Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy

The β-cell mitogenic effects of ANGPTL8 have been subjected to substantial debate. The original findings suggested that ANGPTL8 overexpression in mice induced a 17-fold increase in β-cell proliferation. Subsequent studies in mice contested this claim, but a more recent report in rats supported the original observations. These conflicting results might be explained by variable ANGPTL8 expression and differing methods of β-cell quantification. To resolve the controversy, three independent labs collaborated on a blinded study to test the effects of ANGPTL8 upon β-cell proliferation. Recombinant human betatrophin (hBT) fused to maltose binding protein (MBP) was delivered to mice by intravenous injection. The results demonstrate that ANGPTL8 does not stimulate significant β-cell proliferation. Each lab employed different methods for β-cell identification, resulting in variable quantification of β-cell proliferation and suggests a need for standardizing practices for β-cell quantification. We also observed a new action of ANGPTL8 in stimulating CD45+ hematopoietic-derived cell proliferation which may explain, in part, published discrepancies. Overall, the hypothesis that ANGPTL8 induces dramatic and specific β-cell proliferation can no longer be supported. However, while ANGPTL8 does not stimulate robust β-cell proliferation, the original experimental model using drug-induced (S961) insulin resistance was validated in subsequent studies, and thus still represents a robust system for studying signals that are either necessary or sufficient for β-cell expansion. As an added note, we would like to commend collaborative group efforts, with repetition of results and procedures in multiple laboratories, as an effective method to resolve discrepancies in the literature

ANGPTL8 treatment in mice has no effect on β-cell proliferation.

<p><b>(a-d)</b> Staining for insulin (yellow) and <b>(a-b)</b> Ki67 (green) or <b>(c-d)</b> EdU (red) for <b>(a,c)</b> MBP and <b>(b,d)</b> MBP+hBT islets. Scale bar: 100 μm. <b>(e-j)</b> β-cell proliferation quantified by insulin⁺ DAPI⁺ cells containing <b>(e-g)</b> Ki67 or <b>(h-j)</b> EdU as a percentage of total β-cells, from two control groups (buffer injection alone or with EdU), MBP, and MBP+hBT samples. One-way ANOVA was performed with Bonferroni’s multiple comparison test. *** <i>p</i> < 0.001 MBP+hBT versus MBP, ** <i>p</i> < 0.01 MBP+hBT versus Buffer, no EdU and buffer, EdU. <b>(k-m)</b> Linear regression analysis of the correlation between EdU⁺ and Ki67⁺ β-cell proliferation. Data are mean ± SEM. Buffer, no EdU and buffer, EdU = 5 animals per group; MBP and MBP+hBT = 10 animals per group.</p

EdU captures all proliferative events detected by Ki67.

<p><b>(a-b)</b> Immunostaining by Lab #2 for insulin (yellow), Ki67 (green), EdU (red), and DAPI (blue). Scale bar: 100 μm. Insets demonstrate <b>(a)</b> Ki67⁺ EdU⁺ co-positive cells and <b>(b)</b> a rare Ki67⁺ EdU- cell. <b>(c-d)</b> Quantification of Ki67⁺ cells co-expressing EdU in <b>(c)</b> all pancreatic cells and <b>(d)</b> β-cells. Data are reported as the mean ± SEM. MBP and MBP+hBT = 7 animals per group.</p

Highly proliferative cells in islets of ANGPTL8 treated mice are not immune related cells.

<p>Immunostaining for EdU (green) and DAPI (blue), with various markers for immune-related cells (red) in <b>(a,c,e,g,i)</b> pancreatic islets or in <b>(b,d,f,h,j)</b> the spleen used as a positive control. <b>(a-b)</b> CD3 (T-cells), <b>(c-d)</b> B220 (B-Cells), <b>(e-f)</b> F480 (macrophages), <b>(g-h)</b> CD11c (dendritic cells), and <b>(i- j)</b> Gr-1 (Neutrophils). Insets indicate an EdU⁺ replicating cell that does not express CD3, B220, F480, CD11c, or Gr-1. Scale bar = 100 μm.</p

ANGPTL8 treatment in mice increases hematopoietic-derived cell proliferation.

<p><b>(a-d)</b> Staining for CD45 (red), EdU (green), insulin (yellow), and DAPI (blue) in <b>(a)</b> pancreatic islets, in <b>(b)</b> spleen used as a positive control, and in islets of <b>(c)</b> MBP or <b>(d)</b> MBP+hBT treated mice. <b>(e)</b> Quantification of CD45⁺ intra- and peri-islet cell proliferation. Intra- and peri-islet cells were identified by dilating insulin area by one cell’s diameter and filling all holes within the object. Scale bar = 100 μm. Student’s <i>t</i> test was performed. * <i>p</i> < 0.05.</p

ANGPTL8 treatment in mice increases non-β-cell proliferation.

<p><b>(a-b)</b> Staining performed by Lab #2 for insulin (yellow), EdU (green), Nkx6.1 (red), and DAPI (blue) for <b>(a)</b> MBP and <b>(b)</b> MBP+hBT islets. White arrowheads indicate a proliferating insulin⁺ Nkx6.1⁺ DAPI⁺ cell [inset <b>(a)</b>] and red arrowheads indicate a proliferating insulin^- Nkx6.1^- DAPI⁺ cell [inset <b>(b)</b>]. Scale bar: 100 μm. <b>(c-e)</b> Quantification of EdU⁺ proliferation for <b>(c)</b> β-cells, <b>(d)</b> total islet cells, and <b>(e)</b> non-β islet cells. <b>(f-h)</b> Quantification of <b>(f)</b> β-cell number, <b>(g)</b> total islet cell number, and <b>(h)</b> non-β islet cell number per islet. Islet cells were identified by dilating insulin area by one cell’s diameter and filling all holes within the object. β-cells were identified by Nkx6.1⁺ cells co-localized with DAPI surrounded by insulin. Non-β islet cells were calculated by subtracting the β-cell counts from the total islet cell counts. <b>(i)</b> Total islet cell proliferation by Ki67 from original stained slides by Lab #2 examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159276#pone.0159276.g003" target="_blank">Fig 3</a>. <b>(j-k)</b> Quantification of pancreatic proliferation by <b>(j)</b> Ki67⁺ or <b>(k)</b> EdU⁺ (% of total non-islet cells) from original stained slides by Lab #2 examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159276#pone.0159276.g003" target="_blank">Fig 3</a>. Data are mean ± SEM. MBP and MBP+hBT = 10 animals per group. Student’s <i>t</i> test was performed. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001.</p

Highly proliferative cells in islets of ANGPTL8 treated mice are not glucagon, endocrine, epithelial, neuronal, myofibroblast, or vascular cells.

<p><b>(a)</b> Immunostaining for insulin (yellow), glucagon (green), EdU (red) and DAPI (blue). <b>(b)</b> Immunostaining for synaptophysin (green), EdU (red), and DAPI (blue). <b>(c-h)</b> Immunostaining for EdU (green) and DAPI (blue) with various makers (red); <b>(c)</b> E-cadherin, <b>(d)</b> N-cadherin, <b>(e)</b> smooth muscle actin α (SMAα), <b>(f)</b> desmin, <b>(g)</b> CD31, <b>(h)</b> CD34. Insets indicate EdU positive cells that do not express glucagon, synaptophysin, E-cadherin, N-cadherin, SMAα, desmin, CD31, or CD34. Scale bar = 100 μm.</p

Schematic design of a collaborative, blinded experiment.

<p>Schematic design of a collaborative, blinded experiment.</p

ANGPTL8 is detected in serum and acutely increases triglycerides.

<p><b>(a-b)</b> SDS-PAGE gel electrophoresis of cell lysates from MBP and MBP+hBT expressing <i>E</i>.<i>coli</i> cells with <b>(a)</b> Coomassie blue staining and <b>(b)</b> western blot detection of MBP (42 kDa) and hBT (64 kDa). <b>(c)</b> Timing of serum collection and MBP or MBP+hBT tail vein injection. <b>(d)</b> Serum triglyceride (mg/del) levels over time (h). Data are reported as the mean ± SEM. MBP and MBP+hBT = 5 animals per group. Two-way ANOVA with a Bonferroni posthoc test was performed. * <i>p</i> < 0.05 versus MBP.</p