FFA2 Contribution to Gestational Glucose Tolerance Is Not Disrupted by Antibiotics

<div><p>During the insulin resistant phase of pregnancy, the mRNA expression of free fatty acid 2 receptor (<i>Ffar2</i>) is upregulated and as we recently reported, this receptor contributes to insulin secretion and pancreatic beta cell mass expansion in order to maintain normal glucose homeostasis during pregnancy. As impaired gestational glucose levels can affect metabolic health of offspring, we aimed to explore the role of maternal <i>Ffar2</i> expression during pregnancy on the metabolic health of offspring and also the effects of antibiotics, which have been shown to disrupt gut microbiota fermentative activity (the source of the FFA2 ligands) on gestational glucose homeostasis. We found that maternal <i>Ffar2</i> expression and impaired glucose tolerance during pregnancy had no effect on the growth rates, <i>ad lib</i> glucose and glucose tolerance in the offspring between 3 and 6 weeks of age. To disrupt short chain fatty acid production, we chronically treated WT mice and <i>Ffar2</i>^<i>-/-</i> mice with broad range antibiotics and further compared their glucose tolerance prior to pregnancy and at gestational day 15, and also quantified cecum and plasma SCFAs. We found that during pregnancy antibiotic treatment reduced the levels of SCFAs in the cecum of the mice, but resulted in elevated levels of plasma SCFAs and altered concentrations of individual SCFAs. Along with these changes, gestational glucose tolerance in WT mice, but not <i>Ffar2</i>^<i>-/-</i> mice improved while on antibiotics. Additional data showed that gestational glucose tolerance worsened in <i>Ffar2</i>^<i>-/-</i> mice during a second pregnancy. Together, these results indicate that antibiotic treatment alone is inadequate to deplete plasma SCFA concentrations, and that modulation of gut microbiota by antibiotics does not disrupt the contribution of FFA2 to gestational glucose tolerance.</p></div

Nerve Guidance Conduits Based on Double-Layered Scaffolds of Electrospun Nanofibers for Repairing the Peripheral Nervous System

Compared to the nerve guidance conduits (NGCs) constructed from a single layer of aligned nanofibers, bilayer NGCs with random and aligned nanofibers in the outer and inner layers are more robust and tear-resistant during surgical procedures thanks to an isotropic mechanical property provided by the random nanofibers. However, it remains unclear whether the random nanofibers will interfere with the aligned nanofibers to alter the extension pattern of the neurites and impede regeneration. To answer this question, we seeded dorsal root ganglia (DRG) on a double-layered scaffold, with aligned and random nanofibers on the top and bottom layers, respectively, and evaluated the outgrowth of neurites. The random nanofibers in the bottom layer exerted a negative impact on the extension of neurites projecting from the DRG, giving neurites a less ordered structure compared to those cultured on a single layer of aligned nanofibers. The negative impact of the random nanofibers could be effectively mitigated by preseeding the double-layered scaffold with Schwann cells. DRG cultured on top of such a scaffold exhibited a neurite outgrowth pattern similar to that for DRG cultured on a single layer of aligned nanofibers. We further fabricated bilayer NGCs from the double-layered scaffolds and tested their ability to facilitate nerve regeneration in a rat sciatic nerve injury model. Both histomorphometric analysis and functional characterization demonstrated that bilayer NGCs with an inner surface that was preseeded with Schwann cells could reach 54%, 64.2%, and 74.9% of the performance of isografts in terms of nerve fiber number, maximum isometric tetanic force, and mass of the extensor digitorum longus muscle, respectively. It can be concluded that the bilayer NGCs hold great potential in facilitating motor axon regeneration and functional motor recovery

Effects of antibiotic treatment on gestational glucose tolerance before and during pregnancy.

<p>(<b>a</b>) Timeline of antibiotic treatment, pregnancy and reconstitution of the gut microbiota of female mice, where the control group was WT (<i>Ffar2</i>^+/+) mice and the experimental group was <i>Ffar2</i>^-/- mice. (<b>b-c</b>) Plasma glucose concentrations during an IPGTT in antibiotic-treated mice at G0 (<b>b</b>) and at G15 (<b>c</b>). (<b>d-e</b>) Plasma glucose concentrations during an IPGTT in mice after gut microbiome reconstitution at G0 (<b>d</b>) and G15 (<b>e</b>). (<b>f-g</b>) Comparison of AUC values for plasma glucose concentrations during an IPGTT administered to control, antibiotic-treated and gut microbiota reconstituted WT and <i>Ffar2</i>^-/- mice prior to pregnancy (<b>f</b>) and at G15 (<b>g</b>). (<b>h</b>) Area under the curve (AUC) values for plasma glucose concentrations during an IPGTT in WT and <i>Ffar2</i>^-/- mice on G15 of their first and second pregnancy. WT, circles and white bars; <i>Ffar2</i>^-/-, triangles and black bars. Data in (<b>b-e</b>) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (<b>f-h</b>) were compared by Student’s t-test. (*, p<0.05; **, p<0.01; ***p<0.001), n = 7–16, mice/group.</p

Antibiotic treatment substantially alters GLP-1 secretion independent of the mouse genotype.

<p>Insulin sensitivity, as measured by the insulin tolerance test in control female WT (<b>a</b>) and female <i>Ffar2</i>^-/- mice (<b>b</b>), treated with antibiotics (open circles) or untreated (filled circles), where the y axis shows the relative glucose level at each time point as compared to the glucose level at 0 min. (<b>c-d</b>) The serum insulin response to a glucose challenge in antibiotic treated mice (open symbols) compared with control mice (filled symbols) in both WT mice (<b>c</b>) and <i>Ffar2</i>^-/- mice (<b>d</b>). Plasma GLP-1 levels in antibiotic treated mice (white bars) compared to control mice (black bars) at 0 min and 30 min for the WT (<b>e</b>) and <i>Ffar2</i>^-/- mice (<b>f</b>). Data are represented as mean ± SEM. Data in (<b>a-d</b>) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (<b>e, f</b>) were compared by Student’s t-test. (*, p<0.05; **, p<0.01; ***p<0.001), n = 5–8 mice/group.</p

<i>WT</i>, <i>Ffar2</i>^-/- and *<i>Ffar2</i>^-/- mice exhibit similar weight gain, random glucose levels and glucose tolerance at 6 weeks of age.

<p>(<b>a</b>) Breeding scheme to generate these offspring is shown. For <b>b-g</b>, changes in body weight (<b>b-c</b>), <i>ad lib</i> plasma glucose concentrations (<b>d-e</b>) and plasma glucose concentrations during an IPGTT (<b>f-g</b>) with male (<b>b, d</b> and <b>f</b>) and female (<b>c, e</b> and <b>g</b>) <i>WT</i>, <i>Ffar2</i>^-/- and *<i>Ffar2</i>^-/- offspring are shown. Inserts for <b>f</b> and <b>g</b> represent the area under the curve for the IPGTT. WT, circles and white bars; <i>Ffar2</i>^-/-, squares and gray bars; *<i>Ffar2</i>^-/-, triangles and black bars. Data are represented as mean ± SEM (*p ≤ 0.05), n = 3–12. Data in (b-g) were compared by 2-way ANOVA with Bonferroni post-hoc analyses.</p

Simultaneous Purification of Pulchinenoside B₄ and B₅ from <i>Pulsatilla chinensis</i> Using Macroporous Resin and Preparative High Performance Liquid Chromatography

Pulchinenoside B₄ and B₅ (PB₄, PB₅) are two major triterpenoid saponins existing in the roots of <i>Pulsatilla chinensis</i> (Bunge) Regel. In this study, a systematic preparative process was developed for the simultaneous purification of PB₄ and PB₅ from the herb. The performance and separation characteristics of nine types of macroporous resins were critically evaluated. Static absorption/desorption experiments revealed that LX17 belonging to the polyacrylate class possessed superior separation properties. Further dynamic absorption/desorption experiments on LX17 column were conducted to obtain the optimal parameters. To obtain both compounds with high purity, a second stage procedure was developed using preparative reversed-phase high performance liquid chromatography with a dynamic axial compression column system. The separation process was high-efficiency and low-cost, which indicated potential for industrial applications

<i>Ffar2</i>^+/- mice exhibit normal glucose tolerance at gestational day 15 (G15).

<p>(<b>a</b>) Plasma glucose concentrations from WT, <i>Ffar2</i>^+/- and <i>Ffar2</i>^-/- female mice during an IPGTT at G15. (<b>b</b>) The corresponding area under the curve (AUC) for the IPGTT. WT circles and white bars; <i>Ffar2</i>^+/-, squares and gray bars; <i>Ffar2</i>^-/-, triangles and black bars. Data in (a) were compared by 2-way ANOVA with Bonferroni post-hoc analyses. Data in (b) were compared by Student’s t-test (*, p<0.05; **, p<0.01; ***p<0.001), n = 4–5 mice/ group.</p

Litter size and morbidity rate

<p>Litter size and morbidity rate</p

Antibiotics alter the relative abundance of individual SCFAs in circulation during pregnancy.

<p>Total plasma SCFA levels which includes acetate, propionate, and butyrate measured in WT and <i>Ffar2</i>^-/- mice at G0 <b>(a)</b> and G15 <b>(b)</b> under control vs antibiotic-treated conditions. (<b>b-h</b>) Relative abundance of individual SCFAs (acetate, <b>c-d</b>; propionate, <b>e-f</b>; and butyrate, <b>g-h</b>) in WT and <i>Ffar2</i>^-/- mice at G0 (<b>c, e</b> and <b>g</b>) and G15 (<b>d, f</b> and <b>h</b>) under control vs antibiotic-treated conditions. WT, white bars; <i>Ffar2</i>^-/-, black bars. Data are represented as mean ± SEM n = 6–15, and were analyzed by Student’s t-test (*p ≤ 0.05).</p

Age-Dependent Sex Hormone-Binding Globulin Expression in Male Rat

<div><p></p><p>Sex hormone binding globulin (SHBG) is known as a carrier protein, classically thought to be mainly synthesized in the liver and then secreted into the circulating system, where it binds to sex steroids with a high affinity and modulates the bioavailability of these hormones. In humans, the organs other than the liver known to produce SHBG include the brain, uterus, testis, prostate, breast and ovary, and the locally expressed SHBG is considered to play an important role in various physiological and pathological processes. A few studies of SHBG in rats were reported, but systemic SHBG studies in consideration of different organs and aging are currently missing. So we examined the SHBG expression in the brain, liver, prostate, and serum in 40 Sprague–Dawley male rats in four different groups (newborn, 2, 6, and 12 months old, respectively) with 10 in each group by immunohistochemistry, immunofluorescience microscopy, qRT-PCR, ELISA, western blotting, and laser confocal microscopy. We discovered that SHBG was increasingly expressed in all the three tissues along with age, and the SHBG protein expression was observed in the cytoplasm and membrane of neurons, hepatocyte, and prostate epithelial cells. The ELISA assay of the sera also supported an increasing SHBG level along with age. It is concluded that the locally synthesized SHBG in the liver, brain, and prostate and the circulating SHBG of male SD rats are positively associated with age, further indicating a potential role of SHBG in aging.</p></div