Combined Treatment of Betulinic Acid,a PTP1B Inhibitor,with Orthosiphon stamineus Extract Decreases Body Weight in High-Fat

Abstract

Leptin resistance is a common feature of obesity and is accompanied by hyperleptinemia. Although leptin sensitizers improve leptin resistance, they also decrease plasma leptin levels that attenuate the leptin-associated antiobesity effect. We hypothesized that the combinational treatment of leptin sensitizer and endogenous leptin expression stimulant would synergistically induce an antiobesity effect in high-fat–fed obese animals. Betulinic acid (BA) isolated from Saussurea lappa suppressed the hypothalamic protein tyrosine phosphatase 1B in mice and enhanced the antiobesity effect of leptin in obese rats. Ethanol extract of Orthosiphon stamineus (OS) induced leptin expressions in both 3T3-L1 adipocytes and mice in a dose-dependent manner. To evaluate our hypothesis, we treated obese mice induced by 6 weeks of high-fat-diet feeding with BA and OS for 2 weeks. Although BA or OS alone did not decrease body weight in obese mice, the combinational treatment of BA and OS decreased body weight significantly compared to either BA- or OS-treated obese mice. These results suggest that combinational treatment of BA and OS would be effective for the treatment of obesity.

Introduction

B ody weight homeostasis is maintained by the balance between energy intake and energy expenditure, which are controlled by the hypothalamus, the center for body weight regulation. The most important circulating peripheral signal messenger to the hypothalamus is leptin, which inhibits appetite and stimulates energy expenditure.¹ However, leptin treatment does not decrease body fat deposition effectively in the obese state because of leptin resistance.² Although the mechanisms of leptin resistance are multifactorial, the most common cause is defective leptin signaling in the hypothalamus.³ Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of the leptin-signaling pathway in the hypothalamus, which was supported by previous reports that PTP1B is elevated in high-fat–fed rats,⁴ and that deficiency of hypothalamic PTP1B results in resistance to diet-induced obesity due to leptin hypersensitivity in mice.⁵ There is evidence that PTP1B inhibitors may enhance leptin sensitivity and act as effective therapeutic targets for treatment of obesity. Several naturally occurring PTP1B inhibitors have been reported.^6
–8 Among them, betulinic acid (BA), a pentacyclic triterpenoid, is a powerful PTP1B inhibitor.⁷ The root of Saussurea lappa has been used in traditional Chinese herbal medicine to relieve various symptoms associated with digestion, respiration, and circulation⁹ and is a source of BA.⁷ In this context, we speculate that BA as a PTP1B inhibitor may improve leptin sensitivity. However, the conditions that improve leptin sensitivity eventually decrease plasma leptin concentration because of compensatory processes, which attenuate their antiobesity effects. Thus, the supplementation of leptin combined with leptin sensitizer could augment its antiobesity effect. Because recombinant leptin is expensive, an endogenous leptin inducer would be recommended in the clinical field.

Orthosiphon stamineus (OS) has been used as a traditional medicinal herb in Southeast Asian countries such as Malaysia, Indonesia, Myanmar, and Vietnam. The tea made from its leaves is believed to have therapeutic effects on various pathologic conditions.¹⁰ Recently, various beneficial effects were reported, such as antidiabetic, anti-inflammatory, antiproliferative, and antiangiogenic effects.^11
–13 We reported recently that OS elevates plasma leptin levels and has an antiobesity effect in Sprague-Dawley rats.¹⁴

The purposes of this study were to evaluate (1) whether BA enhances leptin sensitivity in high-fat–fed obese animals with leptin resistance; (2) whether OS elevates leptin mRNA expression dose dependently in vitro and in vivo; and (3) whether the combinational treatment of BA and OS decreases visceral fat deposition additively in a high-fat–fed obese animal model.

Materials and Methods

Animal care

Male C57BL/6N mice (body weights, 18–20g) and Sprague-Dawley rats (body weights, 280–300 g) were purchased from Jung-Ang Experimental Animals (Seoul, Korea). The mice and rats were housed individually with a 12 h light–12 h dark cycle (7:00 a.m. to 7:00 p.m.) and were cared for in accordance with the Guide to the Care and Use of Experimental Animals provided by the Yeungnam Medical Center. This experimental protocol was approved by the Ethics Committee of Yeungnam University.

Experimental design

Experiment 1 (acute study)

To evaluate the leptin-stimulating effect of OS, various dosages (0, 10, 50, and 100 μg/mL) of OS ethanol extract were used to treat 3T3-L1 adipocytes in vitro for 6 h. The OS-treated cells were analyzed for leptin mRNA expression by real-time polymerase chain reaction (PCR). We also measured leptin mRNA expression in mouse adipose tissue and plasma leptin concentration after treatment with various dosages (0, 100, and 1000 μg/mouse daily for 2 consecutive days, orally) of OS ethanol extract.

We also attempted to confirm the PTP1B-suppressing effect of BA in the hypothalamus. Various dosages (0, 250, and 500 μg/mouse, orally) of BA were administered daily for 2 consecutive days in 10-week-old C57BL/6N mice. The hypothalamus was excised according to the method established by Park et al.,¹⁵ quickly frozen with liquid nitrogen, and stored in a deep freezer.

Experiment 2

To evaluate the leptin-sensitizing effect of BA, we measured body weight and food intake for 7 days after infusion of BA and/or leptin into the lateral ventricle using an osmotic minipump in rats fed chow or high-fat diets for 8 weeks. A brain infusion kit (Alzet, Cupertino, CA, USA) was placed into the left lateral ventricle and connected with an osmotic minipump (Alzet) as described previously.² Briefly, a brain infusion cannula was stereotaxically placed into the lateral ventricle of rats under anesthesia with xylazine hydrochloride (8 mg/kg subcutaneously) and ketamine (90 mg/kg intraperitoneally). The coordinates were 1.3 mm posterior to bregma, 1.9 mm lateral to the midsagittal suture, and to a depth of 4.0 mm. Then, the osmotic minipump inserted in the dorsal subcutaneous pocket was connected with the brain infusion cannula. The infusion rate used was 1 μL/h.

Experiment 3 (chronic study)

To evaluate the antiobesity effect of combinational treatment with OS and BA, OS and/or BA was administered in the diet-induced obese mice or chow-fed normal mice. Obesity was induced by 6 weeks of high-fat feeding. The diets used in this study were the HFD60%cal high-fat diet and the AIN93G control diet. The HFD60%cal diet was composed of protein, carbohydrate, and fat (20%, 20%, and 60%, respectively, of total calories) supplemented with vitamins (1%) and minerals (3.5%), while the energy composition of AIN93G was 20%, 64%, and 16% (protein, carbohydrate, and fat, respectively). The diets were provided by Feedlab Korea Co. (Seoul, Korea).

BA (500 μg/day in 100 μL) and/or OS (1 mg/day in 100 μL) was administered orally using gavage-feeding needles. Control mice were treated with the same volume of saline. After 2 weeks of the treatment, mice were anesthetized with intraperitoneal injection of Tiletamine and Zolazepam (25 mg/kg body weight, Zoletil^®; Virbac, Carros, France). Blood was collected rapidly through the abdominal aorta using a heparin-coated syringe and then separated from plasma by centrifugation at 4°C. Retroperitoneal white adipose tissue (RT-WAT) was excised and weighed.

Extraction of OS and isolation of BA from S. lappa root

Dried OS powder (78.6 g) was extracted with 70% ethanol by reflux for 12 h, and the ethanol solution was evaporated to dryness. Dried OS leaves imported from Indonesia were kindly donated by Dongbang FTL Co. (Seoul, Korea). A voucher specimen (OS201103) was deposited at the College of Pharmacy, Yeungnam University, Korea. BA was isolated from the S. lappa root extract as described in a previous report.⁷ BA powder was dissolved in a mixture of ethanol, Tween 80, and water (10%/10%/80%) and orally administered to mice. The OS extract was dissolved in physiological saline.

Cell culture

3T3-L1 mouse embryo fibroblasts were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) until confluent. For differentiation, cultures were maintained for 2 days after confluency and then stimulated with DMEM containing 10% FBS, 1 μg/mL insulin, 0.5 mM isobutylmethylxanthine, and 0.25 μM dexamethasone for 2 days. Subsequently, the medium was replaced with 10% FBS/DMEM containing 1 μg/mL insulin and incubated for 2 days, followed by culturing with 10% FBS/DMEM for an additional 4 days, at which time >90% of the cells were mature adipocytes with accumulated fat droplets. Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.

RNA extraction and real-time PCR analysis

The total RNA was isolated from the hypothalamus and adipose tissue using Trizol (Sigma, St. Louis, MO, USA) and RNeasy Lipid Tissue kit (Qiagen, Barcelona, Spain), respectively. The integrity of the RNA was verified and quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

RNA was reverse transcribed to cDNA from 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed using the Light-Cycler (Roche, Mannheim, Germany) and Power SYBR Green PCR master mix (Applied Biosystems), according to the manufacturer's instructions. The expression level of GAPDH was used as an internal control. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 56°C for 4 sec, and 72°C for 7 sec. Primers for mouse leptin, PTP1B, and GAPDH were based on information obtained from the NCBI nucleotide database and designed using the Primer Express program (Applied Biosystems): leptin (forward, 5′-CAG GAT GAC ACC AAA ACC CTC-3′; reverse, 5′-TCC AAG CCA GTG ACC CTC TG-3′); PTP1B (forward, 5′-CAA AAA CCG GAA CAG GTA CC-3′ and reverse, 5′-TTT TTA TCA AGC TGG CAT TGA T-3′); and GAPDH (forward, 5′-AAC GGC ACA GTC AAG GCC-3′; reverse, 5′-CGC TCC TGG AAG ATG GTG AT-3′).

Measurement of plasma leptin concentration

Plasma leptin was analyzed using the mouse leptin ELISA kit (Millipore, Billerica, MA, USA) according to the manufacturer's instruction.

Data analysis

The data were expressed as means±SEM. One-way analysis of variance (ANOVA) and repeated-measured ANOVA were used for comparisons of dose-dependent and serial changes among the experimental groups. Post hoc comparisons were performed using Duncan's test. Statistical comparisons of body weight and plasma leptin concentrations between control and BA- and/or OS-treated groups were evaluated by Student's t-test. Values of P<.05 were considered statistically significant.

Results

OS treatment stimulated leptin mRNA expression dose dependently in 3T3-L1 cells (Fig. 1A). OS also stimulated leptin mRNA expression in adipose tissue and elevated plasma leptin concentrations in mice (Fig. 1B, C). BA treatment suppressed hypothalamic PTP1B mRNA expression dose dependently in mice (Fig. 1D). To evaluate the leptin-sensitizing effect of BA, we infused BA and/or leptin intracerebroventricularly and found that not only did BA decrease body weight, but it also potentiated the effects of leptin on weight loss in high-fat–fed rats (P<.05, Fig. 2). The effect of leptin on body weight loss (mean body weight difference between leptin-treated rats and control rats at day 7) was blunted in high-fat–fed rats compared to chow-dieted rats (13.6 g vs. 33.6 g, respectively), which suggested an induction of leptin resistance. However, intracerebroventricular infusion of BA failed to reduce body weight and to change the leptin's effect in chow-fed rats. Leptin treatment decreased cumulative food intake and visceral fat mass in chow-fed rats, but not in high-fat–fed rats, while BA treatment did not change them in both chow- and high-fat–fed rats. However, the combined treatment of BA and leptin suppressed food intake and reduced visceral fat mass compared to high-fat–fed rats, suggesting the leptin-sensitizing effect of BA.

FIG. 1.

The dose-dependent effect of Orthosiphon stamineus (OS) extract on leptin mRNA expression in 3T3-L1 cells (A) and adipose tissue (B) of C57BL/6N mice. OS treatment increased leptin mRNA expression in vitro and in vivo in a dose-dependent manner. (C) OS treatment also increased plasma leptin concentration in mice. (D) Betulinic acid (BA) treatment decreased hypothalamic protein tyrosine phosphatase 1B (PTP1B) mRNA expression dose-dependently in mice. Bars represent mean±SEM of 7–9 experimental animals per group. ^abcValues that do not share a common superscript are significantly different at P<.05.

FIG. 2.

Effect of intracerebroventricular infusion of BA and/or leptin (Lept) for 7 days on body weight loss in chow-dieted (A) and high-fat–fed (B) rats. Leptin treatment decreased body weight significantly, while BA treatment did not affect body weight in chow-dieted mice. However, BA treatment decreased body weight as well as enhanced the body weight loss effect of leptin in high-fat–fed rats. Cumulative food intake for 7 days was suppressed by leptin treatment in chow-dieted rats (C), but not in high-fat–fed rats (D), while it was decreased by the combined treatment of BA and leptin in high-fat–fed rats. (E) The changes of visceral fat mass exhibited similar patterns with the food intake changes. Values are mean±SEM of 6–7 experimental animals per group. *P<.05, vs. N-Cont; ^# P<.05 vs. H-Cont; ^& P<.05 vs. H-BA; ^$ P<.05, vs. H-Lept. N-, normal-dieted; H-, high-fat–fed; Cont, control.

We treated mice with a high-fat diet for 8 weeks and showed the induction of obesity. The high-fat–fed mice exhibited higher body weight and RT-WAT weight compared with chow-fed mice (P<.05; Table 1 and Fig. 3). Moreover, the plasma leptin concentration was elevated in high-fat–fed mice, more than fourfold compared to chow-dieted mice (P<.05). Treatment with OS alone for 2 weeks did not result in any specific changes in body weight, RT-WAT, average daily energy intake, or plasma leptin concentrations in chow-fed mice compared to their controls. However, an increasing trend of plasma leptin concentration was shown in OS-treated high-fat–fed mice compared to high-fat–fed controls, which was accompanied with slightly increased RT-WAT mass. BA treatment for 2 weeks decreased RT-WAT in chow-fed mice compared to the controls (P<.05). The combinational treatment of OS and BA did not induce further decrease in RT-WAT or plasma leptin concentrations in chow-diet mice compared to BA-treated mice. However, combined treatment induced statistically significant decreases in body weight, RT-WAT, and plasma leptin concentration in high-fat–fed mice compared to the control mice (P<.05; Table 1 and Fig. 3).

FIG. 3.

The effect of BA and/or O. stamineus (OS) treatment for 2 weeks on changes of body weight (A, C) and retroperitoneal white adipose tissue (RT-WAT) (B, D) in chow- (A, B) and high-fat–fed (C, D) mice. In chow-fed mice, body weight differences were differed significantly in all experimental groups compared to controls, while RT-WAT was decreased significantly only in BA-treated mice. In high-fat–fed mice, the body weight difference was differed significantly only in BA+OS-treated group accompanying with a significant reduction of RT-WAT. Bars represent mean±SEM of 7–9 experimental animals per group. ^abValues that do not share a common superscript are significantly different at P<.05.

Table 1.

Effects of Betulinic acid and/or Orthosiphon stamineus Extract on Body Weights, Average Daily Food Intake, and Plasma Leptin Concentration in Chow- or High-Fat–Fed Mice

	Chow diet				High-fat diet
Treatment	Control	OS	BA	OS+BA	Control	OS	BA	OS+BA
Body weights (g)
Initial, 0 weeks	17.2±0.58	17.2±0.18	17.2±0.31	17.2±0.52	17.2±0.39	17.2±0.36	17.2±0.32	17.2±0.34
Start tx, 6 weeks	26.6±1.17	26.5±1.07	26.7±1.01	26.9±1.24	29.6±0.98^*	29.6±0.80	29.8±0.94	29.8±1.18
End point, 8 weeks	27.9±1.11	26.3±0.87	26.4±1.02	26.7±0.69	30.3±1.05	30.1±0.79	29.7±0.87	28.6±0.83
Average daily caloric intake (Cal)	10.3±0.34	10.1±0.33	11.3±0.20	11.0±0.33	11.0±0.34	11.6±0.16	11.6±0.37	11.8±0.31
Plasma leptin concentration (ng/mL)	3.52±1.08	5.58±1.66	2.05±0.49	3.70±2.51	15.73±6.32^*	28.45±7.50	8.77±4.22	6.17±2.55^#

Values are mean±SEM of 7–9 experimental animals per group.

P<.05, vs. chow-dieted control mice; ^# P<.05 vs. high-fat–fed control mice.

BA, betulinic acid; OS, Orthosiphon stamineus ; start tx, at starting treatment of BA and/or OS.

Discussion

In the present study, we demonstrated that OS treatment stimulated leptin expression in vitro as well as in vivo, whereas BA treatment suppressed the hypothalamic PTP-1B expression in mice and increased leptin sensitivity in high-fat–fed rats with leptin resistance. Moreover, the combinational treatment of OS and BA resulted in a significant loss of body weight and visceral fat mass in high-fat–fed mice.

We determined the leptin-stimulating effect of OS in vitro as well as in an acute in vivo study, which was supported by our previous report¹⁴ that OS treatment elevated plasma leptin concentrations in normal rats. In our chronic study, however, we could not find statistically significant changes in plasma leptin concentration in OS-treated mice, because long-term leptin regulation is associated more closely with body fat mass as demonstrated in our previous study.² Moreover, OS treatment alone failed to reduce body weights in both chow- and high-fat–fed mice, which differed from our previous results in that OS treatment decreased visceral fat mass in chow-fed rats. This different response to OS treatment may be the result of different dosages. A 450 mg/kg dose of OS was administered daily to Sprague-Dawley rats in our previous study, which was about 13.5× higher than that of the present study. Several studies discovered that OS contains physiologically active compounds, such as terpenoids, polyphenols, sterols, orthosiphols, saponins, flavonoids, caffeic acid, and oleanolic acid.^10,16 However, it is not yet known which compound in OS is responsible for the leptin stimulation. Further study is needed to identify the biologically active compounds in OS.

PTP1B is known as a negative regulator of the insulin signaling pathway¹⁷ and is expressed in insulin-sensitive tissue such as skeletal muscle, adipose tissues, and the liver.¹⁸ Recently, it was reported that PTP1B is also involved in the leptin signaling pathway via dephosphorylation of the leptin receptor-associated Janus kinase 2 and signal transducers and activators of transcription 3 (STAT3), which leads to leptin resistance.¹⁹ High-fat feeding elevates hypothalamic PTP1B, which is associated by an elevation of inflammatory cytokines such as TNF-α.²⁰ Furthermore, a selective inhibition of PTP1B by trodusquemine increases phosphorylation of hypothalamic STAT3 and causes weight loss in high-fat–fed mice.²¹ Although numerous PTP1B inhibitors that are synthesized chemically or isolated from natural resources have been introduced in the literature,²² Choi et al. ⁷ described in vitro PTP1B inhibitory activity of BA isolated from S. lappa, a plant widely grown and used throughout Asian countries. We used the BA isolated from S. lappa in the present study and found that BA treatment decreased body weight and retroperitoneal fat in chow-fed mice, while there was a decreasing tendency of body weight in high-fat–fed mice. These results are partly supported by a report from de Melo et al.,²³ which demonstrated that BA treatment for 15 weeks decreased abdominal fat accumulation in high-fat–fed mice through modulation of fat and carbohydrate metabolism. They also demonstrated that BA treatment elevated leptin concentrations, which was inconsistent with our present study. We found that leptin concentrations were slightly decreased, but not statistically significantly. Although the reason of this discrepancy cannot be explained here, we believed that the decreasing tendency of leptin concentration may result from BA's leptin-sensitizing effect and fat-lowering effect, which is supported by observations of Lantz et al. ²⁰ that trodusquemine decreased leptin concentration compared to controls as well as pair-feeding mice. Another possible mechanism for fat reduction by BA treatment may be an inhibition of triglyceride formation by suppression of diacylglycerol acetyltransferase.²⁴ Further study is needed to elucidate the mechanism of action.

The combinational treatment of OS and BA in high-fat–fed mice decreased body weight, visceral fat mass, and plasma leptin concentration compared to high-fat–fed control mice. Moreover, the combined effect of BA and OS on the visceral fat mass of high-fat–fed mice was revealed in a synergistic manner. The significant decrease in plasma leptin concentrations in OS+BA-treated high-fat–fed mice, even with the usage of leptin inducer, is probably due to the sharp decrease in fat mass, because leptin secretion is sensitively regulated with the changes in the body fat mass.²⁵ The fat reduction in this study was not associated with decreased energy intake, but may be due to elevated energy expenditure. Although it was not statistically significant, an obvious tendency for elevation of plasma leptin concentration by OS was shown in high-fat–fed mice, but we could not observe any fat loss effect of OS alone in this study. We considered the possibility that the stimulated plasma leptin level by OS worsens the leptin resistance induced by high-fat feeding and eventually promotes obesity, because the elevated leptin²⁶ or over-reactivity of the leptin receptor signaling system²⁷ can induce obesity. Therefore, the fat loss effect of the combinational treatment in the present study supports our hypothesis that the simultaneous treatment of leptin sensitizer and inducer would be an effective strategy for the treatment of obesity.

Although relatively short term, less than half a year, dietary interventions result in clinically significant weight reduction, but weight regain is common.²⁸ Thus, a long-term approach to weight regulation using medicinal plants would be a good strategy. In this aspect, OS has several beneficial characteristics, such as being an established, well-known medicinal herb throughout Southeast Asia,²⁹ with a wide spectrum of beneficial effects,^10,11,29 and has no demonstrated harmful side effects (even a 5 g/kg dose of OS extract did not cause any severe toxicity in rats).^30,31 BA also has additional anti-inflammatory effects,³² anticancer effects,³³ and inhibits pancreatic lipase.³⁴ The maternal plant of BA, S. lappa roots, have a wide variety of beneficial effects, such as antiulcer, anti-inflammatory, and antitumor effects.^35
–37 In the present study, we did not find any specific behavioral change or derangement of liver histology and enzymes associated with the administration of OS or BA for a 2-week time period.

In summary, OS treatment stimulated leptin expression in vitro as well as in vivo, while BA treatment suppressed the hypothalamic PTP-1B expression in mice and increased leptin sensitivity in high-fat–fed rats with leptin resistance. Moreover, the combinational treatment of OS and BA caused significant decreases in body weight, visceral fat mass, and plasma leptin concentration in high-fat–fed mice. These results suggested that combinational treatment of BA and OS would be effective for the treatment of obesity.

Footnotes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2009-0069055).

Author Disclosure Statement

The authors declare that they have no conflicts of interest associated with this work.

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Combined Treatment of Betulinic Acid,a PTP1B Inhibitor,with Orthosiphon stamineus Extract Decreases Body Weight in High-Fat–Fed Mice

Abstract

Introduction

Materials and Methods

Animal care

Experimental design

Experiment 1 (acute study)

Experiment 2

Experiment 3 (chronic study)

Extraction of OS and isolation of BA from S. lappa root

Cell culture

RNA extraction and real-time PCR analysis

Measurement of plasma leptin concentration

Data analysis

Results

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References