Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation

Engineering of Insulin Receptor Isoform-Selective Insulin Analogues

BACKGROUND: The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits

The Morphogenesis of Cranial Sutures in Zebrafish

<div><p>Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA <i>in situ</i> hybridization for <i>col1a1a</i>, <i>col2a1a</i>, <i>col10a1</i>, <i>bglap/osteocalcin</i>, <i>fgfr1a</i>, <i>fgfr1b</i>, <i>fgfr2</i>, <i>fgfr3</i>, <i>foxq1</i>, <i>twist2</i>, <i>twist3</i>, <i>runx2a</i>, <i>runx2b</i>, <i>sp7/osterix</i>, and <i>spp1/ osteopontin</i>, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.</p></div

The growth pattern of calvaria bones as revealed by sequential staining with Alizarin red and Calcein green.

<p>(A) Initial directions of frontal bone growth (white arrows) and radial growth of the parietal bone are depicted after each vital staining by red and white dotted lines. The supraoccipital bone is depicted by the red dotted line. (B-C) The white, blue and purple arrows indicate developing sutures: interfrontal, sagittal and coronal, respectively. (B) The contour of frontal and parietal bones growth is outlined by red dotted lines labeling the first vital staining with Alizarin and by white-dotted lines the second treatment by Calcein green. (C) Posterior frontal bone advancement (long white arrows) revealed after second vital staining with Calcein green, and lateral growth of frontal bone (small arrow on the left side). Note the interdigitation of the frontal and parietal bones. Skulls were dissected and mounted for imaging from a dorsal view.</p

RNAscope in situ hybridization and IHC for GFP performed for adults at 12 wpf.

<p>(A-F) Sequential sections (4 μm) of interfrontal suture expression. Names of genes tested by RNAscope in situ hybridization are labeled on each image. (A, B) Representative images of the skull taken at low magnification (5x) that visualize entire expression pattern of (A) <i>col1a1a</i> and (B) <i>col10a1a</i>. (C-F) Images of interfrontal sutures and the expression of representative genes as labeled on each image. (G) Immunohistochemistry for sp7 observed in the interfrontal suture using a GFP reporter in the Tg(sp7:EGFP)^b1212 transgenic line. Transverse paraffin section, GFP positive cells (green) and nuclei stained with DAPI (blue).</p

Histological examination of cranial sutures in adult zebrafish.

<p>H&E stained transverse sections (A-C) and sagittal sections (D-E) of skulls. (A) The interfrontal suture (boxed) and presented in (B) higher magnification; green arrows indicate the interfrontal suture between overlapping frontal bones. (C) The sagittal suture (yellow arrow) observed between two parietal bones. (D) The sagittal plane of sectioning revealed the coronal (left box) and lambdoid sutures (right box, black arrow). (E) The posterior frontal bone overlapping the anterior portion of the parietal bone, with the coronal suture formed between them (red arrow). (F) The lambdoid suture (black arrow) separates the parietal (upper plate) and the supraoccipital bone (lower plate).</p