Berberine chloride ameliorated PI3K/Akt-p/SIRT-1/PTEN signaling pathway in insulin resistance syndrome-induced rats

Insulin resistance is one of dangerous factors as it leads to numerous metabolic disorders such as non-insulin dependent diabetes mellitus. It affects most tissues mainly adipose tissue, liver and muscle. Nowadays, berberine has several medical applications against diseases. The current study was carried out to identify the effect of berberine chloride (BER-chloride) on phosphatidyl inositol-3-kinase/ phosphorylated protein kinase B/ sirtuin type 1/ phosphatase and tension homologue (PI3K/Akt-p/SIRT-1/PTEN) pathway during insulin resistance phenomena. Insulin resistance model was performed in experimental rats by using high fat diet. Plasma glucose, serum insulin, lipid profiles, hepatic oxidative stress markers were estimated. Serum transaminases activities and kidney function tests were determined. Further, hepatic PI3K, AKt-p, SIRT-1; PTEN levels were assayed. The concentration of adiponectin in serum, hepatic tissue and white adipose tissue was determined. Moreover, fold change in hepatic insulin, insulin receptor and retinol binding protein-4 (RBP4) at molecular level was performed. Histopathological study of white adipose tissue was also determined. The results showed increase the rats’ body weights, blood glucose, homeostatic model assessment, glycated hemoglobin, insulin and lipid profiles levels in group of rats fed on high fat diet for eight weeks and this elevation was decreased after administration of BER-chloride for two weeks. Further, BER-chloride administration exhibited improvement of oxidative stress parameters, PI3K, AKt-p, SIRT-1 and PTEN. This was associated with down-regulation of RBP4. According to these data we conclude that, BER-chloride mediated several insulin signaling pathways that could be of therapeutic significance to insulin resistance.


44
The increased prevalence of obesity between individuals has become a serious health 45 problem worldwide. Under normal conditions, β-cell of pancreas maintains the 46 normal glucose tolerance by increasing insulin release to overcome the reduction of 47 insulin efficiency. One of the predisposing risk factor to obesity is the amount of fat in 48 the diet due to modern life styles. Obesity usually accompanied by insulin resistance 49 and hyperglycemia [1]. Insulin resistance defined as a disease condition in which 50 insulin is secreted from β-cell of pancreas but its function is impaired in peripheral 51 tissues such as liver, adipose tissue and skeletal muscle. Insulin resistance usually 52 associated with metabolic disorders such as hyperlipidemia, type II diabetes mellitus, 53 non-alcoholic fatty liver and cardiovascular disease and early mortality is considered 54 one of insulin resistance prognosis in some individuals [2]. Phosphatidyl inositol-3-55 kinase/ phosphorylated protein kinase B (PI3K/Akt) pathway is one of the most 56 important signaling pathways which involved in metabolic effect of insulin [3]. 57 Therefore any treatment strategy of insulin resistance should be associated with 58 targeting of insulin signaling pathway complications. Nowadays, using of herbal 59 compounds occupied a huge importance in medical field. 60 Berberine (BER) is a natural isoquinoline alkaloid isolated from different plants such 61 as Berberis vulgaris [4]. BER is a strong base which is usually unstable when present 62 in free form so it usually accompanied with chloride ion in form of BER-chloride [5, 63 6]. BER has several pharmacological activities, it acts as anticancer [6,7], anti- Parkinson , s diseases as it has the ability to pass the blood brain barrier [4]. 69 To investigate whether BER-chloride has a protective effect on insulin resistance, we 70 set up in vivo model for insulin resistance by High fat diet (HFD) feeding. The effects 71 of BER-chloride on various insulin signaling pathway were investigated. Labcaire, Germany).

160
Total RNA was extracted from hepatic tissue by using total RNA extraction Kit and PCR conditions (denaturation temperature/ denaturation times/ annealing temperature/ annealing times/ extension temperature/ extension times/ 174 number of cycles of PCR program) and gene specific primers which used in PCR program were illustrated in Table 1. 11 175

Histopathological preparation of white adipose tissue:
176 White adipose tissue of each rat from each group was excised and immediately fixed 177 at 10% neutral buffered formalin solution after washing with ice cold normal saline.

178
The resultant fixed tissue samples were used for histological examination in the    (Table 2). Moreover, HFD up regulated insulin gene expression in liver 203 tissue and the treatment with BER-chloride for two weeks did not showed any 204 positive effect on insulin expression (Fig 1). Values represent the mean ± SD of six rats. ANOVA (one way) followed by Student-Newman-keuls test.

208
Means with letters (a), (b) and (c) were statistical represented compared to sham control group as follow: a= p < 0.001, b= p < 0.01, c= p < 0.05.

Lipid profiles parameters 210
Lipid profile in this study showed significant increase in HFD group than that of sham control where TC increased 1.2-fold and TG, LDL-c and 211 vLDL-c were 0.9-fold increase, while HDL-c was significantly decreased by 43%. The disturbance which occurred in lipid profiles was partially 212 repaired after using BER-chloride as treatment for two weeks (Table 3). Values represent the mean ± SD of six rats. ANOVA (one way) followed by Student-Newman-keuls test.  (Table 4).

224
Also, HFD intake increased both liver function parameters and kidney function test  Values represent the mean ± SD of six rats. ANOVA (one way) followed by Student-Newman-keuls test.   Values represent the mean ± SD of six rats. ANOVA (one way) followed by Student-Newman-keuls test.

258
The biochemical results were confirmed by the histological studies in white adipose 259 tissue ( Figs. 2A-2E). Control rat's white adipose tissue revealed normal tissue ( Fig   260   2A). Both PEG and BER-chloride administrated groups after feeding LFD were 261 similar to control rats (Fig 2B and 2C). However, adipose tissue of HFD rats revealed 262 multiple fibrosis and degeneration for the architecture of the adipocytes (Fig 2D).

263
Treatment of HFD rats with BER-chloride for two weeks lead to regeneration of the 264 cells and reduction of the lipids droplets inside it (Fig 2E).  representation was designed (Fig 3) to summarize the modification of insulin 375 resistance by BER-chloride.

595
Blue arrows means inhibition and violet arrows means activation.