Background: Type 1 diabetes (T1D) is an autoimmune disease where autoreactive T cells infiltrate pancreatic islets, resulting in beta-cell destruction. Antigen-specific immunotherapy with tolerogenic peptides to induce peripheral tolerance has shown promise in preclinical studies but has not shown clinical efficacy. Methods: Here, we develop peptide-nanobubbles (NBs) as an image-guided platform for induction of peripheral immune tolerance in mouse models of T1D. Sub-micron sized NB ultrasound contrast agents can passively accumulate in pancreatic islets of non-obese diabetic (NOD) mice during the development of diabetes as a result of increased microvascular permeability. We incorporated an insulin B:9-23 peptide mimotope into NBs to target peptides to pancreatic islets for expansion of islet-resident regulatory T cells. Results: NBs retained normal size distribution and acoustic properties following peptide incorporation. Peptide-NBs accumulated in islets of NOD mice and this accumulation could be visualized in real time using contrast enhanced ultrasound. This resulted in an increased proportion of islet insulin-reactive regulatory T cells. Further, peptide-NBs prepared with a hybrid insulin peptide (HIP) expanded islet HIP-reactive regulatory T cells and substantially delayed diabetes onset in an adoptive transfer mouse model of autoimmune diabetes. Conclusions: Peptide-NBs offer a promising 'theranostic' approach for induction of peripheral tolerance in T1D while monitoring delivery and action via ultrasound contrast.

Cardiorespiratory Fitness (CRF) a universal predictor of cardiovascular and all-cause mortality, is lower in people with diabetes. Skeletal muscle is a key determinant of CRF, in particular mitochondrial content, function and oxygen extraction. Hyperglycemia is implicated in the dysfunction of microvascular blood flow. Our previous work demonstrated lower mitochondrial density/fiber and capillary mean flow velocity (MFV) with 4 weeks of hyperglycemia in males and females. Treadmill running resulted in greater capillary MFV and mitochondrial density in only hyperglycemic females. We hypothesize that along with alterations in mitochondrial density and blood flow, there is a disruption in skeletal muscle mitochondrial function with hyperglycemia, and that these parameters will respond to nitrite (NO2-) treatment in a sex-specific manner. We used a model of short-term hyperglycemia with tamoxifen inducible β-cell deletion of glucokinase. Two weeks after induction, blood flow in the gastrocnemius (gastroc) was measured using intravital microscopy, and the gastroc was collected from male (M) and female (F) mice with blood glucose >400mg/dl (HG) and euglycemic controls (EG). A subset of mice received NO2- treatment for 1-2 weeks prior to gastroc measurements. Immunofluorescence imaging of gastroc were analyzed for expression of COX1 (mitochondrial marker), Dystrophin (muscle fiber sarcolemma boundary), and CD31 (capillary marker). Mitochondrial respiration was measured in gastroc fibers using Oroboros O2k. Mitochondrial respiration was lower in HG compared to EG (p<0.0009), but only respiration in M appeared to respond to NO2- treatment (p=0.0252). No difference was observed in mitochondrial density in EG versus HG. Capillary MFV was greater with HG and mitigated by NO2-treatment. NO2- targets mitochondrial function by improving capillary blood flow, but only in males. Our combined data sets suggest HG-associated mitochondrial defects need to be treated in a sex-specific manner. Disclosure N.A.Hulett: None. L.Knaub: None. G.Pott: None. D.Ramirez: None. A.Johnston: None. R.L.Scalzo: None. J.E.B.Reusch: Advisory Panel; Medtronic. Funding U.S. Department of Veterans Affairs (BX002046, CX001532); National Institutes of Health (P30DK116073, 5T32AG000279-20)

Abstract Diabetes is a life-threatening and debilitating disease with pathological hallmarks, including glucose intolerance and insulin resistance. Plant compounds are a source of novel and effective therapeutics, and the flavonoid (−)-epicatechin, common to popular foods worldwide, has been shown to improve carbohydrate metabolism in both clinical studies and preclinical models. We hypothesized that (−)-epicatechin would alleviate thermoneutral housing-induced glucose intolerance. Male rats were housed at either thermoneutral (30 °C) or room temperature (24 °C) for 16 weeks and gavaged with either 1 mg/kg body weight or vehicle for the last 15 days before sacrifice. Rats housed at thermoneutrality had a significantly elevated serum glucose area under the curve (p < 0.05) and reduced glucose-mediated insulin secretion. In contrast, rats at thermoneutrality treated with (−)-epicatechin had improved glucose tolerance and increased insulin secretion (p < 0.05). Insulin tolerance tests revealed no differences in insulin sensitivity in any of the four groups. Pancreatic immunohistochemistry staining showed significantly greater islet insulin positive cells in animals housed at thermoneutrality. In conclusion, (−)-epicatechin improved carbohydrate tolerance via increased insulin secretion in response to glucose challenge without a change in insulin sensitivity.

People with T2D have lower cardiorespiratory fitness (CRF) than those without diabetes contributing to increased cardiovascular morbidity and mortality. People with T2D have impaired oxidative flux associated with lower CRF. Yet, reports on muscle mitochondrial dysfunction in T2D are mixed. As diabetes duration and age are predictors of poor outcomes, we hypothesized that T2D and increased age associate with impaired mitochondrial function. We assessed post-exercise calf mitochondrial function in overweight sedentary participants (45 with and 54 without T2D) ages 30-50 and 51-70y. Mitochondrial function was measured in vivo by 31Phosphorous magnetic resonance spectroscopy (31P-MRS) before, during and after 90 seconds of isotonic calf press at 70% maximal force and ex vivo by mitochondrial respiration in muscle fibers using the Oroboros Oxygraph-2k. There was no significant effect of age or T2D on ex vivo measures. MRS-measured phosphocreatine synthesis, apparent maximum rate of oxidative ATP synthesis and ADP time constant were worse in those with T2D or age >50 (Figure) . Age and T2D interact to significantly decrease apparent maximum rate of oxidative ATP synthesis. These data suggest that age and T2D decrease in vivo oxidative capacity. Given that impairment is only found in in vivo mitochondrial measures, factors such as micro- and macrovascular function and blood flow, combined with cardiac function, are likely implicated. Disclosure L.Abushamat: None. J.G.Regensteiner: None. R.L.Scalzo: None. D.Ramirez: None. I.E.Schauer: None. L.Knaub: None. E.Clark: None. Y.Garcia reyes: None. M.Cree-green: None. J.Reusch: Advisory Panel; Medtronic. Funding American Diabetes Association (1-21-CMF-003) (1-12-CT-64) ; Department of Veterans Affairs (BX002046, CX001532)

In type 1 diabetes (T1D), immune-cell infiltration into islets of Langerhans (insulitis) and β-cell decline occur years before diabetes presents. There is a lack of validated clinical approaches for detecting insulitis and β-cell decline, to diagnose eventual diabetes and monitor the efficacy of therapeutic interventions. We previously determined that contrast-enhanced ultrasound measurements of pancreas perfusion dynamics predict disease progression in T1D pre-clinical models. Here, we test whether these measurements predict therapeutic prevention of T1D. We performed destruction-reperfusion measurements with size-isolated microbubbles in non-obese diabetic (NOD)–severe combined immunodeficiency (SCID) mice receiving an adoptive transfer of diabetogenic splenocytes. Mice received vehicle control or the following treatments: (i) anti-CD3 to block T-cell activation; (ii) anti-CD4 to deplete CD4+ T cells; (iii) verapamil to reduce β-cell apoptosis; or (iv) tauroursodeoxycholic acid (TUDCA) to reduce β-cell endoplasmic reticulum stress. We compared measurements of pancreas perfusion dynamics with subsequent progression to diabetes. Anti-CD3, anti-CD4, and verapamil delayed diabetes development. Blood flow dynamics was significantly altered in treated mice with delayed/absent diabetes development compared with untreated mice. Conversely, blood flow dynamics in treated mice with unchanged diabetes development was similar to that in untreated mice. Thus, measurement of pancreas perfusion dynamics predicts the successful prevention of diabetes. This strategy may provide a clinically deployable predictive marker for therapeutic prevention in asymptomatic T1D.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies, which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilizing an expanding gas surface by employing the lung surfactant replacement, Beractant. Lung surfactant contains a suite of lipids and surfactant proteins that provides efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that Beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized Beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and re-solidification. We then harnessed this unique spreading capability to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca2+ homeostasis. Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6–12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.

Objective: Diabetes occurs because of insufficient insulin secretion due to (3-cell dysfunction within the islet of Langerhans. Elevated glucose levels trigger (3-cell membrane depolarization, action potential generation, and slow sustained free-Ca2+ ([Ca2+]) oscillations, which trigger insulin release. Nuclear factor of activated T-cell (NFAT) is a transcription factor, which is regulated by the increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems and regulates proliferation and insulin granule biogenesis within the (3-cell. However, the link between the regulation of (3-cell electrical activity and oscillatory [Ca2+] dynamics with NFAT activation and downstream transcription is poorly understood. Here, we tested whether dynamic changes to (3-cell electrical activity and [Ca2+] regulate NFAT activation and downstream transcription. Methods: In cell lines, mouse islets, and human islets, including those from donors with type 2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate (3-cell electrical activity. We measured the dynamics of [Ca2+] and NFAT activation as well as performed whole transcriptome and functional analyses. Results: Both glucose-induced membrane depolarization and optogenetic stimulation triggered NFAT activation as well as increased the transcription of NFAT targets and intermediate early genes (IEGs). Importantly, slow, sustained [Ca2+] oscillation conditions led to NFAT activation and downstream transcription. In contrast, in human islets from donors with type2 diabetes, NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. NFAT activation regulated GJD2 expression and increased Cx36 gap junction permeability upon elevated oscillatory [Ca2+] dynamics. However, it is unclear if NFAT directly binds the GJD2 gene to regulate expression. Conclusions: This study provides an insight into the specific patterns of electrical activity that regulate NFAT activation, gene transcription, and islet function. In addition, it provides information on how these factors are disrupted in diabetes. (c) 2022 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Objective: Diabetes occurs because of insufficient insulin secretion due to β-cell dysfunction within the islet of Langerhans. Elevated glucose levels trigger β-cell membrane depolarization, action potential generation, and slow sustained free-Ca2+ ([Ca2+]) oscillations, which trigger insulin release. Nuclear factor of activated T-cell (NFAT) is a transcription factor, which is regulated by the increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems and regulates proliferation and insulin granule biogenesis within the β-cell. However, the link between the regulation of β-cell electrical activity and oscillatory [Ca2+] dynamics with NFAT activation and downstream transcription is poorly understood. Here, we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulate NFAT activation and downstream transcription. Methods: In cell lines, mouse islets, and human islets, including those from donors with type 2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. We measured the dynamics of [Ca2+] and NFAT activation as well as performed whole transcriptome and functional analyses. Results: Both glucose-induced membrane depolarization and optogenetic stimulation triggered NFAT activation as well as increased the transcription of NFAT targets and intermediate early genes (IEGs). Importantly, slow, sustained [Ca2+] oscillation conditions led to NFAT activation and downstream transcription. In contrast, in human islets from donors with type2 diabetes, NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. NFAT activation regulated GJD2 expression and increased Cx36 gap junction permeability upon elevated oscillatory [Ca2+] dynamics. However, it is unclear if NFAT directly binds the GJD2 gene to regulate expression. Conclusions: This study provides an insight into the specific patterns of electrical activity that regulate NFAT activation, gene transcription, and islet function. In addition, it provides information on how these factors are disrupted in diabetes.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Significance Methods to detect type 1 diabetes (T1D) progression prior to clinical diagnosis are needed. T1D results from autoreactive T cells infiltrating the islets of Langerhans, destroying insulin-producing β-cells. Overt disease takes years to present, and at diagnosis, there is substantial β-cell loss. Therapeutic intervention to preserve β-cell mass is hampered by an inability to follow presymptomatic T1D progression. Several immunotherapies can delay T1D development. However, identifying “at-risk” individuals and tracking whether therapeutic interventions are impacting disease progression, are lacking. We present ultrasound imaging of nanodroplet (ND) contrast-agent accumulation within the islet. ND accumulation is dependent on immune infiltration; therefore, it tracks presymptomatic T1D development and progression to diabetes. This provides an opportunity to guide therapeutic treatments to prevent T1D.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

In recent work, oxygen microbubbles (OMB) have been shown to oxygenate hypoxic tumors, increase radio-sensitivity and improve tumor control by radiation therapy. Compared to intra-tumoral injection, intravenous delivery of adjuvant agents such as OMBs for radiotherapy offers an attractive means of achieving true theranostic function in a minimally invasive manner via contrast-enhanced ultrasound (CEUS), while reducing the risk of injury, infection or displacing tumor cells. However, short intravascular circulation times with conventional DSPC-lipid OMBs may lead to premature off-target dissolution of OMBs with an associated reduction in tumoral oxygen delivery. Prior work on microbubble stability and gas exchange suggests that increasing phospholipid acyl-chain length of the encapsulating shell and OMB size may increase circulation persistence, delivery and dissolved oxygen content. In the following studies, we investigate the effect of two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0), as well as three size distributions (0.5-2 µm, 2-10 µm and polydisperse) on OMB circulation persistence utilizing CEUS in the kidneys of live C57B1/6 male and female mice, six weeks of age. DBPC OMB formulations demonstrated increased circulation half-lives versus DSPC formulations (2.4 ± 1.0 vs. 0.6 ± 0.5 s, p<0.01 for 2-10 µm), as well as an increased maximum intensity by over tenfold (p<0.01). Size-dependent effects remained consistent across both formulations with larger 2-10 µm microbubbles demonstrating significantly increased half-lives (2.4 ± 1.0 vs. 0.3 ± 0.2 s, p < 0.01) compared to smaller 0.5-2 µm formulations of DBPC. These studies indicate that DBPC 2-10 µm OMBs may be improved adjuvant agents for radiotherapy with significant potential for CEUS interrogation.

In type 1 diabetes (T1D) immune-cell infiltration into the islets of Langerhans (insulitis) and β-cell decline occurs many years before diabetes presents. Non-invasively detecting insulitis and β-cell decline would allow diagnosis of eventual diabetes and provide a means to monitor the efficacy of therapeutic intervention. However, there is a lack of validated clinical approaches for non-invasively imaging disease progression leading to T1D. Islets have a dense microvasculature that reorganizes during diabetes. We previously demonstrated contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics could predict disease progression in T1D pre-clinical models. Here we test whether these measurements can predict successful therapeutic prevention of T1D. We performed destruction-reperfusion measurements using a small-animal ultrasound machine and size-isolated microbubbles, in NOD-scid mice receiving an adoptive transfer of diabetogenic splenocytes (AT mice). Mice received vehicle control or either of the following treatments: 1) antiCD4 to deplete CD4+ T cells; 2) antiCD3 to block T cell activation, 3) Verapamil to reduce β-cell apoptosis and 4) TUDCA to reduce ER stress. We compared measurements of pancreas blood-flow dynamics with subsequent progression to diabetes. In AT mice blood-flow dynamics were altered >2 weeks after splenocyte transfer. AntiCD4, antiCD3 and verapamil provided a significant delay in diabetes development. Treated AT mice with delayed or absent diabetes development showed significantly altered blood flow dynamics compared to untreated AT mice. Conversely, treated AT mice that developed diabetes, despite therapy, showed similar blood-flow dynamics to untreated AT mice. Thus, contrast-enhanced ultrasound measurement of pancreas blood-flow dynamics can predict the successful or unsuccessful delay or prevention of diabetes upon therapeutic treatments that target both immune activity or β-cell protection. This strategy may provide a clinically deployable predictive marker for disease progression and therapeutic reversal in asymptomatic T1D.

AbstractDiabetes results from insufficient insulin secretion as a result of dysfunction to β-cells within the islet of Langerhans. Elevated glucose causes β-cell membrane depolarization and action potential generation, voltage gated Ca2+channel activation and oscillations in free-Ca2+activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the β-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of β-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes.

Diabetes results from insufficient insulin secretion as a result of dysfunction to β-cells within the islet of Langerhans. Elevated glucose causes β-cell membrane depolarization and action potential generation, voltage gated Ca2+ channel activation and oscillations in free-Ca2+ activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the β-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of β-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes. ### Competing Interest Statement The authors have declared no competing interest.

In type1 diabetes (T1D) autoreactive T-cells infiltrate the islets of Langerhans, depleting insulin-secreting β-cells (insulitis). Insulitis arises during an asymptomatic phase, prior to clinical diagnosis of T1D. Methods to diagnose insulitis and β-cell mass changes during this asymptomatic phase are limited, precluding early therapeutic intervention. During T1D the islet microvasculature increases permeability, allowing nanoparticles to access the microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide acoustic backscatter in vasculature. Here, we report that sub-micron sized ‘nanobubble’ ultrasound contrast agents can be used to measure increased islet microvasculature permeability and indicate asymptomatic T1D. Through CEUS and histological analysis, pre-clinical models of T1D show accumulation of nanobubbles specifically within pancreatic islets, correlating with insulitis. Importantly, accumulation is detected early in disease progression and decreases with successful therapeutic intervention. Thus, sub-micron sized nanobubble ultrasound contrast agents provide a predicative marker for disease progression and therapeutic reversal early in asymptomatic T1D.

Here we report on an oxygen microbubble that has increased circulation and contrast persistence to treat tumor hypoxia during X-ray therapy. In recent work, oxygen microbubbles injected directly into tumors were shown to oxygenate hypoxic tumors and improve radiation therapy [1]. Here, we investigate oxygen microbubbles designed for intravenous, rather than intra-tumoral, injection. Oxygen microbubbles were designed with two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0). DBPC microbubbles showed a significant increase in ultrasound contrast persistence in vivo in the mouse kidney, and this composition may be ideal for an intravascular microbubble injection to treat tumor hypoxia in radiation oncology.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.

In type 1 diabetes (T1D) the decline in beta-cell mass occurs many years prior to clinical presentation. A non-invasive method to diagnose insulitis and beta-cell mass decline prior to diabetes does not exist. During T1D progression, the islet microvasculature increases permeability due to insulitis. Iron oxide nanoparticle MRI contrast accumulation has previously been suggested as a measure of ongoing insulitis. Contrast enhanced ultrasound (CEUS) using gas-filled microbubbles (MBs) measures acoustic backscatter at sub-harmonic frequencies and is clinically approved. Sub-micron sized ‘nanobubbles’ (NBs) have previously been developed and show extravasation in tumors. Here we tested whether these sub-micron sized NBs show disease-mediated extravasation in mouse models of T1D. We performed CEUS measurements following NB infusion in NOD mice and control (NOD;Rag1ko and C57Bl6) mice. We also examined fluorescent coverage (FC) following rhodamine-labeled bubble delivery in these animals, through isolating pancreata for histology and quantifying FC and insulitis scores. We observed progressive increased CEUS signal accumulation in pancreas of 4w and 10w NOD mice, which was absent in NOD kidneys (p < 0.001). Rag1ko controls showed minimal CEUS signal in pancreas and kidney. The FC was limited to the pancreata of NOD mice and was substantially higher in endocrine compared to exocrine tissue (p = 0.002). NOD mouse islets also had substantially more FC compared to Rag1ko (p < 0.0001), and showed a correlation between NOD insulitis score and FC (p=0.043). Thus, CEUS with NB contrast agents can detect disease progression prior to T1D onset, which will be important for early T1D diagnosis and monitoring of disease prevention or reversal. Disclosure D. Ramirez: None. R.K. Benninger: None.