High-throughput mediation analysis of human proteome and metabolome identifies mediators of post-bariatric surgical diabetes control

To improve the power of mediation in high-throughput studies, here we introduce High-throughput mediation analysis (Hitman), which accounts for direction of mediation and applies empirical Bayesian linear modeling. We apply Hitman in a retrospective, exploratory analysis of the SLIMM-T2D clinical trial in which participants with type 2 diabetes were randomized to Roux-en-Y gastric bypass (RYGB) or nonsurgical diabetes/weight management, and fasting plasma proteome and metabolome were assayed up to 3 years. RYGB caused greater improvement in HbA1c, which was mediated by growth hormone receptor (GHR). GHR’s mediation is more significant than clinical mediators, including BMI. GHR decreases at 3 months postoperatively alongside increased insulin-like growth factor binding proteins IGFBP1/BP2; plasma GH increased at 1 year. Experimental validation indicates (1) hepatic GHR expression decreases in post-bariatric rats; (2) GHR knockdown in primary hepatocytes decreases gluconeogenic gene expression and glucose production. Thus, RYGB may induce resistance to diabetogenic effects of GH signaling

Sources of beta cells inside the pancreas

The generation of beta(-like) cells to compensate for their absolute or relative shortage in type 1 and type 2 diabetes is an obvious therapeutic strategy. Patients first received grafts of donor islet cells over 25 years ago, but this procedure has not become routine in clinical practice because of a donor cell shortage and (auto) immune problems. Transplantation of differentiated embryonic and induced pluripotent stem cells may overcome some but not all the current limitations. Reprogramming exocrine cells towards functional beta(-like) cells would offer an alternative abundant and autologous source of beta(-like) cells. This review focuses on work by our research group towards achieving such a source of cells. It summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s0012-5016-3892-9, and by Heiko Lickert and colleagues, DOI: 10.1007/s00125-016-3949-9) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2)

Estrogen receptor α regulates β-cell formation during pancreas development and following injury

Identifying pathways for beta-cell generation is essential for cell therapy in diabetes. We investigated the potential of 17 beta-estradiol (E-2) and estrogen receptor (ER) signaling for stimulating beta-cell generation during embryonic development and in the severely injured adult pancreas. E-2 concentration, ER activity, and number of ER alpha transcripts were enhanced in the pancreas injured by partial duct ligation (PDL) along with nuclear localization of ER alpha in beta-cells. PDL-induced proliferation of beta-cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and beta-cell growth in PDL pancreas were impaired when ER was turned off chemically or genetically (ER alpha(-/-)), whereas in situ delivery of E-2 promoted beta-cell formation. In the embryonic pancreas, beta-cell replication, number of Ngn3(+) progenitor cells, and expression of key transcription factors of the endocrine lineage were decreased by ER alpha inactivation. The current study reveals that E-2 and ER alpha signaling can drive beta-cell replication and formation in mouse pancreas

Combining MK626, a Novel DPP-4 Inhibitor, and Low-Dose Monoclonal CD3 Antibody for Stable Remission of New-Onset Diabetes in Mice

<div><p>Combining immune intervention with therapies that directly influence the functional state of the β-cells is an interesting strategy in type 1 diabetes cure. Dipeptidyl peptidase-4 (DPP-4) inhibitors elevate circulating levels of active incretins, which have been reported to enhance insulin secretion and synthesis, can support β-cell survival and possibly stimulate β-cell proliferation and neogenesis. In the current study, we demonstrate that the DPP-4 inhibitor MK626, which has appropriate pharmacokinetics in mice, preceded by a short-course of low-dose anti-CD3 generated durable diabetes remission in new-onset diabetic non-obese diabetic (NOD) mice. Induction of remission involved recovery of β-cell secretory function with resolution of destructive insulitis and preservation of β-cell volume/mass, along with repair of the islet angioarchitecture via SDF-1- and VEGF-dependent actions. Combination therapy temporarily reduced the CD4-to-CD8 distribution in spleen although not in pancreatic draining lymph nodes (PLN) and increased the proportion of effector/memory T cells as did anti-CD3 alone. In contrast, only combination therapy amplified Foxp3+ regulatory T cells in PLN and locally in pancreas. These findings open new opportunities for the treatment of new-onset type 1 diabetes by introducing DPP-4 inhibitors in human CD3-directed clinical trials.</p></div

Anti-CD3+MK626 treatment rescued islet β-cell function, resolved destructive insulitis and preserved β-cell volume/mass in new-onset diabetic NOD mice.

<p>Mice treated as indicated were fasted and then challenged with glucose load for 15 minutes. (A–C) Changes in fasting and glucose-stimulated blood glucose values, glucose-stimulated plasma insulin and C-peptide levels (A), severity of insulitis and destructive lesions (B), pancreatic β-cell volume (µl) and insulin content (pmol/mg pancreas)(C) for the control groups and anti-CD3+placebo and anti-CD3+MK626 treated mice at 2 and 7 weeks after therapy. Pancreas sections were classified as with no (score 0), peri- (score 1), <50% (score 2), >50% (score 3) and destructive (score 4) insulitis. Only animals that normalized their blood glucose (<200 mg/dl) during reversal of diabetes by anti-CD3+placebo or anti-CD3+MK626 treatment are shown in the figure. All box plots are depicted with Tukey whiskers from minimum to maximum (values from at least 5 to 16 animals per group). * vs. new-onset diabetic mice; † vs. longstanding diabetic mice; ‡ vs. age-matched normoglycemic NOD-scid mice. One symbol <i>p</i><0.05; two symbols <i>p</i><0.01; three symbols <i>p</i><0.001 and four symbols <i>p</i><0.0001.</p

Anti-CD3+MK626 treatment temporarily reduced the CD4-to-CD8 distribution and enlarged the proportion of effector/memory T cells.

<p>(A) Frequency of CD4+ T cells (left panel) and CD8+ T cells (middle panel) as well as CD4-to-CD8 ratio (right panel) in spleens and PLN of new-onset diabetic NOD mice that remained protected for 2 and 7 weeks following each of the treatments. (B) Frequency of naïve (CD44loCD69-CD62L+), effector (CD44hiCD69+CD62L-) and memory (CD44hiCD69-CD62L+) T cell subsets in CD4+ (upper) and CD8+ (lower) gate from spleens and PLN of new-onset diabetic NOD mice that remained protected for 2 and 7 weeks following each of the treatments. Each dot represents an individual mouse. * vs. new-onset diabetic mice. One symbol p<0.05; two symbols p<0.01; three symbols p<0.001.</p

Anti-CD3+MK626 treatment allowed marginal β-cell replication in new-onset diabetic NOD mice.

<p>(A) Confocal microscopy images of insulin (gray), CldU (green), IdU (red) (left). Green arrowheads indicate CldU+ positive β-cells, red arrowheads indicated IdU+ positive β-cells and orange arrowheads indicate CldU+IdU+ β-cells. Quantitative analysis of β-cell proliferation after thymidine analog labeling, as measured by insulin+ cells that co-stained for CldU+, IdU+, or both (right). Box plots are depicted with Tukey whiskers from minimum and maximum. (B) Representative images from confocal microscopy of Ki67 (red) and insulin (green) staining of pancreatic sections from anti-CD3+MK626 treated (2 weeks after therapy) mice. Arrow heads indicate Ki67+ β-cells. The box plots represent the percentage of insulin+Ki67+ cells in at least 80 islets and are depicted with Tukey whiskers from minimum and maximum. For each figure set, at least 5,000 insulin+ cells were counted from 20 non-serial sections for the control and treated mice (5 to 6 per group), respectively. * vs. new-onset diabetic mice; † vs. longstanding diabetic mice. One symbol p<0.05; two symbols p<0.01.</p

Anti-CD3+MK626 treatment stimulated expression of endocrine β-cell markers in new-onset diabetic NOD mice.

<p>PDX-1, NeuroD, GLUT2 and INS-2 gene expression locally in pancreata of untreated new-onset and long-standing diabetic controls as well as in those retrieved from mice 7 weeks after anti-CD3+placebo or anti-CD3+MK626 therapy. * vs. new-onset diabetic mice; † vs. longstanding diabetic mice. One symbol p<0.05.</p

Anti-CD3+MK626 treatment induced stable diabetes reversal in new-onset diabetic NOD mice.

<p>New-onset diabetic NOD mice (2 consecutive readings of blood glucose values>200 mg/dl), received treatments as indicated in figures. Random blood glucose values were monitored until 7 weeks after therapy. The number of mice assigned to each treatment group is shown in the figure. The gray area represents the treatment period. (A) Percentage of diabetic mice per treatment group. † indicates dead or moribund mice. (B) Therapeutic efficacy of anti-CD3+placebo (left) and anti-CD3+MK626 treated (right) NOD mice was stratified according to blood glucose values at diabetes diagnosis. * p<0.05, ** p<0.01.</p

Anti-CD3+MK626 treatment rejuvenated pancreatic islets from new-onset diabetic NOD mice.

<p>Confocal microscopy images of GLUT-2 (red) and insulin (green) staining of pancreatic sections from anti-CD3+MK626 treated mice. The box plots represent the percentage of GLUT-2+insulin- (right) and GLUT-2+insulin+ (left) pancreatic islets from mice treated with anti-CD3+placebo or anti-CD3+MK626 2 weeks after therapy stop as well as untreated new-onset and longstanding diabetic controls and are depicted with Tukey whiskers from minimum and maximum. Islets (at least 80) from each experimental group (5 to 7 mice) from 2 to 4 non-serial sections per mouse were examined. * vs. new-onset diabetic mice; † vs. longstanding diabetic mice; ‡ vs. age-matched normoglycemic NOD-scid mice. One symbol p<0.05; two symbols p<0.01.</p