Insulin-Mimicking Bioactivities of Acylated Inositol Glycans in Several Mouse Models of Diabetes with or without Obesity

<div><p>Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity.</p></div

The effects of IG-1 on plasma glucose concentrations (A), glycogen contents in the liver (B) and skeletal muscles (C), and intraperitoneal GTT curve (D) and AUC values (E) after 7 days in the C57B/6N mice fed with high fat diets.

<p><b>A</b>; Mice fed with high fat diets received an injection of IG-1 (red square; 1 mg/day, green square; 0.3 mg/day, blue square; 0.1 mg/day, black square; 0.03 mg/day), DMSO (black open square) or human regular insulin (red open square; 1 units/kg body wt) from tail veins, and blood glucose was assayed immediately before and at 30, 60, 90, 120, 180, 240 and 300 min after injection. Values are expressed as % change from preadministraion of IG-1 of each group. <b>B</b>,<b>C</b>; Glycogen contents of the liver and skeletal muscles were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100466#s2" target="_blank">Methods and Materials</a>. Basal glycogen contents in the liver and skeletal muscles were 8.42±1.46 and 1.33±0.29 mg/g tissue, respectively. <b>D</b>,<b>E</b>; C57B/6N mice fed with high fat diets (body weight 24.3±2.1 g) received intraperitoneal injection of IG-1 (0.1–1.0 mg/day), DMSO or human regular insulin (2 units/kg body wt) for 7 days. IG-1 doses per body weights were 41.2±3.5 mg/Kg (1 mg group), 12.3±1.1 mg/Kg (0.3 mg) and 4.12±0.35 mg/Kg (0.1 mg). Overnight-fast mice were given intraperitoneal glucose (2 g/kg body wt), and blood glucose was assayed immediately before and at 15, 30, 60, and 120 min after administration. Values are expressed as AUC based on weighted means of all glucose measurements (t = 0, 15, 30, 60 and 120 min). Similar representative results were obtained from 3 experiments, and the data are presented as means±SEM (n = 4). *p<0.05, **p<0.01, ^#p<0.001 (vs. DMSO), assessed by one-way ANOVA followed by Tukey-Kramer post hoc test.</p

The effects of IG-1 on glycogen synthesis (A) and lipogenesis (B) in 3T3-L1 adipocytes.

<p>3T3-L1 adipocytes were treated with various concentrations of IG-1 in DMSO or 1–1000 nM insulin for 30 min. Then 1 µCi of [¹⁴C]-glucose (approximately 220 cpm/nmol) was added and glycogen and lipid synthesis were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100466#s2" target="_blank">Methods and Materials</a>. Values are expressed as fold change vs. basal. Graphs show the means±SEM of 3 independent experiments. *p<0.05, **p<0.01, ^#p<0.001, ^##p<0.0001 (vs. insulin 0 nM), assessed by one-way ANOVA followed by Tukey-Kramer post hoc test.</p

The effects of IG-1 on plasma glucose concentrations (A), glycogen contents in the liver (B) and skeletal muscles (C), and intraperitoneal GTT curve (D) and AUC values (E) after 7 days in the C57B/6N mice fed with normal diets.

<p><b>A</b>. C57B/6N mice fed with normal diets received an injection of IG-1 (red square; 1 mg/day, green square; 0.3 mg/day, blue square; 0.1 mg/day, black square; 0.03 mg/day), DMSO (black open square) or human regular insulin (red open square; 1 units/kg body wt) from tail veins, and blood glucose was assayed immediately before and at 30, 60, 90, 120, 180, 240 and 300 min after injection. Values are expressed as % change from preadministraion of IG-1 of each group. <b>B</b>,<b>C</b>; C57B/6N mice fed with normal diets received an injection of 0.03–1 mg of IG-1, DMSO or human regular insulin (1 units/kg body wt) from tail veins, and glycogen contents of the liver and skeletal muscles were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100466#s2" target="_blank">Methods and Materials</a>. Basal glycogen contents in the liver and skeletal muscles were 9.28±1.04 and 1.51±0.29 mg/g tissue, respectively. <b>D</b>,<b>E</b>; C57B/6N mice fed with normal diets (body weight 19.3±1.6 g) received intraperitoneal injection of IG-1 (0.1–1.0 mg/day), DMSO or human regular insulin (1 units/kg body wt) for 7 days. IG-1 doses per body weights were 51.8±4.3 mg/Kg (1 mg group),15.5±1.3 mg/Kg (0.3 mg) and 5.18±0.43 mg/Kg (0.1 mg). Overnight-fast mice were given intraperitoneal glucose (2 g/kg body wt), and blood glucose was assayed immediately before and at 15, 30, 60, and 120 min after administration. Values are expressed as AUC based on weighted means of all glucose measurements (t = 0, 15, 30, 60, 120 min). Similar representative results were obtained from 3 experiments, and the data are presented as means±SEM (n = 4). *p<0.05, **p<0.01, ^#p<0.001 (vs. DMSO), assessed by one-way ANOVA followed by Tukey-Kramer post hoc test.</p

Metabolic parameters in the C57B/6N mice on high fat diets after the IG-1 treatment for 7 days.

<p>Mean ± SEM (n = 6).</p

The effects of IG-13, IG-14 and IG-15 on glycogen synthesis (A) and lipogenesis (B) in 3T3-L1 adipocytes.

<p>3T3-L1 adipocytes were treated with 1–100 nM insulin in the presence or absence of 100 µM IG-13, IG-14, or IG-15 in DMSO for 30 min. Then 1 µCi of [¹⁴C]-glucose (approximately 220 cpm/nmol) was added and glycogen and lipid synthesis were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100466#s2" target="_blank">Methods and Materials</a>. Values are expressed as fold change vs. basal. Graphs show the means±SEM of 3 independent experiments. *p<0.05 (vs. insulin 0 nM), assessed by one-way ANOVA followed by Tukey-Kramer post hoc test.</p

The effects of IG-1 on plasma glucose (A), glycogen contents in the liver (B) and skeletal muscles (C), and intraperitoneal GTT curve (D) and AUC values (E) after 7 days in the db/db obese diabetic mice.

<p><b>A</b>; Overnight-fasting db/db mice received an injection of IG-1 (red square; 1 mg/day, green square; 0.3 mg/day. blue square; 0.1 mg/day, black square; 0.03 mg/day), DMSO (black open square) or human regular insulin (red open square; 2 units/kg body wt) from tail veins, and blood glucose was assayed immediately before and at 30, 60, 90, 120, 180, 240 and 300 min after injection. Values are expressed as % change from preadministraion of IG-1 of each group. <b>B</b>,<b>C</b>; Glycogen contents of the liver and skeletal muscles were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100466#s2" target="_blank">Methods and Materials</a>. Basal glycogen contents in the liver and skeletal muscles were 8.24±4.14 and 1.33±0.29 mg/g tissue, respectively. <b>D</b>,<b>E</b>; The db/db mice (body weight 30.1±4.0 g) received intraperitoneal injection of IG-1 (0.1–1.0 mg/day), DMSO or human regular insulin (2 units/kg body wt) for 7 days. IG-1 doses per body weights were 33.2±4.4 mg/Kg (1 mg group), 10.0±1.3 mg/Kg (0.3 mg) and 3.32±0.44 mg/Kg (0.1 mg). Overnight-fast mice were given intraperitoneal glucose (2 g/kg body wt), and blood glucose was assayed immediately before and at 15, 30, 60, and 120 min after administration. Values are expressed as AUC based on weighted means of all glucose measurements (t = 0, 15, 30, 60 and 120 min). Similar representative results were obtained from 3 experiments, and the data are presented as means±SEM (n = 4). *p<0.05, **p<0.01 (vs. DMSO), assessed by two-way ANOVA followed by Tukey-Kramer post hoc test.</p

Metabolic parameters in the ob/ob obese mice after the IG-1 treatment for 28 days.

<p>Mean ± SEM (n = 6).</p

Nucleic acid sensing by T cells initiates Th2 cell differentiation

While T-cell responses are directly modulated by Toll-like receptor (TLR) ligands, the mechanism and physiological function of nucleic acids (NAs)-mediated T cell costimulation remains unclear. Here we show that unlike in innate cells, T-cell costimulation is induced even by non-CpG DNA and by self-DNA, which is released from dead cells and complexes with antimicrobial peptides or histones. Such NA complexes are internalized by T cells and induce costimulatory responses independently of known NA sensors, including TLRs, RIG-I-like receptors (RLRs), inflammasomes and STING-dependent cytosolic DNA sensors. Such NA-mediated costimulation crucially induces Th2 differentiation by suppressing T-bet expression, followed by the induction of GATA-3 and Th2 cytokines. These findings unveil the function of NA sensing by T cells to trigger and amplify allergic inflammation