Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of G _i and G _q GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of G _i signaling reduced insulin and glucagon secretion, while activation of G _q signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca ²⁺ . The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca²⁺ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca²⁺-binding protein, with Ca²⁺-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca²⁺ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca²⁺ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Ca²⁺release from the endoplasmic reticulum is an important component of Ca²⁺signal transduction that controls numerous physiological processes in eukaryotic cells. Release of Ca²⁺from the endoplasmic reticulum is coupled to the activation of store-operated Ca²⁺entry into cells. Store-operated Ca²⁺entry provides Ca²⁺for replenishing depleted endoplasmic reticulum Ca²⁺stores and a Ca²⁺signal that regulates Ca²⁺-dependent intracellular biochemical events. Central to connecting discharge of endoplasmic reticulum Ca²⁺stores following G protein-coupled receptor activation with the induction of store-operated Ca²⁺entry are stromal interaction molecules (STIM1 and STIM2). These highly homologous endoplasmic reticulum transmembrane proteins function as sensors of the Ca²⁺concentration within the endoplasmic reticulum lumen and activators of Ca²⁺release-activated Ca²⁺channels. Emerging evidence indicates that in addition to their role in Ca²⁺release-activated Ca²⁺channel gating and store-operated Ca²⁺entry, STIM1 and STIM2 regulate other cellular signaling events. Recent studies have shown that disruption of STIM expression and function is associated with the pathogenesis of several diseases including autoimmune disorders, cancer, cardiovascular disease, and myopathies. Here, we provide an overview of the latest developments in the molecular physiology and pathophysiology of STIM1 and STIM2.Impact statement: Intracellular Ca²⁺signaling is a fundamentally important regulator of cell physiology. Recent studies have revealed that Ca²⁺-binding stromal interaction molecules (Stim1 and Stim2) expressed in the membrane of the endoplasmic reticulum (ER) are essential components of eukaryote Ca²⁺signal transduction that control the activity of ion channels and other signaling effectors present in the plasma membrane. This review summarizes the most recent information on the molecular physiology and pathophysiology of stromal interaction molecules. We anticipate that the work presented in our review will provide new insights into molecular interactions that participate in interorganelle signaling crosstalk, cell function, and the pathogenesis of human diseases.

Stromal interaction molecule 1 (STIM1) regulates store-operated Ca²⁺ entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca²⁺-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting β-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 β-cells with RNA interference inhibits SOCE and ATP-sensitive K⁺ (K_ATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1. Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the K_ATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and K_ATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca²⁺ signaling events in β-cells.

Background: The free hormone (FH) hypothesis states that hormone action and the corresponding biological effects are mediated by the unbound (free) fraction of hormone in circulation. The in vivo relationship between protein-bound and FH is complex and dynamic. In most individuals, measurement of total hormone (TH) is usually adequate to reflect the hormone status; however, certain physiological conditions and/or medications can affect protein binding and alter FH concentration. In these cases, measurement of FH will provide a better measure of the bioactive hormone status than measurement of the TH. Measurement of FH presents many challenges, as the concentrations are very low and there are number of pitfalls, which may affect the measured concentrations. Content: In this review, we discuss techniques used in the separation and direct quantitation of FH concentrations in biological samples using mass spectrometry for analysis. We also highlight clinical situations in which FH analysis is warranted and when mass spectrometry should be the preferred methodology over immunoassays. Summary: Equilibrium dialysis, ultrafiltration, or size-exclusion separation coupled with liquid chromatography–tandem mass spectrometry provides a sensitive and specific method to measure FH concentrations. These direct methods are useful in iatrogenic or physiological states that alter hormone binding or metabolism.

Context: There are currently 2 classes of thyroid-stimulating hormone receptor (TSHR) antibody assays, namely TSHR antibody (TRAb) assays and thyroid-stimulating immunoglobulin (TSI) assays. Clinical guidelines do not currently specify appropriate use of TSHR autoantibody tests in the diagnosis of hyperthyroidism, which may result in paired orders for both tests with the possibility of discordant results and excessive costs. Objective: This work aimed to evaluate the clinical and analytical redundancy of paired TSHR autoantibody testing—specifically TRAb and TSI assays—in the diagnosis of autoimmune-mediated hyperthyroidism, by analyzing the frequency of paired orders, assay concordance, and clinical correlation across a large cohort of patient encounters. Methods: Over 189 000 patient encounters with TRAb and TSI bioassay (TSI-BA) or TSI bridge immunoassay (TSI-Br) test orders were examined to assess the frequency of paired orders and qualitative agreement of TRAB/TSI. A chart review was performed on a subset of patients for clinical correlation. Lastly, a cost analysis was performed to estimate the financial burden of unnecessary paired testing. Results: TRAb and TSI were co-ordered on the same encounter in 14.3% of TRAb/TSI-BA orders and 17.4% of TRAb/TSI-Br orders. Qualitative comparison showed discordance in 12.5% (1590) of TRAb and TSI-BA paired orders and 6.6% (1149) of TRAb and TSI-Br paired orders. Based on patient free thyroxine and TSH concentrations and disease status, the TSI assays aligned better with hyperthyroidism and confirmed Graves disease diagnoses. Paired orders resulted in a 31% to 325% increase in potentially unnecessary testing costs. Conclusion: We observed good clinical and analytical agreement between TRAb and TSI assays, suggesting that paired orders for TRAb and TSI are redundant in the assessment of autoimmune-mediated hyperthyroidism. Use of a single test to assess TRAb would be appropriate in most scenarios and may lead to considerable savings of health care dollars.