In Vivo

The leaf of Elaeagnus lanceolata and Elaeagnus henryi as well as Elaeagnus pungens has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional clinical medicine. This study was aimed at evaluating the antiasthmatic, antitussive, and expectorant activities of the water extracts from the three plants in vivo and analyzing their chemical components by HPLC-DAD. At the medium and high doses, the water extracts of three Elaeagnus leaves significantly prolonged the preconvulsive time (P < 0.01) in guinea pigs, lengthened the latent period of cough (P < 0.01) and decreased the cough frequency caused by aqueous ammonia in mice (P < 0.01), and enhanced tracheal phenol red output in mice (P < 0.01). There were no significant differences in the pharmacological actions between the three Elaeagnus leaves. Moreover, there was more similarity on overlap peaks in the range of retention time from 10 to 40 min by HPLC and many peaks that belonged to flavonoids compounds. It suggested that the main constituents of the three Elaeagnus leaves were flavonoid for the pharmacological activities. These effects were the important evidence for the traditional use of E. henryi leaf and E. lanceolata leaf as well as E. pungens to treat asthma and chronic bronchitis

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation

Berberine Regulated Gck, G6pc, Pck1 and Srebp-1c Expression and Activated AMP-activated Protein Kinase in Primary Rat Hepatocytes

The effects of hormonal and dietary stimuli on hepatic glucose and lipid homeostasis include regulation of gene expression. Berberine, an effective compound in certain Chinese medicinal herbs, has been reported to lower plasma glucose and lipid levels in diabetic and hypercholesterolemic patients. We hypothesized that it may affect the expression of hepatic genes involved in glucose and lipid metabolism. The effects of berberine hydrochloride on viability, gene expression, and activation of AMP activated protein kinase (AMPK) in primary hepatocytes from Sprague-Dawley (SD), Zucker lean (ZL) or fatty (ZF) rats were examined with MTT assay, real-time PCR, and western blotting, respectively. Berberine hydochloride at 50 &#181;M or higher caused cytotoxic effects on hepatocytes. In SD and ZL hepatocytes, it induced Gck and suppressed G6pc expression at 10 and 25 &#181;M, but not as potent as 1 nM insulin. Its effects on Pck1, and insulin-regulated Gck and G6pc expression depended on the hepatocyte sources and the dosage used. In ZF hepatocytes, it increased Gck, and suppressed Pck1 and G6pc expression without insulin. Its effects on Gck and G6pc, but not Pck1 expression, were additive with insulin. Berberine hydrochloride at 25 &#181;M attenuated insulin-suppressed Pck1 (ZL/ZF cells), and insulin-induced Srebp-1c expression (SD/ZL/ZF cells), suggesting modulation of insulin action. Berberine hydrochloride did not alter these genes' mRNA stability. Its treatment caused a dose-dependent increase of phosphorylation of AMPK&#945;, and its substrate, acetyl-CoA carboxylase, in primary hepatocytes. We conclude that berberine hydrochloride regulated the transcription of hepatic genes involved in glucose and fatty acid metabolism.</p

The effects of compactin and insulin on the mRNA levels of <i>Srebp-1c</i> (A) and <i>Pck1</i> (B) in hepatocytes from <i>ad libitum</i> Zucker lean and fatty rats.

<p>Primary hepatocytes after pretreatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021342#s2" target="_blank">Materials and Methods</a> were incubated in medium A without or with 50 µM compactin in the absence or presence of 1 nM insulin for 6 hours. Total RNA was extracted and subjected to real-time PCR analysis. The expression level of the indicated transcripts in the control hepatocytes (lean or fatty) was arbitrarily assigned a value of one for its corresponding cell type. Results were plotted as fold induction and presented as means ± SD of four independent hepatocyte isolations for both lean and fatty rats (n = 4 for hepatocyte isolations; for <i>Srebp-1c</i>, b>a>c, b>d and b>b′, using one-way ANOVA; for <i>Pck1</i>, e/g>f, and f′>f, using one-way ANOVA; all <i>P</i><0.05).</p

The mRNA levels of <i>Srebp-1c</i> (A) and <i>Pck1</i> (B) in response to insulin, T1317 and insulin+T1317 treatments in hepatocytes from ZL or ZF rats in <i>ad libitum</i> or after fasting for overnight.

<p>Primary hepatocytes from rats on different feeding conditions were isolated and pre-treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021342#s2" target="_blank">Materials and Methods</a>. Hepatocytes were incubated in medium A without or with 1 µM T1317 in the absence or presence of 1 nM insulin for 6 hours. Total RNA was extracted and subjected to real-time PCR analysis. The expression level of the indicated transcripts in the control hepatocytes from lean or fatty rats in <i>ad lib</i> or overnight fasting was arbitrarily assigned a value of one for its corresponding cell type and feeding condition. Results were plotted as fold induction and presented as means ± SD of indicated numbers (in parenthesis after animal) of independent hepatocyte isolations for both lean and fatty rats (for <i>Srebp-1c</i>: d>b>c>a, g>a/f, j>h>i>a, and m>k/l>a, b>e, c>f, d>g, h>b, h>k, j>m, k>e, l>f, and m>g, using one-way ANOVA; for <i>Pck1</i>: a′/b′>c′/d′, a′/h′>i′/j′, a′/k′>l′/m′, f′>c′, g′>d′, f′>l′, and g′>m′, using one-way ANOVA; all <i>P</i><0.05).</p

The effects of insulin and glucagon on the mRNA levels of <i>Srebp-1c</i> (A) and <i>Pck1</i> (B) in hepatocytes from <i>ad libitum</i> Zucker lean and fatty rats.

<p>Primary hepatocytes after pretreatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021342#s2" target="_blank">Materials and Methods</a> were incubated in medium A without or with 10 nM glucagon in the absence or presence of 100 nM insulin for 6 hours. Total RNA was extracted and subjected to real-time PCR analysis. The expression level of the indicated transcripts in the control hepatocytes (lean or fatty) was arbitrarily assigned a value of one for its corresponding cell type. Results were plotted as fold induction and presented as means ± SD of five independent hepatocyte isolations for both lean and fatty rats (n = 5 for hepatocyte isolations; for <i>Srebp-1c</i>, b>a>c, b>d, b>b′, and c′>c, using one-way ANOVA; for <i>Pck1</i>, g>h>e>f, g′>h′>e′>f′, and e′>e, using one-way ANOVA; all <i>P</i><0.05).</p

Immunoblot analysis of phospho-Akt and total Akt levels in primary hepatocyes treated with increasing concentrations (A) or 1 nM (B) of insulin.

<p>After overnight pretreatment, primary hepatocytes from <i>ad libitum</i> ZL and ZF rats were incubated in medium A containing indicated concentrations of insulin for 10 minutes. After which, hepatocytes were washed once with 3 ml PBS and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021342#s2" target="_blank">Materials and Methods</a>. Total protein (30 µg/lane) was separated on 8% SDS/PAGE gels, detected by specific primary antibodies as indicated, and visualized by chemiluminescence. <b>A</b>. The levels of phoshpo-Akt(Thr308), phosphor-Akt(Ser473), and total Akt in <i>ad libitum</i> lean and fatty hepatocytes treated with increasing concentration of insulin (0 to 100 nM). Graph was the representative of three independent experiments with similar results using hepatocytes isolated from three different <i>ad libitum</i> ZL or ZF rats in different days. <b>B</b>. The levels of phospho-Akt(Ser473) and total Akt in <i>ad libitum</i> lean and fatty hepatocytes treated without or with 1 nM insulin. Graph represented three independent isolations of hepatocytes from different <i>ad libitum</i> ZL or ZF rats in different days, which were run side by side.</p