IAPP Marks Mono-hormonal Stem-cell Derived ß Cells that Maintain Stable Insulin Production in vitro and in vivo.

Islet transplantation for treatment of diabetes is limited by availability of donor islets and requirements for immunosuppression. Stem cell-derived islets might circumvent these issues. SC-islets effectively control glucose metabolism post transplantation, but do not yet achieve full function in vitro with current published differentiation protocols. We aimed to identify markers of mature subpopulations of SC-ß cells by studying transcriptional changes associated with in vivo maturation of SC-ß cells using RNA-seq and co-expression network analysis. The ß cell-specific hormone islet amyloid polypeptide (IAPP) emerged as the top candidate to be such a marker. IAPP+ cells had more mature ß cell gene expression and higher cellular insulin content than IAPP- cells in vitro. IAPP+ INS+ cells were more stable in long-term culture than IAPP- INS+ cells and retained insulin expression after transplantation into mice. Finally, we conducted a small molecule screen to identify compounds that enhance IAPP expression. Aconitine up-regulated IAPP and could help to optimize differentiation protocols.

Glucose Response by Stem Cell-Derived ß Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis.

Stem cell-derived ß (SC-ß) cells could provide unlimited human ß cells toward a curative diabetes treatment. Differentiation of SC-ß cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-ß cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-ß cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-ß cells. This activity can be rescued by challenging SC-ß cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.

A Nutrient-Sensing Transition at Birth Triggers Glucose-Responsive Insulin Secretion.

A drastic transition at birth, from constant maternal nutrient supply in utero to intermittent postnatal feeding, requires changes in the metabolic system of the neonate. Despite their central role in metabolic homeostasis, little is known about how pancreatic ß cells adjust to the new nutritional challenge. Here, we find that after birth ß cell function shifts from amino acid- to glucose-stimulated insulin secretion in correlation with the change in the nutritional environment. This adaptation is mediated by a transition in nutrient sensitivity of the mTORC1 pathway, which leads to intermittent mTORC1 activity. Disrupting nutrient sensitivity of mTORC1 in mature ß cells reverts insulin secretion to a functionally immature state. Finally, manipulating nutrient sensitivity of mTORC1 in stem cell-derived ß cells in vitro strongly enhances their glucose-responsive insulin secretion. These results reveal a mechanism by which nutrients regulate ß cell function, thereby enabling a metabolic adaptation for the newborn.

Live Cell Monitoring and Enrichment of Stem Cell-Derived ß Cells Using Intracellular Zinc Content as a Population Marker.

Our laboratory and others have developed protocols to generate glucose-responsive stem cell-derived ß cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell-based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell-derived ß cells and gene-editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell-derived ß cells. This purification of SC-ß cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors. Basic Protocol: Fluorescent labeling and isolation of stem cell-derived ß cells.

Apolipoprotein E is a pancreatic extracellular factor that maintains mature ß-cell gene expression.

The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key ß-cell transcription factors necessary for ß-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance ß-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain ß-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key ß-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature ß-cell gene expression profile.

IV acetaminophen: Efficacy of a single dose for postoperative pain after hip arthroplasty: subset data analysis of 2 unpublished randomized clinical trials.

Inadequate control of postoperative pain after orthopedic procedures may trigger complications that increase morbidity. Multimodal analgesia is used to manage pain effectively after surgical procedures and reduce the need for rescue analgesia. Intravenous (IV) acetaminophen (OFIRMEV; Cadence Pharmaceuticals, Inc.), an analgesic that has been studied and used in the multimodal management of acute pain after major orthopedic procedures, combines the safety seen with oral and rectal formulations with a preferred route of administration. Two double-blind, randomized, placebo-controlled clinical trials were conducted (total 130 patients) to determine the efficacy and safety of single-dose IV acetaminophen in patients following total hip arthroplasty. Although both studies were stopped prematurely, overlap in patient populations, study design, and methodologies in the single-dose phase of these studies allowed for analysis of their results to be presented concurrently. Both trials demonstrated IV acetaminophen having greater efficacy than placebo in terms of primary endpoints [pain intensity differences from T0.5 to T3 (P < 0.05 in both studies)]. The use of IV acetaminophen also reduced the need for rescue opioid consumption, with patients receiving IV acetaminophen consuming, on average, less than half the amount of rescue medication as those receiving placebo. IV acetaminophen was effective in treating moderate-to-severe pain after total hip arthroplasty and reduced the need for rescue opioid consumption.

Reversal of ß cell de-differentiation by a small molecule inhibitor of the TGFß pathway.

Dysfunction or death of pancreatic ß cells underlies both types of diabetes. This functional decline begins with ß cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate ß cell stress nor prevent, let alone reverse, ß cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature ß cells, is down-regulated in the early stages of T2D in mice and when ß cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature ß cell state, we screen for factors that reverse ß cell de-differentiation. We find that a small molecule inhibitor of TGFß receptor I (Alk5) protects cells from the loss of key ß cell transcription factors and restores a mature ß cell identity even after exposure to prolonged and severe diabetes.

Induction of spermatogenic synchrony by retinoic acid in neonatal mice.

Retinoic acid (RA) is required for the successful differentiation and meiotic entry of germ cells in the murine testis. The availability of RA to undifferentiated germ cells begins in a variable, uneven pattern during the first few days after birth and establishes the asynchronous pattern of germ cell differentiation in adulthood. It has been shown that synchronous spermatogenesis can be induced in 2 d postpartum mice, but not in adult mice, by treating vitamin A sufficient males with RA. In this study, neonatal males were treated at different ages with a single dose of RA and spermatogenesis was examined after recovery to adulthood. The failure of exogenous RA to alter asynchrony correlates with the appearance of meiotic preleptotene spermatocytes within the seminiferous epithelium.

Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males.

Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that Rdh10 loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of Rdh10 in postnatal mouse testes and found that an RDH10 deficiency in Sertoli cells, but not in germ cells, results in a mild germ cell depletion phenotype. A deficiency of RDH10 in both Sertoli and germ cells in juvenile mice results in a blockage of spermatogonial differentiation, similar to that seen in vitamin A-deficient animals. This defect in spermatogenesis arises from a complete deficiency in juvenile testicular RA synthesis and can be rescued by retinoid administration. Thus, in juvenile mice, the primary, but not exclusive, source of RA in the testes is Sertoli cells. In contrast, adult Rdh10-deficient mice exhibit phenotypically normal spermatogenesis, indicating that during development a change occurs in either the cellular source of RA or the retinaldehyde dehydrogenase involved in RA synthesis.

Exposure to retinoic acid in the neonatal but not adult mouse results in synchronous spermatogenesis.

Retinoic acid (RA) is required for germ cell differentiation, the regulation of which gives rise to a constant production of mature sperm. In testes from 3-day postpartum (dpp) RARE-hsplacZ mice, periodic regions positive for beta-galactosidase activity were observed along the length of the seminiferous tubules. Periodicity was abolished by treatment of neonates with exogenous RA at 2 dpp. To assess the consequences, 2-dpp mice were treated with RA, and the long- and short-term effects were assessed. Long-term effects of neonatal RA exposure included a delay in the appearance of advanced germ cells and the absence of a spermatogenic wave (synchronous spermatogenesis) in the adult. In contrast, RA exposure in vitamin A-sufficient adults did not result in synchronous spermatogenesis but rather induced apoptosis in a subset of spermatogonia. Shortly after (24 h) neonates were exposed, altered expression of known germ cell differentiation and the (Stra8, Kit, Sycp3, and Rec8) meiosis markers and an increase in the number of STRA8 and SYCP3 immunopositive cells were observed relative to those of vehicle controls. However, 48 and 72 h after exposure, a significant reduction in the number of STRA8 and SYCP3 immunopositive cells occurred. Immunohistochemical analysis of a marker for apoptosis demonstrated neonatal exposure resulted in increased germ cell apoptosis, as observed in the adult. Additionally, RA exposure resulted in increased Cyp26a1 expression of the RA-degrading enzyme. Thus, while RA treatment of neonatal and adult mice resulted in apoptosis of spermatogonia, synchronous spermatogenesis occurred only after neonatal RA exposure.

Efficacy and safety of a once-daily morphine formulation in chronic, moderate-to-severe osteoarthritis pain: results from a randomized, placebo-controlled, double-blind trial and an open-label extension trial.

A randomized, 4-week, double-blind trial followed by an open-label extension trial assessed the efficacy and safety of a once-daily, extended-release morphine formulation (Avinza (previously referred to as Morphelan)) in 295 patients with chronic, moderate-to-severe osteoarthritis pain who had failed to obtain adequate pain relief with NSAIDs and acetaminophen. Participants received one of four treatments: Avinza 30 mg once daily (QAM or QPM), MS Contin(R) 15 mg twice daily, or placebo twice daily. Patients (n =181) received Avinza QAM or QPM during the 26-week open-label extension trial and could increase their dose to optimize pain control. Avinza and MS Contin reduced pain and improved several sleep measures versus placebo. Analgesic efficacy was comparable between Avinza and MS Contin; however, Avinza QAM demonstrated greater improvements in overall quality of sleep. The most common adverse events were constipation and nausea. The majority of AEs occurred at a similar incidence among the active treatment groups.

Nitrogen and phosphorus uptake in two Idaho (USA) headwater wilderness streams.

Nitrate and phosphate solutions were released into two reaches of two central Idaho streams to determine within- and between-stream variability in uptake lengths, uptake rates, and mass transfer coefficients. Physical and biotic stream characteristics and periphyton nitrate-uptake rates in recirculating chambers were measured to determine their influence on nutrient dynamics. Phosphate uptake length did not differ among the four reaches. There were no within-stream differences in nitrate uptake lengths but they did differ between the two streams. Long nitrate uptake lengths likely were due to instream concentrations above saturation but also may have been influenced by differences in active surface area and algal abundance. Nitrate and phosphate uptake lengths were longer, and uptake rates higher, than most other published values. However, mass transfer coefficients were comparable to measurements in other streams. Mass transfer coefficients may be a better parameter for temporal and spatial comparisons of instream nutrient dynamics, and for determining the underlying causes of variability in uptake length.