Mapping the single-cell transcriptomic response of murine diabetic kidney disease to therapies.

Diabetic kidney disease (DKD) occurs in ∼40% of patients with diabetes and causes kidney failure, cardiovascular disease, and premature death. We analyzed the response of a murine DKD model to five treatment regimens using single-cell RNA sequencing (scRNA-seq). Our atlas of ∼1 million cells revealed a heterogeneous response of all kidney cell types both to DKD and its treatment. Both monotherapy and combination therapies targeted differing cell types and induced distinct and non-overlapping transcriptional changes. The early effects of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on the S1 segment of the proximal tubule suggest that this drug class induces fasting mimicry and hypoxia responses. Diabetes downregulated the spliceosome regulator serine/arginine-rich splicing factor 7 (Srsf7) in proximal tubule that was specifically rescued by SGLT2i. In vitro proximal tubule knockdown of Srsf7 induced a pro-inflammatory phenotype, implicating alternative splicing as a driver of DKD and suggesting SGLT2i regulation of proximal tubule alternative splicing as a potential mechanism of action for this drug class.

Discovery and Optimization of 7-Alkylidenyltetrahydroindazole-Based Acylsulfonamide EP3 Antagonists.

A novel series of 7-alkylidenyltetrahydroindazole-based acylsulfonamides were discovered as potent EP3 antagonists. The initial lead compound 7 exhibited potent in vitro EP3 inhibitory activity and good selectivity against other EP receptors. In addition, compound 7 demonstrated in vivo activity in a rat ivGTT model, reversing the suppressive effect of the EP3-specific agonist sulprostone on glucose-stimulated insulin secretion. Further optimization to improve the pharmacokinetic profile led to the discovery of compounds 26 and 28 with potent in vitro activity and significantly lower in vivo clearance and higher oral exposure than compound 7.

Optimization of physicochemical properties of pyridone-based EP3 receptor antagonists.

A novel series of pyridone-based EP3 receptor antagonists was optimized for good physical properties and oral bioavailability in rodents. The lead compounds 3h, 3l and 4d displayed good in vitro profiles, moderate to good metabolic stability and good rodent PK profiles with low clearance, high oral exposure and acceptable half-life.

GRK Inhibition Potentiates Glucagon-Like Peptide-1 Action.

The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) whose activation results in suppression of food intake and improvement of glucose metabolism. Several receptor interacting proteins regulate the signaling of GLP-1R such as G protein-coupled receptor kinases (GRK) and ß-arrestins. Here we evaluated the physiological and pharmacological impact of GRK inhibition on GLP-1R activity leveraging small molecule inhibitors of GRK2 and GRK3. We demonstrated that inhibition of GRK: i) inhibited GLP-1-mediated ß-arrestin recruitment, ii) enhanced GLP-1-induced insulin secretion in isolated islets and iii) has additive effect with dipeptidyl peptidase 4 in mediating suppression of glucose excursion in mice. These findings highlight the importance of GRK to modulate GLP-1R function in vitro and in vivo. GRK inhibition is a potential therapeutic approach to enhance endogenous and pharmacologically stimulated GLP-1R signaling.

Discovery of a Novel Series of Pyridone-Based EP3 Antagonists for the Treatment of Type 2 Diabetes.

A novel series of pyridones were discovered as potent EP3 antagonists. Optimization guided by EP3 binding and functional assays as well as by eADME and PK profiling led to multiple compounds with good physical properties, excellent oral bioavailability, and a clean in vitro safety profile. Compound 13 was identified as a lead compound as evidenced by the reversal of sulprostone-induced suppression of glucose-stimulated insulin secretion in INS 1E ß-cells in vitro and in a rat ivGTT model in vivo. A glutathione adduction liability was eliminated by replacing the naphthalene of structure 13 with the indazole ring of structure 43.

Tamoxifen suppresses pancreatic ß-cell proliferation in mice.

Tamoxifen is a mixed agonist/antagonist estrogen analogue that is frequently used to induce conditional gene deletion in mice using Cre-loxP mediated gene recombination. Tamoxifen is routinely employed in extremely high-doses relative to typical human doses to induce efficient gene deletion in mice. Although tamoxifen has been widely assumed to have no influence upon ß-cells, the acute developmental and functional consequences of high-dose tamoxifen upon glucose homeostasis and adult ß-cells are largely unknown. We tested if tamoxifen influences glucose homeostasis in male mice of various genetic backgrounds. We then carried out detailed histomorphometry studies of mouse pancreata. We also performed gene expression studies with islets of tamoxifen-treated mice and controls. Tamoxifen had modest effects upon glucose homeostasis of mixed genetic background (F1 B6129SF1/J) mice, with fasting hyperglycemia and improved glucose tolerance but without overt effects on fed glucose levels or insulin sensitivity. Tamoxifen inhibited proliferation of ß-cells in a dose-dependent manner, with dramatic reductions in ß-cell turnover at the highest dose (decreased by 66%). In sharp contrast, tamoxifen did not reduce proliferation of pancreatic acinar cells. ß-cell proliferation was unchanged by tamoxifen in 129S2 mice but was reduced in C57Bl6 genetic background mice (decreased by 59%). Gene expression studies revealed suppression of RNA for cyclins D1 and D2 within islets of tamoxifen-treated mice. Tamoxifen has a cytostatic effect on ß-cells, independent of changes in glucose homeostasis, in mixed genetic background and also in C57Bl6 mice. Tamoxifen should be used judiciously to inducibly inactivate genes in studies of glucose homeostasis.

Glucagon Receptor Antagonist-Stimulated α-Cell Proliferation Is Severely Restricted With Advanced Age.

Glucagon-containing α-cells potently regulate glucose homeostasis, but the developmental biology of α-cells in adults remains poorly understood. Although glucagon receptor antagonists (GRAs) have great potential as antidiabetic therapies, murine and human studies have raised concerns that GRAs might cause uncontrolled α-cell growth. Surprisingly, previous rodent GRA studies were only performed in young mice, implying that the potential impact of GRAs to drive α-cell expansion in adult patients is unclear. We assessed adaptive α-cell turnover and adaptive proliferation, administering a novel GRA (JNJ-46207382) to both young and aged mice. Basal α-cell proliferation rapidly declined soon after birth and continued to drop to very low levels in aged mice. GRA drove a 2.4-fold increase in α-cell proliferation in young mice. In contrast, GRA-induced α-cell proliferation was severely reduced in aged mice, although still present at 3.2-fold the very low basal rate of aged controls. To interrogate the lineage of GRA-induced α-cells, we sequentially administered thymidine analogs and quantified their incorporation into α-cells. Similar to previous studies of ß-cells, α-cells only divided once in both basal and stimulated conditions. Lack of contribution from highly proliferative "transit-amplifying" cells supports a model whereby α-cells expand by self-renewal and not via specialized progenitors.

Bioanalysis of sulprostone, a prostaglandin E2 analogue and selective EP3 agonist, in monkey plasma by liquid chromatography-tandem mass spectrometry.

Sulprostone is a potent prostaglandin E2 (PGE2) analogue and one of the first identified selective G-protein-coupled receptor 3 (EP3) agonists. It has been investigated as a potential antiulcer agent and frequently used in the research of EP3 antagonist. To assist pharmacokinetic and pharmacodynamic studies, a rapid and sensitive LC-MS/MS method was developed and qualified for the quantitation of sulprostone in monkey plasma. Using electrospray ionization mass spectrometry, an ammonium adduct in positive mode was chosen for analysis which had seven times of the sensitivity of the depronated ion in negative mode. Latanoprost, a prostaglandin F2α analogue, was used as the internal standard while good sensitivity and chromatography were obtained on a 2.6 µm core-shell column with pentafluorophenyl stationary phase. An assay dynamic range of 2 to 4000 ng/mL was achieved with a sample volume of 25 µL plasma on a Sciex API4000 instrument with simple protein precipitation. Several esterase inhibitors including sodium fluoride (NaF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), paraoxon and dichlorvos as well as wet ice conditions were explored for the stabilization of sulprostone in monkey plasma. The developed method was successfully applied for the evaluation of pharmacokinetics of sulprostone after intravenous administration of 0.5 mg/kg to cynomolgus monkey.

Role of CuO in improving NH3 and SO2 capture on nanoporous Fe2O3 sorbents.

In this work, mixed Fe/Cu oxides as sorbents for SO2 and NH3 removal were investigated. Nanoporous iron oxide mixed with 10, 20 and 30 at.% CuO were prepared by thermal decomposition of the corresponding oxalates at 250 °C for 5 h in air. The mixed Fe/Cu oxalates were obtained from the co-precipitation of iron/copper sulfate and ammonium oxalate during ultrasonication. The physical properties of the oxalate precursors and the resulting mixed Fe/Cu oxides were characterized with SEM, TGA-DSC, FTIR, powder XRD and Mössbauer spectroscopy. The porosity was studied by N2 adsorption-desorption isotherms and small angle X-ray scattering. Evenly dispersed CuO hindered the crystallization of Fe2O3, which significantly increased the specific BET surface area from 211 m2/g for Fe2O3 to 354 m2/g for Fe0.8Cu0.2Ox. As a result, SO2 and NH3 adsorption on Fe0.8Cu0.2Ox were enhanced by about 70% compared to Fe2O3. Compared to Fe2O3-impregnated activated carbons, nanoporous Fe0.8Cu0.2Ox could capture five times more SO2 per unit weight, which will be attractive for applications in respirators with lower weight and smaller size.

Highly Proliferative α-Cell-Related Islet Endocrine Cells in Human Pancreata.

The proliferative response of non-ß islet endocrine cells in response to type 1 diabetes (T1D) remains undefined. We quantified islet endocrine cell proliferation in a large collection of nondiabetic control and T1D human pancreata across a wide range of ages. Surprisingly, islet endocrine cells with abundant proliferation were present in many adolescent and young-adult T1D pancreata. But the proliferative islet endocrine cells were also present in similar abundance within control samples. We queried the proliferating islet cells with antisera against various islet hormones. Although pancreatic polypeptide, somatostatin, and ghrelin cells did not exhibit frequent proliferation, glucagon-expressing α-cells were highly proliferative in many adolescent and young-adult samples. Notably, α-cells only comprised a fraction (∼1/3) of the proliferative islet cells within those samples; most proliferative cells did not express islet hormones. The proliferative hormone-negative cells uniformly contained immunoreactivity for ARX (indicating α-cell fate) and cytoplasmic Sox9 (Sox9Cyt). These hormone-negative cells represented the majority of islet endocrine Ki67+ nuclei and were conserved from infancy through young adulthood. Our studies reveal a novel population of highly proliferative ARX+ Sox9Cyt hormone-negative cells and suggest the possibility of previously unrecognized islet development and/or lineage plasticity within adolescent and adult human pancreata.

ß Cells Persist in T1D Pancreata Without Evidence of Ongoing ß-Cell Turnover or Neogenesis.

Context: The cellular basis of persistent ß-cell function in type 1 diabetes (T1D) remains enigmatic. No extensive quantitative ß-cell studies of T1D pancreata have been performed to test for ongoing ß-cell regeneration or neogenesis. Objective: We sought to determine the mechanism of ß-cell persistence in T1D pancreata. Design: We studied T1D (n = 47) and nondiabetic control (n = 59) pancreata over a wide range of ages from the Juvenile Diabetes Research Foundation Network of Pancreatic Organ Donors with Diabetes via high-throughput microscopy. Intervention and Main Outcome Measures: We quantified ß-cell mass, ß-cell turnover [via Ki-67 and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)], islet ductal association, and insulin/glucagon coexpression in T1D and control pancreata. Results: Residual insulin-producing ß cells were detected in some (but not all) T1D cases of varying disease duration. Several T1D pancreata had substantial numbers of ß cells. Although ß-cell proliferation was prominent early in life, it dramatically declined after infancy in both nondiabetic controls and T1D individuals. However, ß-cell proliferation was equivalent in control and T1D pancreata. ß-cell death (assessed by TUNEL) was extremely rare in control and T1D pancreata. Thus, ß-cell turnover was not increased in T1D. Furthermore, we found no evidence of small islet/ductal neogenesis or α-cell to ß-cell transdifferentiation in T1D pancreata, regardless of disease duration. Conclusion: Longstanding ß-cell function in patients with T1D appears to be largely a result of ß cells that persist, without any evidence of attempted ß-cell regeneration, small islet/ductal neogenesis, or transdifferentiation from other islet endocrine cell types.

Incretin Therapies Do Not Expand ß-Cell Mass or Alter Pancreatic Histology in Young Male Mice.

The impact of incretins upon pancreatic ß-cell expansion remains extremely controversial. Multiple studies indicate that incretin-based therapies can increase ß-cell proliferation, and incretins have been hypothesized to expand ß-cell mass. However, disagreement exists on whether incretins increase ß-cell mass. Moreover, some reports indicate that incretins may cause metaplastic changes in pancreatic histology. To resolve these questions, we treated a large cohort of mice with incretin-based therapies and carried out a rigorous analysis of ß-cell turnover and pancreatic histology using high-throughput imaging. Young mice received exenatide via osmotic pump, des-fluoro-sitagliptin, or glipizide compounded in diet for 2 weeks (short-term) on a low-fat diet (LFD) or 4.5 months (long-term) on a LFD or high-fat diet (HFD). Pancreata were quantified for ß-cell turnover and mass. Slides were examined for gross anatomical and microscopic changes to exocrine pancreas. Short-term des-fluoro-sitagliptin increased serum insulin and induced modest ß-cell proliferation but no change in ß-cell mass. Long-term incretin therapy in HFD-fed mice resulted in reduced weight gain, improved glucose homeostasis, and abrogated ß-cell mass expansion. No evidence for rapidly dividing progenitor cells was found in islets or pancreatic parenchyma, indicating that incretins do not induce islet neogenesis or pancreatic metaplasia. Contrasting prior reports, we found no evidence of ß-cell mass expansion after acute or chronic incretin therapy. Chronic incretin administration was not associated with histological abnormalities in pancreatic parenchyma; mice did not develop tumors, pancreatitis, or ductal hyperplasia. We conclude that incretin therapies do not generate ß-cells or alter pancreatic histology in young mice.

Extreme obesity induces massive beta cell expansion in mice through self-renewal and does not alter the beta cell lineage.

AIMS/HYPOTHESIS: Understanding the developmental biology of beta cell regeneration is critical for developing new diabetes therapies. Obesity is a potent but poorly understood stimulus for beta cell expansion. Current models of obesity are complicated by developmental compensation or concurrent diabetes, limiting their usefulness for identifying the lineage mechanism(s) of beta cell expansion. We aimed to determine whether acute inducible obesity stimulates beta cell expansion and to determine the lineage mechanism of beta cell growth in obesity. METHODS: We created whole-body tamoxifen-inducible leptin receptor (LepR)-deficient mice (Ubc-Cre (ERT2) LepR (loxP/loxP) ) as a novel model of acute obesity. Beta cell mass and proliferation were quantified after short-term LepR deletion. Clonal analysis of beta cell expansion using the Brainbow2.1 reporter was performed 6 months post tamoxifen initiation. RESULTS: LepR deficiency induced a doubling of body mass within 3 weeks, with moderate glucose intolerance (unlike typical LepR mutant mice [db/db], which have frank diabetes). Beta cell mass expanded threefold through increased beta cell proliferation, without evidence for contribution from specialised progenitors or stem cells (via sequential thymidine labelling and Brainbow2.1 reporter). Thus, self-renewal is the primary lineage mechanism in obesity-induced beta cell expansion. However, even the rapid beta cell proliferation could not exceed the restrictions of the replication refractory period. CONCLUSIONS/INTERPRETATION: In summary, we created a novel model of inducible obesity demonstrating that even extreme metabolic demand does not alter beta cell lineage.

Novel nanoporous MnOx (x=∼1.75) sorbent for the removal of SO2 and NH3 made from MnC2O4·2H2O.

In this work, nanoporous manganese oxides (MnOx) were prepared by thermal decomposition of MnC2O4·2H2O at 225°C for 6h in air. The manganese oxalate dihydrate precipitate was made from manganese sulfate and ammonium oxalate during ultrasonication and stirring. The physical properties of the oxalate precursors and the resulting MnOx samples were characterized with SEM, TGA-DSC, FTIR and powder XRD. The specific surface areas and porosity of MnOx were studied by single-point BET and multi-point N2 adsorption-desorption measurements. The amorphous MnOx from oxalate prepared by sonication showed a specific surface area as large as 499.7m(2)/g. Dynamic SO2 and NH3 flow tests indicated that the adsorption capacity of MnOx, especially for SO2, can be increased by increased surface area. Compared to the best Mn3O4-impregnated activated carbon adsorbent, nanoporous MnOx could remove approximately three times as much SO2 and a comparable amount of NH3 per gram of adsorbent. This could lead to respirators of lower weight and smaller size which will be attractive to users.

Lifecycle Greenhouse Gas Analysis of an Anaerobic Codigestion Facility Processing Dairy Manure and Industrial Food Waste.

Anaerobic codigestion (AcoD) can address food waste disposal and manure management issues while delivering clean, renewable energy. Quantifying greenhouse gas (GHG) emissions due to implementation of AcoD is important to achieve this goal. A lifecycle analysis was performed on the basis of data from an on-farm AcoD in New York, resulting in a 71% reduction in GHG, or net reduction of 37.5 kg CO2e/t influent relative to conventional treatment of manure and food waste. Displacement of grid electricity provided the largest reduction, followed by avoidance of alternative food waste disposal options and reduced impacts associated with storage of digestate vs undigested manure. These reductions offset digester emissions and the net increase in emissions associated with land application in the AcoD case relative to the reference case. Sensitivity analysis showed that using feedstock diverted from high impact disposal pathways, control of digester emissions, and managing digestate storage emissions were opportunities to improve the AcoD GHG benefits. Regional and parametrized emissions factors for the storage emissions and land application phases would reduce uncertainty.

Extreme Beta-Cell Deficiency in Pancreata of Dogs with Canine Diabetes.

The pathophysiology of canine diabetes remains poorly understood, in part due to enigmatic clinical features and the lack of detailed histopathology studies. Canine diabetes, similar to human type 1 diabetes, is frequently associated with diabetic ketoacidosis at onset or after insulin omission. However, notable differences exist. Whereas human type 1 diabetes often occurs in children, canine diabetes is typically described in middle age to elderly dogs. Many competing theories have been proposed regarding the underlying cause of canine diabetes, from pancreatic atrophy to chronic pancreatitis to autoimmune mediated ß-cell destruction. It remains unclear to what extent ß-cell loss contributes to canine diabetes, as precise quantifications of islet morphometry have not been performed. We used high-throughput microscopy and automated image processing to characterize islet histology in a large collection of pancreata of diabetic dogs. Diabetic pancreata displayed a profound reduction in ß-cells and islet endocrine cells. Unlike humans, canine non-diabetic islets are largely comprised of ß-cells. Very few ß-cells remained in islets of diabetic dogs, even in pancreata from new onset cases. Similarly, total islet endocrine cell number was sharply reduced in diabetic dogs. No compensatory proliferation or lymphocyte infiltration was detected. The majority of pancreata had no evidence of pancreatitis. Thus, canine diabetes is associated with extreme ß-cell deficiency in both new and longstanding disease. The ß-cell predominant composition of canine islets and the near-total absence of ß-cells in new onset elderly diabetic dogs strongly implies that similar to human type 1 diabetes, ß-cell loss underlies the pathophysiology of canine diabetes.

GATA factors promote ER integrity and ß-cell survival and contribute to type 1 diabetes risk.

Pancreatic ß-cell survival remains poorly understood despite decades of research. GATA transcription factors broadly regulate embryogenesis and influence survival of several cell types, but their role in adult ß-cells remains undefined. To investigate the role of GATA factors in adult ß-cells, we derived ß-cell-inducible Gata4- and Gata6-knockout mice, along with whole-body inducible Gata4 knockouts. ß-Cell Gata4 deletion modestly increased the proportion of dying ß-cells in situ with ultrastructural abnormalities suggesting endoplasmic reticulum (ER) stress. Notably, glucose homeostasis was not grossly altered in Gata4- and Gata6-knockout mice, suggesting that GATA factors do not have essential roles in ß-cells. Several ER stress signals were up-regulated in Gata4 and Gata6 knockouts, most notably CHOP, a known regulator of ER stress-induced apoptosis. However, ER stress signals were not elevated to levels observed after acute thapsigargin administration, suggesting that GATA deficiency only caused mild ER stress. Simultaneous deletion of Gata4 and CHOP partially restored ß-cell survival. In contrast, whole-body inducible Gata4 knockouts displayed no evidence of ER stress in other GATA4-enriched tissues, such as heart. Indeed, distinct GATA transcriptional targets were differentially expressed in islets compared with heart. Such ß-cell-specific findings prompted study of a large meta-analysis dataset to investigate single nucleotide polymorphisms harbored within the human GATA4 locus, revealing several variants significantly associated with type 1 diabetes mellitus. We conclude that GATA factors have important but nonessential roles to promote ER integrity and ß-cell survival in a tissue-specific manner and that GATA factors likely contribute to type 1 diabetes mellitus pathogenesis.

ß-Cells are not generated in pancreatic duct ligation-induced injury in adult mice.

The existence of adult ß-cell progenitors remains the most controversial developmental biology topic in diabetes research. It has been reported that ß-cell progenitors can be activated by ductal ligation-induced injury of adult mouse pancreas and apparently act in a cell-autonomous manner to double the functional ß-cell mass within a week by differentiation and proliferation. Here, we demonstrate that pancreatic duct ligation (PDL) does not activate progenitors to contribute to ß-cell mass expansion. Rather, PDL stimulates massive pancreatic injury, which alters pancreatic composition and thus complicates accurate measurement of ß-cell content via traditional morphometry methodologies that superficially sample the pancreas. To overcome this potential bias, we quantified ß-cells from the entire pancreas and observed that ß-cell mass and insulin content are totally unchanged by PDL-induced injury. Lineage-tracing studies using sequential administration of thymidine analogs, rat insulin 2 promoter-driven cre-lox, and low-frequency ubiquitous cre-lox reveal that PDL does not convert progenitors to the ß-cell lineage. Thus, we conclude that ß-cells are not generated in injured adult mouse pancreas.

Terminal phosphinidene formation via tantalaziridine complexes.

Terminal, 4-coordinate phosphinidenes of Ta supported by bulky anilide ligands are prepared by an apparent reaction sequence involving metallaziridine phosphanide complexes.