Agonist-independent Gαz activity negatively regulates beta-cell compensation in a diet-induced obesity model of type 2 diabetes.

The inhibitory G protein alpha-subunit (Gαz) is an important modulator of beta-cell function. Full-body Gαz-null mice are protected from hyperglycemia and glucose intolerance after long-term high-fat diet (HFD) feeding. In this study, at a time point in the feeding regimen where WT mice are only mildly glucose intolerant, transcriptomics analyses reveal islets from HFD-fed Gαz KO mice have a dramatically altered gene expression pattern as compared with WT HFD-fed mice, with entire gene pathways not only being more strongly upregulated or downregulated versus control-diet fed groups but actually reversed in direction. Genes involved in the "pancreatic secretion" pathway are the most strongly differentially regulated: a finding that correlates with enhanced islet insulin secretion and decreased glucagon secretion at the study end. The protection of Gαz-null mice from HFD-induced diabetes is beta-cell autonomous, as beta cell-specific Gαz-null mice phenocopy the full-body KOs. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed beta cell-specific Gαz-null mice is significantly improved as compared with islets from HFD-fed WT controls, which, along with no impact of Gαz loss or HFD feeding on beta-cell proliferation or surrogates of beta-cell mass, supports a secretion-specific mechanism. Gαz is coupled to the prostaglandin EP3 receptor in pancreatic beta cells. We confirm the EP3γ splice variant has both constitutive and agonist-sensitive activity to inhibit cAMP production and downstream beta-cell function, with both activities being dependent on the presence of beta-cell Gαz.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

A patient-centered medical home model for comprehensive sickle cell care in infants and young children.

BACKGROUND: The patient-centered medical home (PCMH) has been proposed as a model for comprehensive care coordination and delivery for children with sickle cell disease (SCD), yet little is known regarding the implementation of PCMH core concepts on adherence to preventative care measures, health care utilization, and parent satisfaction. PROCEDURE: We implemented the newborn cohort clinic (NCC) to explore the application of the PCMH model for infants and children with SCD from birth to age 3 years in 2011. In July 2017, we conducted a retrospective chart review to evaluate subjects currently or previously followed in the clinic. We surveyed parents in the NCC to assess their satisfaction with their experience. RESULTS: A total of 112 patients have been managed in the NCC. All patients received penicillin prophylaxis, while 70% and 73% of patients, respectively, received the 23-valent pneumococcal vaccine and an initial transcranial Doppler by age 36 months. Most (92 of 112) of the subjects utilized the emergency department (569 encounters), with 86% of encounters for fever or other sickle cell-related complications. The majority of parents indicated satisfaction with the clinic, with 71% saying clinic providers always or usually spent enough time with their child, listened carefully to them (81%) and were sensitive to family values and customs (77%). CONCLUSIONS: A comprehensive sickle cell clinic as a component of a PCMH is feasible and can achieve high levels of preventative care. Parents are largely satisfied with this model of care.

Ssm1b expression and function in germ cells of adult mice and in early embryos.

Ssm1b (Strain-specific modifier of DNA methylation 1b) is a Krüppel-associated box (KRAB) zinc finger gene that promotes CpG methylation in the mouse transgene HRD (Heavy chain enhancer, rearrangement by deletion). We report here that Ssm1b expression and concomitant HRD methylation are also present in the male and female germ cells of adult mice. Ssm1b is expressed in both diploid (2N) and haploid (1N) oocytes, as well as in 1N spermatids and spermatozoa, but not in 2N spermatogonia. Interestingly, Ssm1b mRNA is not detected in any other adult mouse organ examined, although Ssm1-family mRNAs are highly expressed in the heart. Reflecting strain specificity, Ssm1b expression and HRD methylation are not observed in early-stage C3H/HeJ mouse embryos; however, an Ssm1b-like gene that closely resembles an Ssm1b-like gene previously found in wild-derived mice is expressed in cultured embryonic stem cells derived from C3H/HeJ embryos, suggesting that culture conditions affect its expression. Collectively, this work demonstrates that HRD methylation by Ssm1b is more temporally restricted during spermatogenesis compared to oogenesis, and is altered when embryonic stem cells are cultured from C3H/HeJ inner cell mass cells.

Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia.

Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.

Sin3a is required by sertoli cells to establish a niche for undifferentiated spermatogonia, germ cell tumors, and spermatid elongation.

Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell-autonomous chromatin modifications have emerged as important determinants for self-renewal and differentiation of stem cells, a role for non-cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Swi-independent 3a (Sin3a) in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell-specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell-associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine-signaling molecules chemokine (C-X-C motif) ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF1) and chemokine (C-X-C motif) receptor 4 (CXCR4), expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell-specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells as well as for tumor initiating cells.

Primary intracranial leiomyosarcoma in an immunocompetent patient: Case report and review of the literature.

Primary leiomyosarcoma is a rare tumor in the CNS, with few reported cases. Here, we describe a case of a primary intracranial leiomyosarcoma of the tentorium cerebelli. A 43-year-old woman presented with headache, acute vision loss, and difficulty speaking. MRI revealed a large heterogeneous-enhancing occipital mass, which was subsequently resected and diagnosed as a primary intracranial leiomyosarcoma. The patient went onto adjuvant radiotherapy delivering 60 Gy in 30 fractions. These tumors are exceedingly rare in immunocompetent individuals. We reviewed the 16 cases that have been reported in the literature. Surgical resection was the most common treatment (92%) with 53% receiving adjuvant radiation. There currently is no standard treatment regimen for intracranial leiomyosarcomas. Additional case reports that include descriptive treatment approaches with patient outcomes may help ascertain the best approach to treating these malignancies.

A single-islet microplate assay to measure mouse and human islet insulin secretion.

One complication to comparing ß-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.