In this extensive study, real-time reverse transcriptase-polymerase chain reaction was used to analyze the expression levels of all 19 Wnt genes and their 11 potential antagonists in mouse blastocysts, pregastrula, gastrula, and neurula stages. By complementing these results with in situ hybridization, we revealed new expression domains for Wnt2b and Sfrp1, respectively, in the future primitive streak at the posterior side and in the anterior visceral endoderm before the initiation of gastrulation. Moreover, the anterior visceral endoderm expresses three secreted Wnt antagonists (Sfrp1, Sfrp5, and Dkk1) in partially overlapping domains. We also identified expression patterns for the Wnt1, Wnt3a, Wnt6, Wnt7b, Wnt9a, Wnt10b, and Sfrp1 genes at the blastocyst stage. In particular, the expression of Wnt1 and Sfrp1 predominantly in the inner cell mass and of Wnt9a in the mural trophoblast and inner cell mass cells surrounding the blastocoele suggests new roles for the Wnt pathway in preimplantation development. This article is the first report on the regional expression of Wnt genes in the mouse blastocyst.

Aims/hypothesisIslet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.MethodsHuman islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1−/− mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.ResultsCFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).Conclusions/interpretationThe ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.Electronic supplementary materialThe online version of this article (10.1007/s00125-018-4672-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Aims/hypothesis We investigated whether heterogeneous nuclear ribonucleoproteins F and K (hnRNP F, hnRNP K) mediate insulin inhibition of renal Agt expression and prevention of hypertension and kidney injury in an Akita mouse model of type 1 diabetes. Methods Adult male Akita mice (12 weeks old) were treated with insulin implants and killed at week 16. Untreated nonAkita littermates served as controls. The effects of insulin on blood glucose, systolic BP (SBP), renal proximal tubular cell (RPTC) gene expression and interstitial fibrosis were studied. We also examined immortalised rat RPTCs stably transfected with control plasmid or with plasmid containing rat Agt promoter in vitro.Results Insulin treatment normalised blood glucose levels and SBP, inhibited renal AGT expression but enhanced hnRNP F, hnRNP K and angiotensin-converting enzyme-2 expression, attenuated renal hypertrophy and glomerular hyperfiltration and decreased urinary albumin/creatinine ratio, as well as AGT and angiotensin II levels, in Akita mice. In vitro, insulin inhibited Agt but stimulated Hnrnpf and Hnrnpk expression in high-glucose media via p44/42 mitogen-activated protein kinase signalling in RPTCs. Transfection with Hnrnpf or Hnrnpk small interfering RNAs prevented insulin inhibition of Agt expression in RPTCs. Conclusions/interpretation These data indicate that insulin prevents hypertension and attenuates kidney injury, at least in part, through suppressing renal Agt transcription via upregulation of hnRNP F and hnRNP K expression in diabetic Akita mice. HnRNP F and hnRNP K may be potential targets in the treatment of hypertension and kidney injury in diabetes.

We investigated the relationship between Ang-(1-7) [angiotensin-(1-7)] action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR [Ang-(1-7) receptor] expression in Type 1 diabetic Akita mice. Ang-(1-7) was administered daily [500 μg/kg of BW (body weight) per day, subcutaneously] to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1-7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1-7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-β1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1-7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.

γ‐Aminobutyric acid (GABA) administration has been shown to increase β‐cell mass, leading to a reversal of type 1 diabetes in mice. Whether GABA has any effect on β cells of healthy and prediabetic/glucose‐intolerant obese mice remains unknown. In the present study, we show that oral GABA administration (ad libitum) to mice indeed increased pancreatic β‐cell mass, which led to a modest enhancement in insulin secretion and glucose tolerance. However, GABA treatment did not further increase insulin‐positive islet area in high fat diet‐fed mice and was unable to prevent or reverse glucose intolerance and insulin resistance. Mechanistically, whether in vivo or in vitro, GABA treatment increased β‐cell proliferation. In vitro, the effect was shown to be mediated via the GABAA receptor. Single‐cell RNA sequencing analysis revealed that GABA preferentially up‐regulated pathways linked to β‐cell proliferation and simultaneously down‐regulated those networks required for other processes, including insulin biosynthesis and metabolism. Interestingly, single‐cell differential expression analysis revealed GABA treatment gave rise to a distinct subpopulation of β cells with a unique transcriptional signature, including urocortin 3 (ucn3), wnt4, and hepacam2. Taken together, this study provides new mechanistic insight into the proliferative nature of GABA but suggests that β‐cell compensation associated with prediabetes overlaps with, and negates, its proliferative effects.—Untereiner, A., Abdo, S., Bhattacharjee, A., Gohil, H., Pourasgari, F., Ibeh, N., Lai, M., Batchuluun, B., Wong, A., Khuu, N., Liu, Y., Al Rijjal, D., Winegarden, N., Virtanen, C., Orser, B. A., Cabrera, O., Varga, G., Rocheleau, J., Dai, F. F., Wheeler, M. B. GABA promotes β‐cell proliferation, but does not overcome impaired glucose homeostasis associated with diet‐induced obesity. FASEB J. 33, 3968–3984 (2019). http://www.fasebj.org

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.

Aims/hypothesisWe investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes.MethodsAdult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied.ResultsOverexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1–7 receptor [MasR]) expression, and normalised urinary Ang 1–7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression.Conclusions/interpretationThese data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3700-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2–related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes.