Leaf Extract from <i>Lithocarpus polystachyus</i> Rehd. Promote Glycogen Synthesis in T2DM Mice

<div><p>The purpose of this study was to investigate the effects of leaf extract from <i>Lithocarpus polystachyus</i> Rehd. on type II diabetes mellitus (T2DM) and the active ingredients of this effect. In addition, this study determined, for the first time, the underlying molecular and pharmacological mechanisms of the extracts on hyperglycemia using long-term double high diet-fed and streptozotocin (STZ) induced type II diabetic mice. In the present study, leaf extract, phloridzin and trilobatin were assessed <i>in vivo</i> (gavage) and <i>in vitro</i> (non-invasive micro-test technique, NMT) in experimental T2DM mice. The biochemical parameters were measured including blood glucose and blood lipid level, liver biochemical indexes, and hepatic glycogen. The relative expression of glycometabolism-related genes was detected. The effect of leaf extracts on physiological glucose flux in liver tissue from control and T2DM mice was also investigated. Body weight of experimental T2DM mice increased significantly after the first week, but stabilized over the subsequent three weeks; body weight of all other groups did not change during the four weeks’ study. After four weeks, all treatment groups decreased blood glucose, and treatment with leaf extract had numerous positive effects: a) promoted in glucose uptake in liver, b) increased synthesis of liver glycogen, c) reduced oxidative stress, d) up-regulation of glucokinase (GK), glucose transporter 2 (GLUT2), insulin receptor (IR) and insulin receptor substrate (IRS) expression in liver, e) down-regulation of glucose-6-phosphatase (G-6-P) expression, and f) ameliorated blood lipid levels. Both treatment with trilobatin or phloridzin accelerated liver glycogen synthesis, decreased oxidative stress and increased expression of GK. IRS and phosphoenolpyruvate carboxykinase (PEPCK) were both up-regulated after treatment with trilobatin. Expression of GLUT2, PEPCK and G-6-P were also increased in liver tissue after treatment with phloridzin. Our data indicate that leaf extract from <i>L</i>. <i>polystachyus</i> Rehd. has a preferable hypoglycemic effects than trilobatin or phloridzin alone. Leaf extract significantly increased glucose uptake and hepatic glycogen synthesis while also inducing a decline of hepatic gluconeogenesis and oxidative stress in T2DM mice. From this study, we draw conclusions that <i>L</i>. <i>polystachyus</i> promoted glycogen synthesis in T2DM mice, and that the active compounds were not only the trilobatin or phloridzin.</p></div

A representative example of glucose flux and concentration, and mean flux.

<p>(A) Representative sample showing glucose influx for a tissue taken from the flavonoid group (no exogenous insulin added). After addition of 20 mM exogenous glucose (shown by a dashed arrow), glucose uptake gradually increased (blue highlighted section). After addition of drug, an immediate and significant increase in glucose flux occurred, and flux stabilized at this level (red highlighted section). (B) Mean flux of glucose in <i>J</i>_<i>basal</i>, <i>J</i>_<i>stim</i>, and <i>J</i>_<i>post</i>. (C) Representative glucose oscillations before and after addition of glucose+extract. Prior to exogenous glucose addition, regular ultradian oscillations in glucose flux, but these waves were dampened after addition of glucose/extract.</p

Lipid levels, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and urea nitrogen (UN) in mice following 4 weeks of treatment with glibenclamide, leaf extract, phloridzin, or trilobatin.

<p>Lipid levels, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and urea nitrogen (UN) in mice following 4 weeks of treatment with glibenclamide, leaf extract, phloridzin, or trilobatin.</p

Effect of leaf extracts from <i>L</i>. <i>polystachyus</i> Rehd. on glucose tolerance.

<p>(A) OGTT tests for treatment groups after four weeks’ gavage; data taken after 12 h fast from tail at 0, 30, 60 and 120 min after glucose loading. (B) AUCG of mean blood glucose for all treatments. The data were analyzed with ANOVA. All values are expressed as the mean ± SEM, n = 10. Columns labeled with different letters are significantly different at <i>P</i> < 0.05.</p

Hepatic biochemical indexes levels of malondialdehyde (MDA), superoxide dismutase (T-SOD), glutamate pyruvate transaminase (GPT), or glutathione (GSH) in mice following 4 weeks of treatment with glibenclamide, leaf extract, phloridzin, or trilobatin.

<p>Hepatic biochemical indexes levels of malondialdehyde (MDA), superoxide dismutase (T-SOD), glutamate pyruvate transaminase (GPT), or glutathione (GSH) in mice following 4 weeks of treatment with glibenclamide, leaf extract, phloridzin, or trilobatin.</p

Effect of leaf extracts from <i>L</i>. <i>polystachyus</i> Rehd. on mean glucose influx of small liver tissue.

<p>A-D shows glucose influx of control mice with different level insulin in Hanks’, E-H shows glucose influx of experimental T2DM mice. Different colors represent four treatment groups. The data were analyzed with ANOVA. The values are expressed as the mean ± SEM, n = 6. Columns labeled with different letters are significantly different at <i>P</i> < 0.05.</p

Leaf extracts from <i>L</i>. <i>polystachyus</i> Rehd. have no effect on body weight of mice after four weeks of gavage.

<p>Body weight of groups control (A), glibenclamide (C), leaf extract (D) and trilobatin (F) were relatively unchanged, while body weight of the T2DM group (B) increased significantly after the first week and stabilized over the subsequent three weeks. As well as phloridzin (E) slightly increased at first week and fluctuated after the continue weeks. The data were analyzed using an ANOVA with repeated measures with a Sphericity Assumed or Greenhouse-Geisser correction, and the mean scores for body weight were statistically reported that control (<i>F</i> (4, 36) = 2.312, <i>P</i> = 0.076), T2DM (<i>F</i> (1.467, 14.675) = 6.556, <i>P</i> = 0.014), glibenclamide (<i>F</i> (2.169, 21.686) = 0.802, <i>P</i> = 0.471), leaf extract (<i>F</i> (1.505, 15.054) = 2.721, <i>P</i> = 0.109), phloridzin (<i>F</i> (4, 40) = 5.01, <i>P</i> = 0.002) and trilobatin (<i>F</i> (2.606, 26.058) = 6.228, <i>P</i> = 0.003), respectively. All values are expressed as the mean ± SEM, n = 10. Columns labeled with different letters are significantly different at <i>P</i> < 0.05.</p

Leaf extracts from <i>L</i>. <i>polystachyus</i> Rehd. decrease blood glucose from tail of experimental T2DM mice after gavage at different weeks.

<p>(A) was Before gavage, (B) was the First week after treatment, (C) was the Second after treatment, (D) was the Third week after treatment and (E) was the Fourth week after treatment. The data were analyzed with ANOVA. All the values are expressed as the mean ± SEM, n = 10. Columns labeled with different letters are significantly different at <i>P</i> < 0.05.</p