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Endocrinology, doi:10.1210/en.2007-1752
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Endocrinology Vol. 149, No. 9 4510-4518
Copyright © 2008 by The Endocrine Society

Berberine Acutely Inhibits Insulin Secretion from β-Cells through 3',5'-Cyclic Adenosine 5'-Monophosphate Signaling Pathway

Libin Zhou1, Xiao Wang1, Li Shao1, Ying Yang, Wenbin Shang, Guoyue Yuan, Boren Jiang, Fengying Li, Jingfeng Tang, Hua Jing and Mingdao Chen

Shanghai Institute of Endocrine and Metabolic Diseases (L.Z., X.W., Y.Y., W.S., G.Y., B.J., F.L., J.T., H.J., M.C.), Department of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, People’s Republic of China; and Department of Geratology (L.S.), East Hospital, Shanghai Tongji University, Shanghai 200120, People’s Republic of China

Address all correspondence and requests for reprints to: Mingdao Chen, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Road II, Shanghai 200025, People’s Republic of China. E-mail: mingdaochensh{at}yahoo.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Berberine, a hypoglycemic agent, has recently been shown to activate AMP-activated protein kinase (AMPK) contributing to its beneficial metabolic effects in peripheral tissues. However, whether berberine exerts a regulatory effect on β-cells via AMPK or other signaling pathways and counteracts glucolipotoxicity remains uncertain. In the present study, the impact of berberine on β-cell function was investigated in vivo and in vitro. In high-fat-fed rats, berberine treatment for 6 wk significantly decreased plasma glucose and insulin levels before and after an oral glucose challenge along with the reduction of body weight and improvement of blood lipid profile. In accordance with the in vivo results, berberine acutely decreased glucose-stimulated insulin secretion (GSIS) and palmitate-potentiated insulin secretion in MIN6 cells and rat islets. However, pretreated with berberine for 24 h augmented the response of MIN6 cells and rat islets to glucose and attenuated the glucolipotoxicity. Berberine acutely increased AMPK activity in MIN6 cells. However, compound C, an AMPK inhibitor, completely reversed troglitazone-suppressed GSIS, not berberine-suppressed GSIS. Otherwise, berberine decreased cAMP-raising agent-potentiated insulin secretion in MIN6 cells and rat islets. These results suggest that the activation of AMPK is required for troglitazone-suppressed GSIS, whereas cAMP signaling pathway contributes, at least in part, to the regulatory effect of berberine on insulin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMP-ACTIVATED PROTEIN KINASE (AMPK) is a phylogenetically conserved intracellular energy sensor implicated in the regulation of glucose and lipid homeostasis (1). The enzyme is activated by phosphorylation on threonine-172 of the {alpha}-subunit in response to a decrease in cellular energy charge and a fall in ATP to AMP ratios (2). Once activated, AMPK may enhance mitochondrial ATP production largely by stimulating fatty acid oxidation through phosphorylation and inactivation of acetyl-coenzyme A carboxylase (ACC). AMPK is now considered as a potentially interesting pharmacological target for the treatment of type 2 diabetes (3) because activation of the enzyme has been shown to decrease gluconeogenesis and increase muscle glucose transport, both in vitro and in vivo (4, 5, 6). It has been shown that metformin and thiazolidinediones (TZDs) increased insulin sensitivity by activating AMPK (7).

In β-cells, AMPK activity is rapidly decreased by elevations in glucose concentration over the physiological range (8, 9). Clamping AMPK activity at the elevated levels at low glucose concentrations by the expression of constitutively active AMPK{alpha} subunits, the use of 5-aminoimidazole-4-carboxamide riboside (AICAR), metformin, or troglitazone suppresses glucose-stimulated insulin secretion (GSIS) from MIN6 cells and islets (6, 10, 11, 12) as well as from INS1 β-cells (13). Conversely, a dominant-negative form of AMPK stimulates insulin release at low glucose concentrations (11, 12). These data suggest that changes in AMPK activity contribute to the regulation of GSIS.

Berberine ([C20H18NO4]+), one of the major constituents of Chinese herb Rhizoma coptidis, has been used to treat type 2 diabetes in China, but its hypoglycemic mechanism remains unknown. Recently we and others found that berberine activated AMPK in adipocytes, myotubes, and liver (14, 15, 16, 17), which contributes to its beneficial metabolic effects in these tissues. However, the effect of berberine on insulin secretion is controversial (18). Moreover, nothing is known about the regulation of AMPK by berberine in β-cells. In addition, cAMP is a potent amplifier of insulin secretion (19). cAMP also plays an important role in lipolysis. It was reported that berberine inhibited lipolysis in 3T3-L1 adipoctyes (15). Pharmacological inhibition of the lipolysis arm is known to inhibit GSIS (20). In the present study, we investigated the effect of berberine on islet function in high-fat-fed rats and further explored whether AMPK or cAMP signaling is involved in the regulatory effect of berberine on insulin secretion in MIN6 cells and isolated rat islets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM and other culture reagents were obtained from Life Technologies, Inc. (Grand Island, NY). The cell culture plates were purchased from Nalge Nunc International (Roskilde, Denmark). Human insulin (Humulin R) was from Eli Lilly S.A.S. (Fegersheim, France). Methylthiotetrazole (MTT), 8-bromoadenosine-cAMP (8-bromo-cAMP), forskolin, 3-isobutyl-1-methylxanthine (IBMX), AICAR, glucagon-like peptide (GLP)-1, collagenase type XI, palmitate, and fatty acid-free BSA were purchased from Sigma (St. Louis, MO). Troglitazone was purchased from Calbiochem (La Jolla, CA). Anti-ACC, anti-AMPK{alpha}, anti-phospho-AMPK (threonine 172), antiphospho-ACC (serine79), antirabbit IgG conjugated with horseradish peroxidase were from Cell Signaling Technology (Beverly, MA). PepTag nonradioactive cAMP-dependent protein kinase A (PKA) assay kit was purchased from Promega (Madison, WI). Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Materials for rat insulin RIA were obtained from Linco Research (St. Charles, MO).

Animal experiments
Male Sprague Dawley rats (~8 wk of age) were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. The animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals of Shanghai Jiaotong University School of Medicine. Animals were kept on a 12-h light, 12-h dark cycle. After 1 wk of adaptation, the rats were randomly assigned to receive either the standard chow diet or high-fat diet for 14 wk. During the subsequent 6-wk experimental period, the high-fat diet rats were randomly allocated to two groups: high-fat control and high-fat berberine treatment. High-fat berberine-treated rats received 150 mg/kg–1·d–1 berberine by gavage once a day. Standard chow and high-fat control rats received the same amount of vehicle (water) by gavage.

Plasma lipid assay
Plasma triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were measured with automated biochemical analyzer. Plasma free fatty acids (FFAs) measurement was performed with commercial kits from Jiancheng Co. (Nanjing, China).

Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)
The OGTT was carried out at 6 wk after berberine treatment. After 12 h of food deprivation, 2.0 g/kg glucose was administered orally to the rats. The ITT was conducted after 6 h of food deprivation. Then 0.5 U/kg insulin (Humulin regular) was ip injected. Blood samples were taken from the tail at the time indicated for measurement of blood glucose and insulin levels.

Cell culture
MIN6 cells (passage 22–30) were cultured in DMEM with 25 mmol/liter glucose, 15% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 µl/liter β-mercaptoethanol at 37 C and 5% CO2. Cells were seeded at 2 x 105 per well in 1 ml DMEM in a 24-well plate for secretory experiment and 1 x 106 per well in a six-well plate for Western blotting. At 24 h before the acute experiment (24 h after seeding), the medium was replaced with DMEM containing 5.6 mmol/liter glucose. The fatty acid coupling procedure was performed as described previously (21). This procedure generated BSA-coupled palmitate in a molar ratio of 5:1 (generally 0.4 mmol/liter to 0.52% BSA, final).

Islet isolation and treatment
Islets of Langerhans were isolated from male Sprague Dawley rats by in situ pancreas collagenase infusion and separated by density gradient centrifugation (22). The concentration of collagenase type XI was 0.5 mg/ml. Freshly isolated rat islets were transferred to 24-well plates (10 islets/well) and cultured overnight in DMEM containing 5.6 mmol/liter glucose, 10 mmol/liter HEPES, 0.5% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C and 5% CO2.

Insulin secretion
Cultured cells or islets were washed once in Krebs-Ringer bicarbonate (KRB) buffer [128.8 mmol/liter NaCl, 4.8 mmol/liter KCl, 1.2 mmol/liter KH2PO4, 1.2 mmol/liter MgSO4, 2.5 mmol/liter CaCl2, 5 mmol/liter NaHCO3, and 10 mmol/liter HEPES (pH 7.4) with 0.1% BSA] containing 2.8 mmol/liter or 3.3 mmol/liter glucose, and then they were preincubated for 30 min in 1 ml of the same medium at 37 C. This buffer was then replaced with 1 ml of prewarmed KRB containing other additions as indicated for a further 60 min at 37 C. An aliquot was then removed for analysis of insulin secretion by RIA. Cells were lysed in 70% acid-ethanol solution for DNA quantification and subsequent normalization.

MTT method
When MIN6 cells were incubated with DMEM containing 0.2% BSA and berberine for 24 h, 100 µl MTT solution (0.5 mg/ml) dissolved in serum-free DMEM were added to each well. After 4 h incubation at 37 C, the MTT medium was replaced with dimethyl sulfoxide. After shaking, the ODs at 570 nm were measured using a Multiskan MS (Labsystems, Vantaa, Finland).

Western blotting
MIN6 cells in six-well plates were washed twice with ice-cold PBS and placed immediately in lysis buffer containing 25 mmol/liter HEPES (pH 7.4), 1% Nonidet P-40, 100 mmol/liter NaCl, 2% glycerol, 5 mmol/liter NaF, 1 mmol/liter EDTA, 1 mmol/liter Na3VO4, 1 mmol/liter NaPPi, 1 mmol/liter phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin. Lysates were gently mixed for 10 min at 4 C and then centrifuged at 13,000 x g for 15 min at 4 C. The protein concentration of the extracts was determined according to the method of Bradford, using BSA as the standard. Samples were separated by SDS-PAGE on 8% polyacrylamide gels and transferred to PVDF-Plus membranes (Bio-Rad, Hercules, CA). Primary antibodies (see above) were detected with donkey antirabbit at 1:2000 for 1 h at room temperature. Visualization was detected with chemiluminescence reagent, using the ECL Western blotting analysis system (Amersham Biosciences, Piscataway, NJ) and exposure to Kodak film (Rochester, NY).

PKA activity assay
The isolated islets treated with different agents were collected in PKA extract buffer containing 25 mmol/liter Tris-HCl (pH 7.4), 0.5 mmol/liter EDTA, 0.5 mmol/liter EGTA, 10 mmol/liter β-mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Protein was extracted and harvested by sonication and centrifugation. The enzymatic activity of PKA in lysates was assessed by measuring phosphorylation of kemptide, a highly specific peptide substrate for PKA, as described in the protocol.

Statistics
Data are presented as means ± SEM. Significance between groups was determined using an unpaired two-tailed Student’s t test or one-way ANOVA when appropriate. Significance was established at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolic effect of berberine on high-fat diet rats
The high-fat diet significantly increased body weight, compared with the standard chow rats at 14 wk after starting the diet (data not shown). The high-fat diet rats were further stratified into high-fat diet control groups and berberine-treated high-fat diet group. After 6 wk treatment, berberine-treated rats gained significantly less body weight, as well as the weight of liver and epididymal fat, than high-fat diet control rats. Plasma TC, LDL-C and FFAs in high-fat diet rats increased significantly, compared with standard chow rats. After berberine treatment, plasma triglycerides, TC, LDL-C and FFAs decreased markedly (Table 1Go).


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  		TABLE 1. Effect of berberine on body weight and plasma lipid in rats fed with high-fat diet

 
To assess glucose homeostasis and insulin sensitivity in high-fat-fed rats treated with berberine, we performed OGTT and ITT. Berberine treatment resulted in a reduction in fasting plasma glucose and insulin in high-fat diet rats to levels comparable with those in standard chow rats. After glucose loading, the plasma glucose and insulin levels of berberine-treated rats at all time points were significantly lower than those of high-fat diet control rats (Fig. 1Go, A and B). In accordance with the result of OGTT, plasma glucose concentrations of berberine-treated rats decreased much more than those of high-fat diet control rats at 15, 60, and 120 min after receiving ip 0.5 U/kg insulin (Fig. 1CGo), suggesting that berberine improved high-fat diet-induced insulin resistance.


Figure 1
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  		FIG. 1. Circulating glucose and insulin levels during oral glucose tolerance test and insulin tolerance test. Male Sprague Dawley rats were fed a high-fat (HF) or a standard chow (SC) diet for 14 wk and high-fat diet rats were treated with berberine (HF+BBR) for another 6 wk. At the end of the period, all animals were fasted overnight (12 h) and then received 2.0 g/kg glucose by gavage. Tail vein blood samples were taken at the indicated times for the measurement of blood glucose and insulin levels. A, Blood glucose levels. B, Blood insulin levels. C, Blood glucose levels were determined at the indicated times after rats were ip challenged with 0.5 U/kg insulin. Results are means ± SEM of nine or 10 animals for each group. *, P < 0.05; **, P < 0.01 vs. a standard chow diet rats. #, P < 0.05; ##, P < 0.01 vs. high-fat diet rats.

 
Acute effect of berberine on insulin secretion in MIN6 cells and rats islets
As mentioned before, berberine decreased fasting insulin secretion and GSIS in high-fat diet rats (Fig. 1BGo). To determine whether berberine exerts a direct effect on insulin secretion in β-cells, we exposed MIN6 cells to berberine in the presence of 25 mmol/liter glucose for 1 h. Berberine significantly decreased insulin secretion from MIN6 cells in a dose-dependent manner (Fig. 2AGo). Consistent with other studies (23, 24), FFAs acutely augmented insulin secretion from MIN6 cells and rat islets as we have previously shown (25). To further investigate whether berberine also has a regulatory effect on FFA-potentiated insulin secretion, MIN6 cells and rat islets were incubated with 2.5 µmol/liter berberine in the presence of low and high glucose level with or without 0.4 mmol/liter palmitate for 1 h. Addition of berberine had no effect on insulin secretion from MIN6 cells at 2.8 mmol/liter glucose and rat islets at 3.3 mmol/liter glucose but resulted in 40% decrease in MIN6 cells at 25 mmol/liter glucose and 53% decrease in rat islets at 16.7 mmol/liter glucose, respectively. Berberine significantly decreased insulin secretion in the presence of palmitate at both 2.8 and 25 mmol/liter glucose in MIN6 cells (P < 0.01; Fig. 2BGo). In isolated rat islets, berberine showed a similar action. Palmitate-potentiated insulin secretion at both 3.3 and 16.7 mmol/liter glucose was markedly inhibited by 32 and 60% in the presence of 2.5 µmol/liter berberine (Fig. 2CGo).


Figure 2
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  		FIG. 2. Acute effect of berberine on insulin secretion. MIN6 cells and isolated rat islets were cultured in KRB buffer with or without berberine containing the indicated concentrations of glucose and 0.4 mmol/liter palmitate (Pal) for 1 h and assayed for insulin secretion. A, Dose-dependent effect of berberine on insulin secretion from MIN6 cells in the presence of 25 mmol/liter glucose. B, MIN6 cells were incubated with 2.5 µmol/liter berberine (BBR) at 2.8 mmol/liter glucose or 25 mmol/liter glucose. C, Isolated rat islets were incubated with 2.5 µmol/liter berberine (BBR) at 3.3 or 16.7 mmol/liter glucose. All values were normalized to total DNA. Results were given as means ± SEM from three independent experiments, each performed in triplicate. *, P < 0.01 vs. corresponding control (CON) values (without berberine).

 
Long-term regulation of insulin secretion by berberine
Cell viability was assayed by the MTT method. As shown in Fig. 3AGo, the OD significantly decreased after MIN6 β-cells were incubated with 2.5 µmol/liter berberine for 24 h, suggesting that berberine exerts a toxic effect on MIN6 cells at this concentration or higher. Therefore, the long-term effect of berberine on insulin secretion was observed at the concentration of 1 µmol/liter. After MIN6 cells were pretreated with 1 µmol/liter berberine for 24 h at 2.8 mmol/liter glucose, 25 mmol/liter glucose, or 25 mmol/liter glucose plus 0.25 mmol/liter palmitate, cells from each group were then stimulated for 1 h in KRB buffer at 2.8 mmol/liter glucose, 25 mmol/liter glucose or 25 mmol/liter glucose plus berberine. In MIN6 cells pretreated with berberine from each group (Fig. 3BGo, bars 4–6, 10–12, and 16–18), the basal insulin secretion and GSIS were all increased in the last hour of incubation without berberine (P < 0.01). Pretreatment with 25 mmol/liter glucose plus palmitate resulted in decreased GSIS (Fig. 3BGo, bars 13–15), which was partly abrogated by berberine, even in the last hour of incubation with berberine (Fig. 3BGo, bars 16–18). After rat islets were preincubated with 1 µmol/liter berberine for 24 h, the response of islets to high glucose significantly increased in the last hour of incubation without berberine (P < 0.01; Fig. 3CGo).


Figure 3
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  		FIG. 3. Long-term effect of berberine on insulin secretion. A, MIN6 cells were cultured in DMEM containing 25 mmol/liter glucose with the indicated concentrations of berberine for 24 h and assayed for cell viability with MTT. B, MIN6 cells were pretreated with 1 µmol/liter berberine (BBR) for 24 h in DMEM containing 2.8 mmol/liter glucose, 25 mmol/liter glucose, or 25 mmol/liter glucose plus 0.25 mmol/liter palmitate, and then cells from each group were stimulated for 1 h in KRB buffer at 2.8 mmol/liter glucose, 25 mmol/liter glucose, or 25 mmol/liter glucose plus berberine for insulin secretion assay. C, Isolated rats islets were pretreated in DMEM containing 5.6 mmol/liter glucose with or without 1 µmol/liter berberine (BBR) for 24 h and then cultured in KRB buffer with the indicated concentrations of glucose for 1 h (without berberine) for insulin secretion assay. All values were normalized to total DNA. Results were given as means ± SEM from three independent experiments, each performed in triplicate. *, P < 0.01 vs. corresponding control (CON) values (without berberine).

 
Effect of AMPK inhibitor on berberine-suppressed insulin secretion
Berberine acutely increased the phosphorylation of AMPK and ACC in MIN6 cells (Fig. 4Go, A and B). To determine whether berberine-suppressed GSIS was mediated through AMPK activation, we further examined the effects of compound C, an AMPK inhibitor, on berberine-suppressed insulin secretion. Unexpectedly, compound C did not reverse berberine-suppressed GSIS in MIN6 cells (Fig. 4CGo). On the contrary, troglitazone-suppressed insulin secretion was completely reversed by compound C (Fig. 4CGo).


Figure 4
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  		FIG. 4. Berberine-suppressed GSIS were not reversed by compound C. A, MIN6 cells were incubated with the indicated concentrations of berberine (BBR) for 1 h. Total cell lysates were analyzed for the phosphorylation (p) of ACC and AMPK by Western blot. B, MIN6 cells were incubated 2.5 µmol/liter berberine for the indicated time. Total cell lysates were analyzed for the phosphorylation of ACC and AMPK by Western blot. C, MIN6 cells were preincubated with 10 µmol/liter compound C for 30 min and then treated with 2.5 µmol/liter berberine and 10 µmol/liter troglitazone at the indicated concentrations of glucose for 1 h for insulin secretion assay. A representative blot from three independent experiments is shown. All three experiments showed similar results. Results were given as means ± SEM from three independent experiments, each performed in triplicate. *, P < 0.01 vs. 25 mmol/liter glucose; #, P < 0.01 vs. troglitazone alone.

 
Berberine decreased cAMP-raising agents-potentiated insulin secretion in MIN6 cells and rat islets
cAMP-raising agents including the phosphodiesterase inhibitor IBMX, the adenylate cyclase activator forskolin, and GLP-1 stimulated insulin secretion at 2.8 mmol/liter glucose in MIN6 cells, which were decreased 70, 38, and 48% by 2.5 µmol/liter berberine, respectively. To further determine whether berberine directly inhibits cAMP-stimulated insulin secretion, MIN6 cells were exposed to 1 mmol/liter 8-bromo-cAMP, a cell-permeant cAMP analog, for 1 h. 8-Bromo-cAMP-augmented insulin secretion was decreased 62% by berberine. To determine whether the loss of responsiveness to glucose or other secretagogues caused by berberine may be due to a general decrease in the viability of cells, we examined the impact of the drug on the nutrient-independent stimulation of secretion elicited by a depolarizing concentration of KCl. As an argument against any loss of cell viability as the underlying cause, berberine had no impact on the stimulation of secretion provoked by 35 mmol/liter KCl from MIN6 cells (Fig. 5AGo). Consistent with the result, MTT results showed that incubation with 2.5 µmol/liter berberine for 1 h also had no toxic effect on MIN6 cells (data not shown). The response of rat islets to cAMP-raising agents was different from MIN6 cells. At the concentration of 3.3 mmol/liter glucose, forskolin, and GLP-1 did not stimulate insulin secretion, and IBMX increased insulin secretion by just 86%, less than that in MIN6 cells. Berberine decreased just IBMX-stimulated insulin secretion and had no effect in the presence of forskolin and GLP-1 as 3.3 mmol/liter glucose alone in rat islets (Fig. 5BGo). However, when the concentration of glucose was 10 mmol/liter, forskolin, IBMX, and GLP-1 all augmented insulin secretion from rat islets, which were decreased by berberine (Fig. 5CGo).


Figure 5
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  		FIG. 5. Berberine decreased cAMP-raising agent-stimulated insulin secretion in MIN6 cells and rats islets. MIN6 cells and isolated rat islets were cultured in KRB buffer with or without 2.5 µmol/liter berberine (BBR) containing the indicated concentrations of glucose and 0.5 mmol/liter IBMX, 10 µmol/liter forskolin (FKL), 10 nmol/liter GLP, 1 mmol/liter 8-bromo-cAMP (cAMP), or 35 mmol/liter KCl for 1 h and assayed for insulin secretion. A, MIN6 cells were incubated with 2.8 mmol/liter glucose. B, Isolated rats islets were incubated with 3.3 mmol/liter glucose. C, Isolated rats islets were incubated with 10 mmol/liter glucose. All values were normalized to total DNA. Results were given as means ± SEM from three independent experiments, each performed in triplicate. *, P < 0.05; **, P < 0.01 vs. corresponding control values (CON).

 
Berberine decreased PKA activity
Because cAMP is a direct activator of PKA, we further investigated whether berberine exerted an action on PKA activity. As shown in Fig. 6Go, forskolin-stimulated PKA activity was decreased by 39% in isolated rat islets in the presence of 2.5 µmol/liter berberine.


Figure 6
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  		FIG. 6. Berberine decreased forskolin-stimulated PKA activity. Isolated rat islets were cultured in KRB buffer with 2.5 µmol/liter berberine and 10 µmol/liter forskolin for 30 min. PKA activity in cell extracts was measured. A representative photograph of the agar gel used for the kemptide assay is shown on the left, with phosphorylated kemptide in the top panel and unphosphorylated kemptide in the bottom panel, and the quantification of the assay on the right. The experiment was repeated three times, and data were expressed as means ± SE. *, P < 0.05 vs. control; #, P < 0.05 vs. forskolin alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is now clear that insulin resistance is not solely a disorder of carbohydrate metabolism but also involves alterations in lipid metabolism (26). Increased adiposity and accumulation of lipid within the liver and skeletal muscle have been shown to be strongly associated with insulin resistance (27). The response of skeletal muscle to insulin greatly influences whole-body insulin sensitivity because it is the main tissue responsible for insulin-stimulated glucose uptake (28). It is generally accepted that high-fat diets can be used to generate a valid rodent model for the metabolic syndrome with insulin resistance and compromised β-cell function, which represents the physiological state more than in genetic model. In this present study, Sprague Dawley rats presented a classical phenotype of metabolic syndrome with obesity, insulin resistance, hyperinsulinemia, and hyperlipidemia after high-fat diet feeding for 20 wk. Berberine treatment decreased blood glucose and lipid levels in high-fat diet-induced insulin-resistant rats. Moreover, berberine reduced basal insulin secretion and GSIS in high-fat diet rats with hyperinsulinemia, suggesting that insulin resistance was markedly improved. In addition, we analyzed the curve of insulin secretion in three groups. The peak of insulin secretion in high-fat diet rats occurred at 60 min after glucose loading, at 30 min in standard chow diet rats. After berberine treatment, serum insulin levels at all time points of OGTT were significantly decreased, but the curve of insulin secretion did not change with the peak at 60 min. Therefore, it manifests lower levels of insulin at 30 min in berberine-treated rats, compared with that of standard chow diet rats. Berberine treatment for 6 wk normalized body weight in high-fat diet rats, not liver and epididymal fat weights. It seems that the berberine intervention is too late to reverse all abnormal characteristics induced by high-fat diet. Another explanation is that berberine exerts different effects depending on the locations of fat. In a randomized, double-blind, and placebo-controlled trial, berberine decreased fasting and postprandial plasma glucose with slightly decreasing postprandial insulin and body weight reduction in type 2 diabetics after 3 months treatment (29), in agreement with the current animal experiment.

The previous studies showed that the effect of berberine on insulin secretion was controversial. In HIT-T15 cells and murine pancreatic islets, Leng et al. (30) described a dose-dependent effect of berberine with a potentiation of insulin secretion at doses of 1–10 µmol/liter. In MIN6 cells, Ko et al. (18) reported that berberine increased GSIS, even at the concentration of 50 µmol/liter. Our previous study showed that berberine had no insulinotropic effect in βTC3 cells (31). In the present study, our in vivo study showed that berberine exerted an insulin-sparing effect. The question remains whether this is due solely to the reduction of peripheral insulin resistance or at least partially caused by a specific berberine effect on pancreatic islet function. For answering this question, we investigated the effect of berberine on insulin secretion in MIN6 β cells and islets isolated from rats in vitro. The results showed that berberine acutely decreased the response of β-cells to high glucose. Whereas after MIN6 cells and rat islets were pretreated with berberine for 24 h, the response of MIN6 cells and rat islets to high glucose markedly increased in the last 1 h incubation without berberine, suggesting that the inhibitory effect of berberine on GSIS is reversible. The distinct results from ours and others may be largely due to the different experiment conditions.

Elevated glucose and FFA levels are hallmarks of adipogenic type 2 diabetes mellitus. Many studies have now confirmed that short-term exposure (<6 h) to elevated fatty acids potentiates GSIS, whereas longer-term exposure (24–48 h) inhibits insulin secretion in isolated islets or β-cell lines (32, 33). Our previous study also showed a similar result (26). Here short-term incubation with palmitate potentiated insulin secretion at both low and high glucose in MIN6 cells and rat islets, which was significantly attenuated by berberine. After MIN6 cells were incubated with high glucose and palmitate for 24 h, GSIS decreased significantly, which was partly abrogated by berberine, even in the last hour of incubation with berberine. These results suggest that berberine exerts a direct protective effect on glucolipotoxicity in β-cells as in peripheral tissues besides its hypoglycemic and hypolipidemic effect.

AMPK is ubiquitously expressed in mammalian tissues (34). In pancreatic islets of Langerhans, AMPK possesses a unique role, connecting cellular energy status to the capacity of β-cells to synthesize and secrete insulin. A number of studies have demonstrated that activation of AMPK in isolated rodent and human islets, as well as clonal β-cells, suppresses glucose metabolism and GSIS (34, 35). The insulin-sparing effects of TZDs and metformin noted in clinical trials have generally been assumed to be secondary to the decreased insulin resistance. Recently there is increasing evidence that these agents directly inhibit insulin release from β-cells (6, 10, 36, 37). Metformin has been shown to activate AMPK in MIN6 cells and rat and human islets (6). We previously found that troglitazone also activated AMPK in MIN6 cells and rat islets (10), which may account for its inhibitory effect on insulin secretion as AICAR (8). It was postulated that berberine was also the case. As expected, berberine markedly increased AMPK activity in MIN6 cells as in other cells. However, compound C did not reverse berberine-suppressed GSIS, in disagreement with the result of troglitazone. Compound C is a potent and selective small-molecule AMPK inhibitor that is competitive with ATP. Incubation of cultured hepatocytes with compound C inhibited ACC inactivation and fatty acid oxidation stimulated by either AICAR or metformin (38). The current study confirmed for the first time that troglitazone acutely decreased GSIS through activating AMPK, which may account for the underlying mechanisms of TZDs on islet β-cell-function. Much attention should be paid to AMPK, not peroxisome proliferator-activated receptor-{gamma} for the study of TZDs on β-cells. It is understandable that although berberine and TZDs share several similar properties, these two chemicals exert distinct action by different pathways. Berberine inhibited adipocyte differentiation via decreasing peroxisome proliferator-activated receptor-{gamma} activity and reduced the body weight of high-fat diet rats (15, 18, 39). However, TZDs promote adipocyte differentiation and increase the body weight of type 2 diabetic patients (40). Therefore, berberine seems to be a desirable agent for treating adipogenic type 2 diabetic patients with hyperinsulinemia.

cAMP is an important modulator of insulin secretion in pancreatic β-cells. cAMP-raising agents including GLP-1, the phosphodiesterase inhibitor IBMX, and the adenylate cyclase activator forskolin stimulate insulin secretion and are believed to act principally via PKA (2). The latter enzyme appears to phosphorylate voltage-sensitive Ca2+ channels as well as undefined components of the exocytotic machinery (41). In addition, cAMP may also enhance insulin secretion via a PKA-independent action on the activity of the guanine nucleotide exchange factor Epac/cAMP-GEF-II and intracellular Ca2+ mobilization (42). There was a great amount of evidence supporting the concept that endogenous β-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists (20)., It has been reported that glucose stimulates lipolytic activity and the antilipolytic agent 3,5-dimethylpyrazole inhibits insulin secretion from isolated islets in response to glucose, cAMP-raising agents, and the mitochondrial fuel {alpha}-ketoisocaproic acid (43). Orlistat, a lipase inhibitor, inhibited cAMP-induced insulin secretion in HIT-T15 cells (20, 44) and GLP-1-stimulated secretion in rat islets (45).

Our in vivo results showed that berberine dramatically decreased circulating FFAs in high-fat-fed rats. In 3T3-L1 adipocytes, we found that berberine markedly inhibited lipolysis induced by IBMX, forskolin and 8-bromo-cAMP (data not shown). Therefore, we determined whether berberine also exerted an inhibitory effect on insulin secretion stimulated by cAMP-raising agents in β-cells. As expected, berberine decreased the augmented insulin secretion by the cAMP-raising agents. 8-Bromo-cAMP-potentiated insulin released from MIN6 cells was also decreased by 62% in the presence of berberine. The response of isolated rat islets to cAMP-raising agents was different from that of MIN6 cells. At the concentration of 3.3 mmol/liter glucose, forskolin, and GLP-1 had no effect on insulin secretion but increased insulin secretion at 10 mmol/liter glucose, which is in agreement with the results from Yamada et al. (46) and Nolan et al. (47), suggesting that forskolin and GLP-1-potentiated insulin secretion is dependent on glucose concentration in rat islets.

The islets of Langerhans contain {alpha}-, β-, {delta}-, and PP cells, and they are able to influence each other through paracrine and autocrine communications. MIN6 is a clonal β-cell line and lacks the paracrine feedback system of other islet hormones, which may contribute to the different response to cAMP-raising agents. IBMX-stimulated insulin secretion at 3.3 mmol/liter glucose was obviously less than that at 10 mmol/liter glucose, which was all suppressed by berberine. Moreover, the absolute magnitude of insulin secretion augmented by IBMX, forskolin, or GLP-1 decreased much more in the presence of berberine than that augmented by 10 mmol/liter glucose alone. In addition, we detected PKA activity in isolated rat islets. The results showed that berberine decreased forskolin-potentiated PKA activity, consistent with the results that berberine suppressed hormone-sensitive lipase phosphorylation stimulated by cAMP-raising agents in 3T3-L1 adipocytes (our unpublished data).

In conclusion, berberine partially inhibits insulin release from β-cells and directly counteracts glucolipotoxicity, which may prevent their functional exhaustion in conditions of chronic exposure to high concentrations of FFAs and glucose. In addition to its effects on peripheral metabolism, berberine also exerts direct actions on β-cells. This insulin-sparing effect might reduce the pathological basal hyperinsulinemia seen in insulin-resistant subjects and in early stages of adipogenic type 2 diabetes. From our results, one would therefore expect that the insulin-sparing effects of berberine would be preferentially useful in prediabetic states. Although both berberine and troglitazone increase AMPK activity in β-cells, only troglitazone is confirmed to inhibit insulin secretion via AMPK activation. cAMP signaling pathway may, at least in part, account for the regulatory effect of berberine on insulin secretion.


   Acknowledgments
 
We thank Dr. J. Miyazaki and K. Inoue for the gift of the MIN6 cell line.


   Footnotes
 
This work was supported by the fund for key laboratory of endocrinology and metabolic diseases from China Ministry of Education (Y0204) and grants from the National Natural Science Foundation of China (30600294 and 30700382) and the Education Commission of Shanghai (06BZ024).

Disclosure Summary: The authors have nothing to declare.

First Published Online May 29, 2008

1 L.Z., X.W., and L.S. contributed equally to this work. Back

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AICAR, 5-aminoimidazole-4-carboxamide riboside; AMPK, AMP-activated protein kinase; 8-bromo-cAMP, 8-bromoadenosine-cAMP; FFA, free fatty acid; GLP, glucagon-like peptide; GSIS, glucose-stimulated insulin secretion; IBMX, 3-isobutyl-1-methylxanthine; ITT, insulin tolerance test; KRB, Krebs-Ringer bicarbonate; LDL-C, low-density lipoprotein cholesterol; MTT, methylthiotetrazole; OGTT, oral glucose tolerance test; PKA, protein kinase A; TC, total cholesterol; TG, triglycerides; TZD, thiazolidinedione.

Received December 19, 2007.

Accepted for publication May 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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