Background/Objectives: Impairment in pulsatile insulin release contributes to insulin resistance and is one of the earliest markers of developing type 2 diabetes. Insulin delivered to the liver in pulses has a stronger glucose-lowering effect than continuous insulin delivery. Whether pulsatility benefits the islet itself is an open question. We previously showed that reducing glucokinase activity with the glucokinase inhibitor D-mannoheptulose (MH) improves function in islets exposed to prolonged hyperglycemic conditions. In this study, we test whether pulsatile vs. continuous delivery impacts the effectiveness of MH in islets. Methods: Islets were exposed to high-glucose conditions (20 mM glucose) for 24 or 48 h to induce early adaptations to hyperglycemia. We then used a specially designed perifusion system to impose pulsatile activity by exposing mouse islets to 3 min of MH in 20 mM glucose and 3 min of only high levels of glucose. Islets given intermittent MH for 18 h were compared with continuous delivery of MH at a full (2.5 mM) or half (1.25 mM) dose. Results: MH delivered by the forced oscillatory system reversed the effects of hyperglycemia and restored glucose sensing more effectively than continuous delivery. Specifically, fura-2AM imaging of intracellular calcium showed that islets given pulsatile MH had greater reductions in the elevated basal calcium caused by hyperglycemic conditions, improved the glucose stimulation index, and improved phase 0 response (indicating glucose-stimulated calcium uptake by the endoplasmic reticulum). Conclusions: These findings suggest that the loss of oscillatory glucose metabolism in islets contributes directly to beta-cell dysfunction.

Background/Objectives: The micronutrient iron is closely connected to inflammation and is among the complex factors contributing to beta-cell failure in diabetes. High levels of dietary iron increase the risk of developing type 2 diabetes, and excessive iron uptake by beta-cells can cause oxidative stress and inhibit function. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)-1beta and IL-6, increase the risk of developing type 2 diabetes, and there is evidence that these low levels of circulating cytokines can lead to islet dysfunction. Methods: In this study, gene microarray and other data were analyzed for expression differences in islets treated for 48 h with 10 pg/mL IL-1beta + 20 pg/mL IL-6 as a model of low-grade inflammation versus untreated. Results: Three iron-associated genes were among the most cytokine-sensitive in the mouse genome: Hamp, Steap4, and Lcn2. These proteins are all involved with increasing/retaining cellular iron. We hypothesized that increased cellular iron would lead to increased susceptibility to ferroptosis. Surprisingly, 24 h pre-exposure to low-grade inflammation, which upregulates this iron-gene network, prevented subsequent erastin-induced ferroptosis. We also found that Steap4 overexpression reduced islet dysfunction caused by high-dose proinflammatory cytokines (10× low-dose), suggesting an overall protective effect. Steap4 overexpression also upregulated Hamp and Lcn2, suggesting Steap4 regulates these cytokine-sensitive iron genes.; in contrast, ferritin and ferroportin gene expression, which are not sensitive to cytokines, were unchanged. Conclusions: These data suggest an inflammation-induced network of genes involved in cellular iron uptake and retention plays a protective role in islets against oxidative stress and ferroptosis.

Administration of parathyroid hormone-related protein (PTHrP) has been shown to increase insulin content and secretion in mice. PTHrP also increases β-cell mass and proliferation. However, the mechanisms for these effects are unknown, and investigations have yet to examine PTHrP in the transcriptome. In this study, we transiently transfected a mouse β-cell line (MIN6) with either full-length PTHrP or DSred vector. Insulin content and glucose-stimulated insulin secretion were measured, and RNA from the cells after incubating in 20 mM glucose was collected. The results showed that PTHrP overexpression increased insulin content and the ratio of insulin secretion between low and high glucose (stimulation index). RNA sequencing showed that PTHrP overexpression downregulated many genes associated with responses to endoplasmic reticulum (ER) stress such as Hspa40, Dnajc3, and Xbp1. Among enriched Kyoto Encyclopedia of Genes and Genomes pathways, the ER stress gene pathway was the most strongly downregulated by far, and the most upregulated pathway was for biosynthesis of amino acids required for protein synthesis. These pathways suggest increased rates of protein biosynthesis. Quantitative polymerase chain reaction supported RNA-sequencing results for several ER stress genes (Xbp1, Bax, Bip). MIN6 cells transfected with PTHrP also had lower proinsulin-to-insulin ratio, indicating that PTHrP enhanced insulin processing in the ER. Our working hypothesis is that PTHrP augments insulin production and ER efficiency, which is consistent with observations of increased insulin content, decreased proinsulin-to-insulin ratio and reduced ER stress markers in MIN6 cells. In conclusion, our findings suggest a previously unknown role for PTHrP in β-cell endoplasmic reticulum, which may have therapeutic implications for enhancing insulin production.

Introduction and Objective: Beta cell exhaustion and T2D are preceded by a left-shift in the glucose-stimulated Ca2+ response by beta cells. Previous studies have shown that modulating the pace of activity in processes upstream of Ca2+ influx can prevent the left-shift, preserving function. Somatostatin (SST) is an established inhibitor of beta cells, so we investigated whether exogenous SST exposure could 1) restore and 2) preserve Ca2+ pulsatility and glucose-stimulated Ca2+ response in islets chronically exposed to elevated glucose. Methods: CD-1 mouse islets were incubated in 28 mM glucose for 48 hours to simulate chronic hyperglycemia. Intracellular Ca2+ was detected using fluorescent microscopy after islets were incubated with the ratiometric indicator Fura-2 AM. ANOVA and 2-tailed T-tests assessed statistical differences between groups, while pulses were detected using deconvolution software. Results: Acute exposure of 37 islets to 200 nM SST elevated mean pulse amplitude by 156 ± 7% and total pulse mass by 127 ± 16% in 11 mM glucose following chronic hyperglycemia (p < 0.001). Chronic exposure to 200 nM SST amidst hyperglycemic conditions did not improve glucose-stimulated Ca2+ response compared to untreated hyperglycemic islets, as assessed by stimulation index (ratio of mean Ca2+ in 28 vs 3 mM glucose). Conclusion: These data demonstrate that exogenous SST can acutely restore Ca2+ pulsatility at stimulatory glucose levels in left-shifted islets, yet long-term exposure to the hormone does not protect islets from the deleterious adaptations arising from chronic hyperglycemia. This refutes the hypotheses that oscillatory Ca2+ activity preserves islet function and that forced beta cell rest can prevent the left shift in glucose sensing. Future studies should investigate whether insulin output reflects the observed Ca2+ patterns. Disclosure B.I. Stiadle: None. B.P. List: None. K. Corbin: None. C.S. Nunemaker: None. Funding Ohio University; National Institutes of Health (R15 DK121247)

Introduction and Objective: Type 1 diabetes (T1D) is a multifactorial disorder defined by pancreatic beta-cell destruction, ultimately causing insulin deficiency. Thus, T1D patients require lifelong insulin therapy to maintain normal glycemic control. T1D treatment requires protecting pancreatic beta cells from cytokine-induced cell death and restoring insulin secretion, two key properties absent in current therapies. MSB-61 is our lead compound as a future T1D therapeutic. MSB-61 augmented insulin secretion starting at ~4h and protected islets from cell death following cytokine treatment. This study uses RNA sequencing to identify genes associated with the effects of MSB-61 and to determine the possible mechanism of action. Methods: To pursue this aim, islets from CD-1 mice were treated with 10 μM MSB-61, 50 μM MSB-61, or vehicle control for one hour (several hours before insulin effects are observed) and processed for RNA isolation to perform RNA sequencing. Results: Using integrated differential expression and pathway analysis (iDEP), we identified 91 differentially expressed genes (DEGs) from 57,010 genes (including non-coding transcripts). By further limiting the data set to genes that were upregulated more than 5-fold, we limited this set to 19 genes and 3 microRNAs. Notably, 12 of these 19 genes and 2 microRNAs were closely associated with Creb (cAMP response element binding). Among its core target genes are transcription factors such as Fos, FosB, Fosl2, and the Nr4a family (Nr4a1, Nr4a2, and Nr4a3), many of which are identified in our study. Conclusion: This suggests that MSB-61 may exert its protective and stimulatory effects on beta cells by modulating Creb-related transcriptional networks. This study shows that RNA sequencing in drug discovery can identify critical cellular and molecular targets, enabling more focused and effective treatments for T1D, P. Khan: None. N. Ajmal: None. K. Corbin: None. X. Tong: None. G. Gu: None. C.S. Nunemaker: None.

Introduction and Objective: Diras2 is a small Ras-related GTPase, primarily expressed in the brain that is implicated in attention-deficit/hyperactivity disorder (ADHD). Our lab has identified Diras2 in mouse pancreatic islets in response to stress conditions. Methods: To elucidate the novel role of Diras2 in pancreatic islets and its physiological significance, we characterized global Diras2-/- knockout male mice on C57B6N/J background compared to C57B6N/J wildtype (WT) mice with functional Diras2. Results: The glucose tolerance test (GTT) and insulin tolerance test (ITT) under chow and high fat diet revealed enhanced glucose tolerance and insulin sensitivity in Diras2-/- mice, while body weight showed no significant differences compared to WT controls. Functional studies on islets isolated through collagenase digestion showed no significant difference in insulin secretion and content between both studied groups under 11mM and 28mM glucose concentrations over 72 hours, as quantified by ELISA. Stress response studies using beta-cell-specific stressors such as thapsigargin (1цM), rotenone (50nM), and proinflammatory cytokines (IL-1β-5ng/mL, TNF-α 10ng/mL and IFN-ϒ, 100ng/mL) demonstrated that Diras2-/- mice were significantly protected against cytokine-induced apoptosis after overnight treatment when measured through cell death using propidium iodide and annexinV. Conclusion: These findings suggest that improved glucose tolerance and insulin sensitivity in the absence of Diras2 may originate from the alterations in neural signals, given the high expression of Diras2 in the brain. Furthermore, Diras2 may possess a proapoptotic role in islets and reducing Diras2 expression may reduce cytokine-induced damage which further offer insights into its potential contribution to metabolic homeostasis, and its relevance as a therapeutic target for diabetes. N. Ajmal: None. K. Corbin: None. N. Cunningham: None. P. Khan: None. M.A. Kalwat: Stock/Shareholder; Eli Lilly and Company, Pfizer Inc. C.S. Nunemaker: None.

Abstract Disclosure: N. Ajmal: None. M. Bogart: None. K. Corbin: None. P. Khan: None. M. Shafqat: None. S. Bergmeier: None. G. Gu: None. X. Tong: None. C.S. Nunemaker: None. Type 1 diabetes (T1D) is a chronic autoimmune disorder caused by proinflammatory cytokines (Interleukin 1-beta; IL1-β, Tumor Necrosis Factor-alpha; TNF-α, and Interferon-gamma; IFN-ϒ) from immune cells that destroy insulin-producing beta cells in pancreatic islets and lead to hyperglycemia. The development of possible treatments to cure T1D is needed besides intensive insulin therapy that does not halt disease progression. We have identified a small molecule called MSB-61 that can protect islet cells from endoplasmic reticulum stressors (66% protection) and cytokines (33% protection) as measured by propidium iodide in islets isolated from mice using 50uM MSB-61. Importantly, we observed large increases in insulin secretion during overnight exposure to 50uM MSB-61. Insulin secretion began to increase in response to MSB-61 at ∼4h, reaching a plateau at ∼8h of greater than 4-fold insulin release compared to controls. The Kegg pathway analysis of RNA-seq data on MSB-61 suggested that multiple genes upregulated by MSB-61 are involved in insulin secretion through an amplifying pathway that includes cAMP/PKA and Pi3k/Akt signaling cascades. Based on RNA sequencing data, this study aimed to test MSB-61 for identifying pathway by which it potentiates insulin secretion. Islets from CD-1 mice and mouse insulinoma Min-6 cell lines were used to test amplifying pathways by using cAMP/PKA and Pi3k/Akt analogs and antagonists with and without MSB-61, and overnight insulin secretion and insulin content will be measured by enzyme-linked immunosorbent assay (ELISA). MSB-61 showed no potential effects on insulin secretion when tested for Pi3k/Akt pathway in the presence of analogs and antagonists such as 740-Y-P and wortmannin. It further suggests MSB-61 might potentiate insulin secretion through the cAMP/PKA pathway, which is still under investigation. RNA sequencing analysis of mouse islets showed several genes that belong to Ras family member-2 and have GTPase activity. Genes with GTPase activity will be further investigated to help identify if MSB-61 stimulates insulin secretion through an amplifying pathway involving the cAMP/PKA downstream signaling cascade. Identifying the mechanism of action and signaling pathway by which MSB-61 potentiates insulin secretion will open new ways of identifying T1D therapeutic interventions that would help replace standard insulin therapy. Presentation: 6/3/2024

Abstract Disclosure: P. Khan: None. K. Corbin: None. N. Ajmal: None. S. Bergmeir: None. G. Gu: None. X. Tong: None. C.S. Nunemaker: None. Abstract Type 1 diabetes (T1D) is a chronic autoimmune disease in which loss of insulin-producing beta-cells leads to hyperglycemia due to which patients eventually require lifelong insulin therapy to maintain normal glycemic control. Insulin remains the gold standard and the only effective treatment for T1D since its discovery 100 years ago. Protecting the pancreatic beta cells from cytokine-induced cell death and restoring insulin secretion are important to treat T1D, two properties that are currently lacking in present therapies. Our lab identified a chemical compound MSB-61 that increases insulin release and protects pancreatic islets against T1D-associated cytokines and endoplasmic reticulum (ER) stress. This study aims to identify the mechanism of action of MSB-61 in protecting pancreatic islets from ER stress-induced cell death. To achieve this aim pancreatic islets obtained from CD-1 strain mice were exposed for one hour to 50uM MSB-61 or vehicle control. After treatment, RNA was collected for RNA sequencing. In addition to RNA extraction for sequencing, MSB-61's ability to secrete insulin was verified by performing ELISA on control,10 and 50uM MSB-61-treated islets, and the activity of protection from cell death was assessed by performing cell death assay on islets treated with ER stressors in the presence and absence of 10 and 50uM MSB-61. Using Genialis, a bioinformatics service, we evaluated 57,010 genes (including non-coding transcripts) in our RNA sequencing data set. The DESeq2 technique of data analysis allowed us to identify 350 differentially expressed genes (DEGs), of which 251 were upregulated and 74 were downregulated. After removing non-coding/undefined transcripts and genes with <5 counts per million, a total of 117 genes remained; 108 up and 9 down. According to the String database (string-db.org), many of the top DEGs formed a common network of genes associated with cell death protection and insulin secretion. A key component of that gene network is a stress-responsive orphan nuclear receptor with known protective effects in beta cells. Testing this orphan nuclear receptor as a possible direct or indirect target of MSB-61 for cell death prevention can aid in identifying the genes and pathways targeted by MSB-61, allowing us to improve our approaches to T1D treatment and develop better medications. Presentation: 6/3/2024

Type 1 diabetes (T1D) is an autoimmune chronic disorder that damages beta cells in the pancreatic islets of Langerhans and results in hyperglycemia due to the loss of insulin. Exogenous insulin therapy can save lives but does not stop disease progression. Thus, an effective therapy may require beta cell restoration and suppression of the autoimmune response. However, currently, there are no treatment options available that can reverse T1D. Within the National Clinical Trial (NCT) database, a majority of over 3000 trials to treat T1D are devoted to insulin therapy. This review focuses on noninsulin pharmacological therapies, specifically immunomodulators. Many investigational new drugs fall under this category, such as the recently FDA-approved CD3 monoclonal antibody teplizumab to delay the onset of T1D. In total, we identified 39 different immunomodulatory investigational drugs. FDA-approved teplizumab for Stage 2 T1D is discussed along with other immunomodulators that have been tested in Phase 3 clinical trials or higher, including otelixizumab (another anti-CD3 monoclonal antibody), daclizumab (an anti-CD25 monoclonal antibody), ladarixin (CXCR1/2 inhibitor), and antithymocyte globulin (ATG). Immunomodulators also play roles in islet transplantation and cellular therapies like FDA-approved Lantidra. Several immunomodulators involved in Phase 3 clinical studies of islet transplantation are also discussed, including alemtuzumab, basiliximab, etanercept, and reparixin, some already FDA-approved for other uses. These include alemtuzumab, basiliximab, etanercept, and reparixin, some of which have been FDA-approved for other uses. This review provides background, mechanism of action, results of completed trials, and adverse effects as well as details regarding ongoing clinical trials for each of these immunomodulators. Trial Registration: ClinicalTrials.gov identifier: NCT03875729, NCT01030861, NCT00129259, NCT00385697, NCT01280682; NCT03929601, NCT04598893, NCT05757713, NCT00678886, NCT01123083, NCT00064714, NCT00468117, NCT04628481, NCT01106157, NCT02215200, NCT00331162, NCT00679042, NCT01220856, NCT01817959.

Enzyme-linked immunosorbent assay (ELISA) is a valuabletool invarious chemical and biological fields for investigating the presenceand quantity of proteins of interest in given samples. The high importanceof the assay makes it widely used in biological research and hencea crucial skill to master for up-and-coming scientists. A major impedimentto teaching the skill to new students in research laboratories isthe high cost of commercial ELISA kits. As a result of the cost ofthe kits, most investigators prefer that more experienced studentsor technicians carry out the assay to prevent errors, thus deprivingless experienced students the chance to actively learn the process.Here, we present a practice ELISA test that can be done in any laboratoryusing different dilutions of trypan blue as the reagents and samples,thereby creating a product at one-hundredth the cost of the leastexpensive ELISA kits. Research has continuously shown that activelearning tools such as this simulation enhance student engagementand self-efficacy. This practice test also generates a numerical scorebased on the students' accuracy in pipetting and attentionto detail, allowing students to identify sources of error. Therefore,the practice ELISA experiment acts as a medium to teach proper techniquebefore performing a real ELISA, thus avoiding wasting expensive reagentsand limited samples.

Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.

Type 1 diabetes (T1D) is a chronic autoimmune disorder that damages beta cells in the pancreatic islets of Langerhans and results in hyperglycemia due to the loss of insulin. Exogenous insulin therapy can save lives but does not halt disease progression. Thus, an effective therapy may require beta-cell restoration and suppression of the autoimmune response. However, currently, there are no treatment options available that can halt T1D. Within the National Clinical Trial (NCT) database, a vast majority of over 3000 trials to treat T1D are devoted to insulin therapy. This review focuses on non-insulin pharmacological therapies. Many investigational new drugs fall under the category of immunomodulators, such as the recently FDA-approved CD-3 monoclonal antibody teplizumab. Four intriguing candidate drugs fall outside the category of immunomodulators, which are the focus of this review. Specifically, we discuss several non-immunomodulators that may have more direct action on beta cells, such as verapamil (a voltage-dependent calcium channel blocker), gamma aminobutyric acid (GABA, a major neurotransmitter with effects on beta cells), tauroursodeoxycholic acid (TUDCA, an endoplasmic reticulum chaperone), and volagidemab (a glucagon receptor antagonist). These emerging anti-diabetic drugs are expected to provide promising results in both beta-cell restoration and in suppressing cytokine-derived inflammation.

Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP & UDelta;/& UDelta;) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at -day 5, are severely stunted in growth, weigh 54% less than control mice at day 1-2 and eventually fail to grow. PTHrP & UDelta;/& UDelta; mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (-10-20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP & UDelta;/& UDelta; mice were smaller in size but secreted more insulin than littermate controls. PTHrP & UDelta;/& UDelta; and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8-20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP & UDelta;/& UDelta; mice (-250 & mu;m2) compared to islets from control mice (-900 & mu;m2), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP & UDelta;/& UDelta; mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function.

Parathyroid Hormone-related protein (PTHrP) is an important polyhormone with multiple functions in development and cell regulation. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have focused on the ability of N-terminal PTHrP to stimulate proliferation in beta cells. We have developed a knock-in mouse model lacking the C-terminal and nuclear localization sequence of PTHrP, retaining only N-terminal PTHrP. These mice die at ~day 5, are severely stunted in growth (weigh 54% less than control mice), have chondrodystrophy, hypoinsulinemia, hypoglycemia (~40-60mg/dL), hypotriglyceridemia, and lack body fat. Percentage stomach weight (including ingested milk) measurements in relation to body weight were consistent among all mouse genotypes indicating that inanition was not the cause of death. To characterize the pancreatic islets in these mice, islets (~10-20) were isolated from 2-5 day-old-mice using collagenase digestion and a glucose-stimulated intracellular calcium assay was conducted at 0, 4, 8, 12, 16 and 20mM glucose. Results showed higher intracellular calcium response in knock-in islets compared to control islets and increased insulin secretion after overnight incubation. Data from immunofluorescence also showed less glucagon staining in knock-in islets compared to control islets. This study revealed that the C-terminus and nuclear localization sequence of PTHrP are crucial to life, including regulation of glucose homeostasis and islet response to glucose metabolism. It also revealed that these portions of PTHrP may be important for the developmental stage of islets and differentiation into alpha or beta cells.

Obesity-related insulin resistance is a highly prevalent and growing health concern, which places stress on the pancreatic islets of Langerhans by increasing insulin secretion to lower blood glucose levels. The glucose transporters GLUT1 and GLUT3 play a key role in glucose-stimulated insulin secretion in human islets, while GLUT2 is the key isoform in rodent islets. However, it is unclear whether other glucose transporters also contribute to insulin secretion by pancreatic islets. Herein, we show that SLC2A6 (GLUT6) is markedly upregulated in pancreatic islets from genetically obese leptin-mutant (ob/ob) and leptin receptor-mutant (db/db) mice, compared to lean controls. Furthermore, we observe that islet SLC2A6 expression positively correlates with body mass index in human patients with type 2 diabetes. To investigate whether GLUT6 plays a functional role in islets, we crossed GLUT6 knockout mice with C57BL/6 ob/ob mice. Pancreatic islets isolated from ob/ob mice lacking GLUT6 secreted more insulin in response to high-dose glucose, compared to ob/ob mice that were wild type for GLUT6. The loss of GLUT6 in ob/ob mice had no adverse impact on body mass, body composition, or glucose tolerance at a whole-body level. This study demonstrates that GLUT6 plays a role in pancreatic islet insulin secretion in vitro but is not a dominant glucose transporter that alters whole-body metabolic physiology in ob/ob mice.

Insulin is secreted from pancreatic islets in a pulsatile manner due to alternating periods of cellular activity and rest. The resulting pulses of insulin are critical for proper hormonal signaling and blood glucose regulation by the liver. To recreate the interaction between the endocrine pancreas and the liver using primary murine tissue, we have designed 3D printed microfluidic platforms that hold hepatocytes downstream of islets. Islets no longer oscillate in sync once isolated from the pancreas, so the cumulative insulin released is non-pulsatile as demonstrated by taking the average intracellular calcium levels of multiple islets (Figure A, top). To force islets to secrete synchronized insulin pulses in vitro, we use programable syringe pumps that flow alternating high (11mM) and low (3mM) glucose solutions over isolated mouse islets. The syringe pump system aligns the oscillations which results in synchronized insulin release (Figure A, bottom). Next, we exposed primary murine hepatocytes to either 100nM pulses of insulin in three-minute periods or constant 50nM insulin in a microfluidic system (Figure B). Both groups were exposed to the same amount of insulin, just in a different pattern. Hepatocytes treated with pulsatile insulin for 4 hours contained significantly more glycogen compared to those treated with continuous 50nM insulin (P<0.05, N=4). Future experiments will add hepatocytes downstream of the islets on the same platform to investigate the effect of pulsatile insulin and glucagon secretion on liver function. Exploring the interaction between pancreatic islets and hepatocytes will lead to a deeper understanding of the loss of pulsatile hormone secretion during the progression of type 2 diabetes.