and chromatin immunoprecipitation studies. RESULTS: Expression of the ChREBP-ß isoform was highly induced in diabetes and by glucose, whereas ChREBP-α was downregulated. Interestingly, ChREBP-ß gain-of-function experiments further revealed that it was ChREBP-ß that downregulated ChREBP-α through a negative feedback loop. On the other hand, ChREBP-ß knockdown led to unabated ChREBP-α activity and glucose-induced expression of target genes, suggesting that one of the physiological roles of this novel ß-isoform is to help keep glucose-induced and ChREBP-α-mediated gene expression under control. CONCLUSIONS: We have identified a previously unappreciated negative feedback loop by which glucose-induced ChREBP-ß downregulates ChREBP-α-signaling providing new insight into the physiological role of islet ChREBP-ß and into the regulation of glucose-induced gene expression.

miR-204 Targets PERK and Regulates UPR Signaling and ß-Cell Apoptosis.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of diabetes and the associated ß-cell apoptosis. Although microRNAs (miRNAs) have been widely studied in various diseases including diabetes, the role of miRNAs in ER stress and ß-cell apoptosis has only started to be elucidated. We recently showed that diabetes increases ß-cell miR-204 and have now discovered that miR-204 directly targets the 3'untranslated region of protein kinase R-like ER kinase (PERK), 1 of the 3 ER transmembrane sensors and a key factor of the unfolded protein response (UPR). In addition, by using primary human islets, mouse islets, and INS-1 ß-cells, we found that miR-204 decreased PERK expression as well as its downstream factors, activating transcription factor 4 and CCAAT enhancer-binding protein homologous protein, whereas it had no effect on the other 2 ER transmembrane sensors, activating transcription factor 6 and inositol-requiring enzyme-1α. Interestingly, we discovered that miR-204 also inhibited PERK signaling in the context of ER stress, and this exacerbated ER stress-induced ß-cell apoptosis. This effect could be mimicked by PERK inhibitors supporting the notion that the miR-204-mediated inhibition of PERK and UPR signaling was conferring these detrimental effects on cell survival. Taken together, we have identified PERK as a novel target of miR-204 and show that miR-204 inhibits PERK signaling and increases ER stress-induced cell death, revealing for the first time a link between this miRNA and UPR.