# Improved Insulin Resistance and Lipid Metabolism by Cinnamon Extract through Activation of Peroxisome Proliferator-Activated Receptors

Authors: Xiaoyan Sheng; Yuebo Zhang; Zhenwei Gong; Cheng Huang; Ying Qin Zang
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptors (PPARs) are transcriptional factors involved in the regulation of insulin resistance and adipogenesis. Cinnamon, a widely used spice in food preparation and traditional antidiabetic remedy, is found to activate PPARγ and α, resulting in improved insulin resistance, reduced fasted glucose, FFA, LDL-c, and AST levels in high-caloric diet-induced obesity (DIO) and db/db mice in its water extract form. In vitro studies demonstrate that cinnamon increases the expression of peroxisome proliferator-activated receptors γ and α (PPARγ/α) and their target genes such as LPL, CD36, GLUT4, and ACO in 3T3-L1 adipocyte. The transactivities of both full length and ligand-binding domain (LBD) of PPARγ and PPARα are activated by cinnamon as evidenced by reporter gene assays. These data suggest that cinnamon in its water extract form can act as a dual activator of PPARγ and α, and may be an alternative to PPARγ activator in managing obesity-related diabetes and hyperlipidemia.


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## Body

## 1. Introduction

Obesity has
become the most common metabolic disorder in the world and is a major risk
factor for insulin resistance in the development of type 2 diabetes mellitus [1]. Peroxisome proliferator-activated receptors (PPARs)
have been recognized as therapeutic targets against dyslipidemia and diabetes since
their discovery in the early 1990s. PPARs are ligand-activated nuclear
hormone receptors that include three isoforms: PPARα, PPARγ, and PPARδ/β. PPARα
is expressed mostly in brown adipose tissue and liver, PPARγ is mainly
expressed in adipose tissue, while PPARδ/β is expressed universally in many
tissues [2]. Activation of PPARα lowers
plasma triglycerides and elevates plasma HDL cholesterol levels [3], while the activation of PPARγ
increases insulin sensitivity and results in antidiabetic effects [4]. Since PPARs play key roles in
regulating carbohydrate and lipid metabolism, most studies have been directed
toward developing synthetic PPAR ligands, and current therapeutic strategies are also based on
separate treatments of insulin resistance and dyslipidaemia. Fibrates, the PPARα agonists, are now
recommended for the treatment of cardiovascular disease and dyslipidemia [5]. Thiazolinediones (TZDs), the
PPARγ agonists, have been demonstrated by a number of clinical trials in the
management of insulin resistance and type 2 diabetes [6–8]. However, these agonists can also
produce moderate to serious side effects such as edema, weight gain, congestive
heart failure, and hepatotoxicity [9, 10]. Thus, the development of drugs of dual or pan
PPAR modulators with less side effects is desirable for the management of obesity-related
diabetes and dyslipidemia.Cinnamon is the
bark ofCinnamoni cassiae and has
been used as traditional folk herbs to treat inflammation for thousands of
years in Asia. It is also used in food
industry as antioxidant and spicy agent. In recent years, several studies have
reported that cinnamon extract has antidiabetic effect on db/db mice and type 2 diabetic patients [11, 12]. Talpur et al. showed that
cinnamon oils can improve insulin sensitivity [13] and Roffey et al. reported that
cinnamon water extract (CE) increases glucose uptake in 3T3-L1 adipocyte [14]. However, the separated compounds
derived from cinnamon displayed little insulin-like or insulin-enhancing
activity [15]. In this report, we demonstrate that
cinnamon water extract can elevate the expression of both PPARγ, α and their target genes in 3T3-L1 adipocyte
possibly through stimulation of the transactivities of both full length and LBD
of PPARγ and PPARα. In vivo
study reveals that cinnamon water extract improves insulin resistance and lipid
metabolism in both high-calorie diet-induced obesity (DIO) mice and db/db mice.


## 2. Materials and Methods

### 2.1. Materials

Dexamethasone
(DEX), 3-isobutyl-1-methylxanthine, insulin, oil red O, troglitazone, and
WY-14643 were purchased from Sigma (St. Louis, Mo). Cinnamon
powder was obtained from Shanghai Traditional Chinese Herbs Pharmacy Co. (Shanghai, China).
The power (10 g) was extracted in (100 mL) double distilled water with
revolving evaporator in vacuum state using vacuum pump till the volume of water
reduced to 50 mL. The supernatant was then filtered through Whatman paper no. 1 to obtain the cinnamon water extract. The final concentration was 0.2 g/mL.


### 2.2. Quantitative RT-PCR

Total RNA extracted
from 3T3-L1 adipocyte or mouse tissues was reverse transcribed into first strand cDNA
with random hexamer primers using a cDNA synthesis kit (Promega, Madison, Wis).
The gene expression levels were analyzed by quantitative real-time RT-PCR conducted with the ABI 7500 (Applied Biosystems, Foster City, Calif). After
an initial incubation for 2 minutes at50°C, the cDNA was denatured at 95°C for 10 minutes followed by 40 cycles of PCR (95°C, 15 seconds, 60°C, 60 seconds). All results were obtained in at
least three independent experiments. The mRNA levels of all genes were
normalized using β-actin as an internal control.


### 2.3. Luciferase Activity Assays

The plasmid
construction and transfection were carried out as described previously [16]. Briefly, 293T cells obtained
from ATCC were cultured in DMEM with 10% FBS. Transfection solution was added
to 293T cells overnight, then CE and PPAR ligands were added into fresh media and
the cells were harvested for the determination of luciferase activity 24 hours
later. The luciferase reporter assays were carried out using the
Dual-Luciferase Reporter Assay System (Promega, Madison, Wis)
and the transfection efficiencies were normalized by renilla luciferase activity.
All transfection experiments were performed in duplicate and repeated at least
three times independently.


### 2.4. 3T3-L1 Adipocyte Differentiation and Oil Red O Staining

3T3-L1 cells were purchased from American Type Culture Collection (Va, USA)
and maintained in DMEM containing 10% FBS. To induce differentiation, two days after
confluence, preadipocytes (designated day 0) were cultured in differentiation
medium (DM, containing 10% FBS plus 1μg/mL insulin, 0.5 μM dexamethasone, 0.5 mM IBMX) or DM + CE at indicated
concentrations for 2 days, then switched to post DM containing 10% FBS and 1 μg/mL insulin. The medium was changed every 2 days. Oil red O staining was
performed on day 5. Briefly, cells were washed twice with PBS and fixed with
7.5% formaldehyde for 15 minutes. After twice washing with PBS, cells were
stained for at least 1 hour in freshly diluted oil red O solution in 37°C.
Cells were photographed using a phase-contrast Olympus IX70 (Tokyo, Japan)
microscope
in combination with a MicroFire digital camera at 200 magnifications.


### 2.5. Western Blot Analysis

Cells were homogenized in 2X SDS sample buffer (Invitrogen, Carlsbad, Calif) and boiled for 5 minutes. 10μg of protein was separated by 10% SDS-PAGE and transferred to PVDF membrane, blocked for 1 hour at room temperature in 5% nonfat dry milk/PBS-T. PPARγ (Upstate, Charlottesville, Va) and β-actin antibodies (Sigma, St. Louis, Mo) were added to 2% BSA in PBS-T and incubated with the membrane for 1 hour at room temperature. The membrane was then washed 3 times with PBS-T for 10 minutes each, incubated with HRP conjugated secondary antibody (Santa Cruz, Calif)
for 30 minutes at room temperature, and washed 3 times with PBS-T for 30 minutes
totally. The signals were detected by ECL Plus Western Blotting Detection
System according to the manufacturer’s specifications (Amersham, Little Chalfont Buckinghamshire, England).


### 2.6. In Vivo Animal Study

Female C57BL/6J mice were purchased from the SLAC Laboratory (Shanghai, China)
anddb/db (C57BL BKS cg-M+/+lepr−/−) mice were purchased from Jackson Lab (Me, USA) and bred in house. All
mice were kept under controlled temperature (22-23°C) and on a 12-hour
light/dark cycle. C57BL/6J mice were placed on a high-fat diet (60% of calories
derived from fat, Research Diets, New Brunswick, NJ, D12492) for induction of
obesity, while control mice were placed on the equivalent chow diet (10% of
calories derived from fat, Research Diets, D12450B). DIO mice with similar body
weight and db/db mice with similar
fasted glucose level were used in the experiments. Mice were gavaged with CE
(correspond to 400 mg cinnamon powder kg•day•) or water only as
control at 2PM each day in a vehicle of water. All animal protocols used in
this study were preapproved by Shanghai Institutes for Biological Sciences of
CAS for animal studies.


### 2.7. Intraperitoneal Glucose Tolerance Test (IPGTT)

C57BL/6J mice
were fasted overnight on day 21 anddb/db mice were fasted for 6 hours on day 15 prior to the blood samples were
collected from tail vein for the determination of baseline glucose values (t=0 minute) before the injection of glucose (intraperitoneal injection 2 g/kg). Additional blood samples were collected with
regular intervals (t=15,30,60,90,120, and 240 minutes) for glucose
measurement. Blood glucose levels were evaluated with a glucose analyzer
(TheraSense, Abbott Park, Ill).


### 2.8. Serum Chemistry Analysis

Serum triglyceride (TG), total cholesterol (TC), HDL-C and LDL-C, AST, and ALT were examined using a Hitachi 7020 Automatic Analyzer in 100μl of heart blood serum. FFA was analyzed with an Ultrospec 2100 Pro UV/Visible spectrophotometer (Amersham, Little Chalfont Buckinghamshire, England) with kits from Jiancheng Bioengineering Ins (Nanjing, China). Serum insulin was measured by ELISA kit (Linco Research, Mo, USA).


### 2.9. Statistical Analysis

The results were
represented as the mean ± SE. Data were analyzed by the statistical analysis
system (SAS) program. Comparison between the control and treated groups was made by ANOVA
variance analysis and their significance was established by Student’st-test.
Differences of P<.05 were considered statistically significant.


## 2.1. Materials

Dexamethasone
(DEX), 3-isobutyl-1-methylxanthine, insulin, oil red O, troglitazone, and
WY-14643 were purchased from Sigma (St. Louis, Mo). Cinnamon
powder was obtained from Shanghai Traditional Chinese Herbs Pharmacy Co. (Shanghai, China).
The power (10 g) was extracted in (100 mL) double distilled water with
revolving evaporator in vacuum state using vacuum pump till the volume of water
reduced to 50 mL. The supernatant was then filtered through Whatman paper no. 1 to obtain the cinnamon water extract. The final concentration was 0.2 g/mL.


## 2.2. Quantitative RT-PCR

Total RNA extracted
from 3T3-L1 adipocyte or mouse tissues was reverse transcribed into first strand cDNA
with random hexamer primers using a cDNA synthesis kit (Promega, Madison, Wis).
The gene expression levels were analyzed by quantitative real-time RT-PCR conducted with the ABI 7500 (Applied Biosystems, Foster City, Calif). After
an initial incubation for 2 minutes at50°C, the cDNA was denatured at 95°C for 10 minutes followed by 40 cycles of PCR (95°C, 15 seconds, 60°C, 60 seconds). All results were obtained in at
least three independent experiments. The mRNA levels of all genes were
normalized using β-actin as an internal control.


## 2.3. Luciferase Activity Assays

The plasmid
construction and transfection were carried out as described previously [16]. Briefly, 293T cells obtained
from ATCC were cultured in DMEM with 10% FBS. Transfection solution was added
to 293T cells overnight, then CE and PPAR ligands were added into fresh media and
the cells were harvested for the determination of luciferase activity 24 hours
later. The luciferase reporter assays were carried out using the
Dual-Luciferase Reporter Assay System (Promega, Madison, Wis)
and the transfection efficiencies were normalized by renilla luciferase activity.
All transfection experiments were performed in duplicate and repeated at least
three times independently.


## 2.4. 3T3-L1 Adipocyte Differentiation and Oil Red O Staining

3T3-L1 cells were purchased from American Type Culture Collection (Va, USA)
and maintained in DMEM containing 10% FBS. To induce differentiation, two days after
confluence, preadipocytes (designated day 0) were cultured in differentiation
medium (DM, containing 10% FBS plus 1μg/mL insulin, 0.5 μM dexamethasone, 0.5 mM IBMX) or DM + CE at indicated
concentrations for 2 days, then switched to post DM containing 10% FBS and 1 μg/mL insulin. The medium was changed every 2 days. Oil red O staining was
performed on day 5. Briefly, cells were washed twice with PBS and fixed with
7.5% formaldehyde for 15 minutes. After twice washing with PBS, cells were
stained for at least 1 hour in freshly diluted oil red O solution in 37°C.
Cells were photographed using a phase-contrast Olympus IX70 (Tokyo, Japan)
microscope
in combination with a MicroFire digital camera at 200 magnifications.


## 2.5. Western Blot Analysis

Cells were homogenized in 2X SDS sample buffer (Invitrogen, Carlsbad, Calif) and boiled for 5 minutes. 10μg of protein was separated by 10% SDS-PAGE and transferred to PVDF membrane, blocked for 1 hour at room temperature in 5% nonfat dry milk/PBS-T. PPARγ (Upstate, Charlottesville, Va) and β-actin antibodies (Sigma, St. Louis, Mo) were added to 2% BSA in PBS-T and incubated with the membrane for 1 hour at room temperature. The membrane was then washed 3 times with PBS-T for 10 minutes each, incubated with HRP conjugated secondary antibody (Santa Cruz, Calif)
for 30 minutes at room temperature, and washed 3 times with PBS-T for 30 minutes
totally. The signals were detected by ECL Plus Western Blotting Detection
System according to the manufacturer’s specifications (Amersham, Little Chalfont Buckinghamshire, England).


## 2.6. In Vivo Animal Study

Female C57BL/6J mice were purchased from the SLAC Laboratory (Shanghai, China)
anddb/db (C57BL BKS cg-M+/+lepr−/−) mice were purchased from Jackson Lab (Me, USA) and bred in house. All
mice were kept under controlled temperature (22-23°C) and on a 12-hour
light/dark cycle. C57BL/6J mice were placed on a high-fat diet (60% of calories
derived from fat, Research Diets, New Brunswick, NJ, D12492) for induction of
obesity, while control mice were placed on the equivalent chow diet (10% of
calories derived from fat, Research Diets, D12450B). DIO mice with similar body
weight and db/db mice with similar
fasted glucose level were used in the experiments. Mice were gavaged with CE
(correspond to 400 mg cinnamon powder kg•day•) or water only as
control at 2PM each day in a vehicle of water. All animal protocols used in
this study were preapproved by Shanghai Institutes for Biological Sciences of
CAS for animal studies.


## 2.7. Intraperitoneal Glucose Tolerance Test (IPGTT)

C57BL/6J mice
were fasted overnight on day 21 anddb/db mice were fasted for 6 hours on day 15 prior to the blood samples were
collected from tail vein for the determination of baseline glucose values (t=0 minute) before the injection of glucose (intraperitoneal injection 2 g/kg). Additional blood samples were collected with
regular intervals (t=15,30,60,90,120, and 240 minutes) for glucose
measurement. Blood glucose levels were evaluated with a glucose analyzer
(TheraSense, Abbott Park, Ill).


## 2.8. Serum Chemistry Analysis

Serum triglyceride (TG), total cholesterol (TC), HDL-C and LDL-C, AST, and ALT were examined using a Hitachi 7020 Automatic Analyzer in 100μl of heart blood serum. FFA was analyzed with an Ultrospec 2100 Pro UV/Visible spectrophotometer (Amersham, Little Chalfont Buckinghamshire, England) with kits from Jiancheng Bioengineering Ins (Nanjing, China). Serum insulin was measured by ELISA kit (Linco Research, Mo, USA).


## 2.9. Statistical Analysis

The results were
represented as the mean ± SE. Data were analyzed by the statistical analysis
system (SAS) program. Comparison between the control and treated groups was made by ANOVA
variance analysis and their significance was established by Student’st-test.
Differences of P<.05 were considered statistically significant.


## 3. Results

### 3.1. CE Improves Hyperlipidemia and Liver Function in DIO C57BL/6J anddb/db Mice

After 5-month induction with high-fat diet, the body weight of C57BL/6J mice was increased to
150% and the serum TG, LDL, AST, and ALT were greatly elevated compared to normal food fed mice as indicated in Table1. Three weeks after gavage treatment with CE, equivalent to cinnamon 400 mg/kg•day, serum FFA and LDL-c were significantly reduced (see Table 1). However, the body weight and food intake remained unchanged (data not shown). Since high-fat diet can usually impair the liver function and elevate AST and ALT levels, we examined AST and ALT in CE-treated DIO mice and found that CE intervention can effectively improve liver function as evidenced by significantly reduced AST and ALT levels in DIO mice (see Table 1). To testify whether CE has similar effects on db/db mice, 11 weeks old db/db mice were treated with CE for 2 weeks. The serum FFA of db/db mice was
decreased markedly after 2 weeks of CE treatment (data not shown), suggesting
that CE can also improve dyslipidemia
in genetically defective obese mice.Table 1
Effects of CE on serum
chemistry of DIO mice. NFC: normal-fat diet
control; HFC: high-fat diet control. The data are represented as mean ± SE,n=5 for each group. P*<.05, P**<.01 compared to HFC.
Serum chemistryNFC (H2O)HFC (H2O)HF + CE (400 mg/kg)FFA (mmol/L)1.31±0.061.67±0.151.08±0.08**TC (mmol/L)1.46±0.152.39±0.332.22±0.31TG (mmol/L)0.13±0.080.14±0.030.11±0.07HDL (mmol/L)1.14±0.142.04±0.272.04±0.24LDL (mmol/L)0.20±0.030.26±0.070.16±0.08*Insulin (ng/mL)0.49±0.071.44±0.751.09±0.25AST (IU/L)164.6±83.7330.8±152.4128.3±27.4**ALT (IU/L)23.28±10.886.43±49.560.31±15.99


### 3.2. CE Reduces Hyperglycemia and Improves Glucose Tolerance in Both DIO Mice anddb/db Mice

To evaluate whether CE has any influence on glucose tolerance, IPGTT determination was carried out
for overnight fasted DIO mice at 0 minute, 15 minutes, 30 minutes, 60 minutes, and 120 minutes following 2 g/kg glucose intraperitoneal injection DIO mice developed severe insulin resistance after 5 months high-fat food inductions, and serum insulin level increased nearly 3 times compared to the control mice on normal diet (see Table1). Three weeks of CE treatment not only reduced fasted glucose level but also improved glucose tolerance markedly (see Figures 1(a) and 1(b)). The serum insulin level was decreased after CE treatment (Table 1). Similar effects were observed on db/db mice as demonstrated in Figures 1(c)
and 1(d). Our results suggested that CE is capable of improving insulin
sensitivities in both high-fat diet-induced diabetic mice and db/db mice.CE reduced the fasted blood glucose level and
improved glucose tolerance in DIO anddb/db mice. Mice were
administered vehicle alone, or CE (equivalent to 400 mg cinnamon powder kg•day•) for 3 weeks
(DIO) or 2 weeks (db/db). The blood glucose levels were
measured in tail vein and the basal glucose levels were shown at 0 minute. (a)
DIO C57BL/6J mice were fasted overnight and glucose levels were measured at 9:00 am at the end treatment. NF: normal food fed mice; HF: high-fat food-induced mice; CE:
cinnamon water extract; (b) fasted DIO mice were intraperitoneally injected with 2 g glucose•kg− body weight and glucose tolerance determined at the time indicated; (c) db/db Mice
were fasted for 6 hours and the fasted blood glucose was measured. CONT: vehicle;
(d) glucose tolerance was
determined following 2 g glucose•kg−1 body weight intraperitoneal injection in db/db mice. The data are
presented as mean ± SE, n=5 for each group. (b) P*<.05 versus HF group or (d) vehicle control (cont).
(a)(b)(c)(d)


### 3.3. CE Promotes 3T3-L1 Preadipocyte Differentiation

In order to
understand the underlying mechanism of CE in hypolipidemia and hypoglycemia, we
used preadipocyte 3T3-L1 as an in
vitro system to study the influence of CE on preadipocyte
differentiation. To analyze the effects of CE on 3T3-L1 preadipocyte
differentiation, CE was added to 3T3-L1 cells during differentiation induction
in differentiation medium (DM). On day 5, we found that the number and the
volume of adipocytes were increased in CE-treated cells compared to those
induced by DM only indicating that CE may promote 3T3-L1 preadipocyte
differentiation (see Figures2(a), 2(b), 2(c), and 2(d)). However,
the number and size of the adipocytes in WAT in CE-treated DIO mice remain
unchanged (Figures 2(e) and 2(f)).CE promoted 3T3-L1 adipocyte differentiation. 3T3-L1 cells were stained with oil red O at day 5. (a) Undifferentiated control cells; (b) DM (insulin 10μg/mL, dexam: 1 μM,
IBMX: 0.5 mM)-induced
differentiated cells; (c) DM-induced differentiated cells + CE 0.2 mg/mL;
(d) DM-induced differentiated cells + CE 0.6 mg/mL; (e) HE
staining of WAT from high-fat diet control (HFC) mice; (f) HE staining of WAT
from HF + CE-treated mice.
(a)(b)(c)(d)(e)(f)


### 3.4. CE Enhances Gene Expression of PPARγ, PPARα, and the Target Genes

Since PPARγ and
PPARα play essential roles in adipocyte differentiation and lipid homeostasis, we
further studied whether CE increases 3T3-L1 adipocyte differentiation through the
activation of PPARs. The above-mentioned CE-treated cells were collected to
determine the expression levels of PPARγ, PPARα and their target genes such as CD36,
FAS, LPL, GLUT4, and ACO. The results showed that CE not only elevated the
expression of PPARγ and its target genes CD36, LPL, FAS, and GLUT4 significantly (see Figure 3(a)), but also increased the expression of PPARα and its target gene ACO markedly (Figure 3(b)). Further examination at the protein level of PPARγ confirmed that CE can significantly increase the PPARγ protein
level in 3T3-L1 adipocyte during differentiation (Figure 3(c)).CE increased
expression of PPARs and their target genes in differentiated 3T3-L1 cells. 3T3-L1 cells were
differentiated in 24-well plate and CE 0.6 mg/mL was added at the same time. On
day 5, cells were collected and total RNA was extracted and reversely
transcribed into the first strand cDNA with random hexamer primers using cDNA synthesis kit.
The gene expression levels were analyzed by quantitative real-time RT-PCR. (a) PPARγ
and its target genes; (b) PPARα and its target gene; (c) Western blot of CE-treated
3T3-L1 differentiated cells from day 5. 1: Control; 2: DM; 3: Rosiglitazone 1 μM; 4: DM + CE 0.2 mg/mL; 5: DM + CE 0.6 mg/mL. For real-time PCR, the results
were repeated in at least 3 independent experiments, and β-actin mRNA was used
as an internal control. Data are presented as mean ± SE. P*<.001.
(a)(b)(c)


### 3.5. CE Increases Transactivities of PPARγ and PPARα

To test whether
CE could increase the transactivities of PPARγ and PPARα, full-length PPARγ and PPARα
expression plasmids were transfected into 293T cells for the reporter gene
assays. We found that CE increased the transactivities of both PPARγ (P<.05) and PPARα (P<.01) in a dose-dependent
manner (see Figures 4(a) and 4(b)). In order to exclude the endogenous factors,
ligand-binding domains (LBD) of PPARγ and PPARα were used to evaluate the reporter
gene activities. Results showed that the LBD activities of both PPARγ and PPARα
were also elevated (P<.05) (see Figures 4(c) and 4(d)). These data
suggest that CE is a dual activator of both PPARγ and PPARα.CE activated transactivities of PPARγ and PPARα. Full-length PPARγ or PPARα was
cotransfected with PPRE-J3-TK-Luc reporter construct to 293T cell and treated
with CE (0.2 mg/mL, 0.6 mg/mL), 1 μM of troglitazone or WY-14643 for 24 hours as positive
controls. Rellina luc was used as a transfection efficiency control and the
relative luciferase activities were measured against renilla luciferase
activities. For LBD activity assay, pMCX-GAL4-LBD of PPARγ or α expression
constructs were cotransfected with USAG×4-TK-Luc into 293T using
the same protocol as described above. The empty vectors were used as control. (a) Full-length
PPARγ. (b) Full-length PPARα. (c) LBD PPARγ. (d) LBD PPARα. The results represent three independent experiments.
Data are presented as mean ± SE. P*<.05, P**<.01.
Tro: troglitazone; WY: WY14643.
(a)(b)(c)(d)


### 3.6. CE Treatment Elevates the Expression of PPARγ/α and Target Genes in DIO Mice

To verify whether CE treatment has any effect at the transcriptional level in related tissues in DIO mice, we examined the gene expression of PPARs and their target genes in white adipocyte tissue (WAT) and liver of CE-treated DIO mice and controls. PPARγ, dominantly expressed in WAT, was slightly elevated, but its target genes, LPL and CD36, were increased markedly (see Figure 5(a)). PPARα and its target gene ACO, mainly expressed in liver, were also elevated significantly in CE-treated mice compared with the controls (Figure
5(b)). In vivo data further suggested that CE is a
dual activator of PPARγ and PPARα.CE promoted gene expression
of PPARγ, PPARα and their target genes in WAT and
liver from DIO mice: (a) real-time PCR of PPARγ and its target gene expression
in white fat tissue; (b) real-time PCR of PPARα and its target gene expression
in liver. HF: high-fat
diet; n=5; data are presented as mean ± SE. P*<.05 compared to control.
(a)(b)


## 3.1. CE Improves Hyperlipidemia and Liver Function in DIO C57BL/6J anddb/db Mice

After 5-month induction with high-fat diet, the body weight of C57BL/6J mice was increased to
150% and the serum TG, LDL, AST, and ALT were greatly elevated compared to normal food fed mice as indicated in Table1. Three weeks after gavage treatment with CE, equivalent to cinnamon 400 mg/kg•day, serum FFA and LDL-c were significantly reduced (see Table 1). However, the body weight and food intake remained unchanged (data not shown). Since high-fat diet can usually impair the liver function and elevate AST and ALT levels, we examined AST and ALT in CE-treated DIO mice and found that CE intervention can effectively improve liver function as evidenced by significantly reduced AST and ALT levels in DIO mice (see Table 1). To testify whether CE has similar effects on db/db mice, 11 weeks old db/db mice were treated with CE for 2 weeks. The serum FFA of db/db mice was
decreased markedly after 2 weeks of CE treatment (data not shown), suggesting
that CE can also improve dyslipidemia
in genetically defective obese mice.Table 1
Effects of CE on serum
chemistry of DIO mice. NFC: normal-fat diet
control; HFC: high-fat diet control. The data are represented as mean ± SE,n=5 for each group. P*<.05, P**<.01 compared to HFC.
Serum chemistryNFC (H2O)HFC (H2O)HF + CE (400 mg/kg)FFA (mmol/L)1.31±0.061.67±0.151.08±0.08**TC (mmol/L)1.46±0.152.39±0.332.22±0.31TG (mmol/L)0.13±0.080.14±0.030.11±0.07HDL (mmol/L)1.14±0.142.04±0.272.04±0.24LDL (mmol/L)0.20±0.030.26±0.070.16±0.08*Insulin (ng/mL)0.49±0.071.44±0.751.09±0.25AST (IU/L)164.6±83.7330.8±152.4128.3±27.4**ALT (IU/L)23.28±10.886.43±49.560.31±15.99


## 3.2. CE Reduces Hyperglycemia and Improves Glucose Tolerance in Both DIO Mice anddb/db Mice

To evaluate whether CE has any influence on glucose tolerance, IPGTT determination was carried out
for overnight fasted DIO mice at 0 minute, 15 minutes, 30 minutes, 60 minutes, and 120 minutes following 2 g/kg glucose intraperitoneal injection DIO mice developed severe insulin resistance after 5 months high-fat food inductions, and serum insulin level increased nearly 3 times compared to the control mice on normal diet (see Table1). Three weeks of CE treatment not only reduced fasted glucose level but also improved glucose tolerance markedly (see Figures 1(a) and 1(b)). The serum insulin level was decreased after CE treatment (Table 1). Similar effects were observed on db/db mice as demonstrated in Figures 1(c)
and 1(d). Our results suggested that CE is capable of improving insulin
sensitivities in both high-fat diet-induced diabetic mice and db/db mice.CE reduced the fasted blood glucose level and
improved glucose tolerance in DIO anddb/db mice. Mice were
administered vehicle alone, or CE (equivalent to 400 mg cinnamon powder kg•day•) for 3 weeks
(DIO) or 2 weeks (db/db). The blood glucose levels were
measured in tail vein and the basal glucose levels were shown at 0 minute. (a)
DIO C57BL/6J mice were fasted overnight and glucose levels were measured at 9:00 am at the end treatment. NF: normal food fed mice; HF: high-fat food-induced mice; CE:
cinnamon water extract; (b) fasted DIO mice were intraperitoneally injected with 2 g glucose•kg− body weight and glucose tolerance determined at the time indicated; (c) db/db Mice
were fasted for 6 hours and the fasted blood glucose was measured. CONT: vehicle;
(d) glucose tolerance was
determined following 2 g glucose•kg−1 body weight intraperitoneal injection in db/db mice. The data are
presented as mean ± SE, n=5 for each group. (b) P*<.05 versus HF group or (d) vehicle control (cont).
(a)(b)(c)(d)


## 3.3. CE Promotes 3T3-L1 Preadipocyte Differentiation

In order to
understand the underlying mechanism of CE in hypolipidemia and hypoglycemia, we
used preadipocyte 3T3-L1 as an in
vitro system to study the influence of CE on preadipocyte
differentiation. To analyze the effects of CE on 3T3-L1 preadipocyte
differentiation, CE was added to 3T3-L1 cells during differentiation induction
in differentiation medium (DM). On day 5, we found that the number and the
volume of adipocytes were increased in CE-treated cells compared to those
induced by DM only indicating that CE may promote 3T3-L1 preadipocyte
differentiation (see Figures2(a), 2(b), 2(c), and 2(d)). However,
the number and size of the adipocytes in WAT in CE-treated DIO mice remain
unchanged (Figures 2(e) and 2(f)).CE promoted 3T3-L1 adipocyte differentiation. 3T3-L1 cells were stained with oil red O at day 5. (a) Undifferentiated control cells; (b) DM (insulin 10μg/mL, dexam: 1 μM,
IBMX: 0.5 mM)-induced
differentiated cells; (c) DM-induced differentiated cells + CE 0.2 mg/mL;
(d) DM-induced differentiated cells + CE 0.6 mg/mL; (e) HE
staining of WAT from high-fat diet control (HFC) mice; (f) HE staining of WAT
from HF + CE-treated mice.
(a)(b)(c)(d)(e)(f)


## 3.4. CE Enhances Gene Expression of PPARγ, PPARα, and the Target Genes

Since PPARγ and
PPARα play essential roles in adipocyte differentiation and lipid homeostasis, we
further studied whether CE increases 3T3-L1 adipocyte differentiation through the
activation of PPARs. The above-mentioned CE-treated cells were collected to
determine the expression levels of PPARγ, PPARα and their target genes such as CD36,
FAS, LPL, GLUT4, and ACO. The results showed that CE not only elevated the
expression of PPARγ and its target genes CD36, LPL, FAS, and GLUT4 significantly (see Figure 3(a)), but also increased the expression of PPARα and its target gene ACO markedly (Figure 3(b)). Further examination at the protein level of PPARγ confirmed that CE can significantly increase the PPARγ protein
level in 3T3-L1 adipocyte during differentiation (Figure 3(c)).CE increased
expression of PPARs and their target genes in differentiated 3T3-L1 cells. 3T3-L1 cells were
differentiated in 24-well plate and CE 0.6 mg/mL was added at the same time. On
day 5, cells were collected and total RNA was extracted and reversely
transcribed into the first strand cDNA with random hexamer primers using cDNA synthesis kit.
The gene expression levels were analyzed by quantitative real-time RT-PCR. (a) PPARγ
and its target genes; (b) PPARα and its target gene; (c) Western blot of CE-treated
3T3-L1 differentiated cells from day 5. 1: Control; 2: DM; 3: Rosiglitazone 1 μM; 4: DM + CE 0.2 mg/mL; 5: DM + CE 0.6 mg/mL. For real-time PCR, the results
were repeated in at least 3 independent experiments, and β-actin mRNA was used
as an internal control. Data are presented as mean ± SE. P*<.001.
(a)(b)(c)


## 3.5. CE Increases Transactivities of PPARγ and PPARα

To test whether
CE could increase the transactivities of PPARγ and PPARα, full-length PPARγ and PPARα
expression plasmids were transfected into 293T cells for the reporter gene
assays. We found that CE increased the transactivities of both PPARγ (P<.05) and PPARα (P<.01) in a dose-dependent
manner (see Figures 4(a) and 4(b)). In order to exclude the endogenous factors,
ligand-binding domains (LBD) of PPARγ and PPARα were used to evaluate the reporter
gene activities. Results showed that the LBD activities of both PPARγ and PPARα
were also elevated (P<.05) (see Figures 4(c) and 4(d)). These data
suggest that CE is a dual activator of both PPARγ and PPARα.CE activated transactivities of PPARγ and PPARα. Full-length PPARγ or PPARα was
cotransfected with PPRE-J3-TK-Luc reporter construct to 293T cell and treated
with CE (0.2 mg/mL, 0.6 mg/mL), 1 μM of troglitazone or WY-14643 for 24 hours as positive
controls. Rellina luc was used as a transfection efficiency control and the
relative luciferase activities were measured against renilla luciferase
activities. For LBD activity assay, pMCX-GAL4-LBD of PPARγ or α expression
constructs were cotransfected with USAG×4-TK-Luc into 293T using
the same protocol as described above. The empty vectors were used as control. (a) Full-length
PPARγ. (b) Full-length PPARα. (c) LBD PPARγ. (d) LBD PPARα. The results represent three independent experiments.
Data are presented as mean ± SE. P*<.05, P**<.01.
Tro: troglitazone; WY: WY14643.
(a)(b)(c)(d)


## 3.6. CE Treatment Elevates the Expression of PPARγ/α and Target Genes in DIO Mice

To verify whether CE treatment has any effect at the transcriptional level in related tissues in DIO mice, we examined the gene expression of PPARs and their target genes in white adipocyte tissue (WAT) and liver of CE-treated DIO mice and controls. PPARγ, dominantly expressed in WAT, was slightly elevated, but its target genes, LPL and CD36, were increased markedly (see Figure 5(a)). PPARα and its target gene ACO, mainly expressed in liver, were also elevated significantly in CE-treated mice compared with the controls (Figure
5(b)). In vivo data further suggested that CE is a
dual activator of PPARγ and PPARα.CE promoted gene expression
of PPARγ, PPARα and their target genes in WAT and
liver from DIO mice: (a) real-time PCR of PPARγ and its target gene expression
in white fat tissue; (b) real-time PCR of PPARα and its target gene expression
in liver. HF: high-fat
diet; n=5; data are presented as mean ± SE. P*<.05 compared to control.
(a)(b)


## 4. Discussion

There are several studies reported that cinnamon
has anti-hyperglycemic and anti-hyperlipidemic effects on diabetic animals and
type 2 diabetic
patients. However, the underlying mechanisms remain illusive. Here, we use early
type 2 diabetic DIO mice, and severe type 2 diabeticdb/db mice, as in vivo models, and 3T3-L1 adipocyte as
in vitro differentiation model
to study the effects of CE on lipid and glucose metabolism and their possible underlying
mechanisms.In vitro studies
demonstrate that CE acts like a dual activator to PPARγ/α based on three evidences: first, oil red O
staining showed that CE stimulated differentiation of 3T3-L1 cells from preadipocytes
into adipocytes. Secondly, CE treatment not only increased mRNA levels of PPARγ and PPARα but their target genes CD36, LPL,
FAS, GLUT4, and ACO increased
up to thousands of folds during 3T3-L1 differentiation compared to those
untreated cells. Finally, the reporter gene assay confirmed that CE increased transactivities
of both full length and LBD of PPARγ and PPARα significantly.Diet-induced obese mouse is an early
type 2 diabetic model. After 5 months induction with 60% calories fat diet, the
body weight and fat weight of C57BL/6J mice were increased remarkably, and the
mice developed insulin resistance and hyperinsulinemia fatty liver, and impairment of liver function which are often
observed in early type 2 diabetes [17, 18]. Our results showed that after 3
weeks gavage with CE, both IPGTT and hyperinsulinemia improved markedly
compared to the untreated ones. Although both increased insulin secretion and
reduced insulin resistance can improve IPGTT, lowered insulin level and
improved IPGTT in our study are beneficial to the reduction of insulin
resistance. Serum contents of FFA, which impair insulin secretion and induce
beta-cell lipotoxicity [19] and inhibit insulin-stimulated
glucose uptake into muscle [20], were also reduced notably in
DIO mice. The gene expression of PPARγ and its target genes CD36, LDL in white
fat tissue, and PPARα and its target gene ACO in liver were also elevated in CE-treated
DIO mice indicating that CE may act as a dual activator of PPARγ and PPARα
resulting in improved insulin resistance and lowered serum lipids. In genetic
diabetic model db/db mice, CE played similar roles in hypoglycemia and
hypolipidemia.Type 2 diabetic patients are at a higher
risk for cardiovascular disease (CVD) than nondiabetic individuals [21]. Current evidence overwhelmingly
confirms the role of low-density lipoprotein cholesterol (LDL-c) in the
pathogenesis of atherosclerosis and the risk of CVD, lowering LDL-c is
associated with a reduction in risk of CVD [22, 23]. Our study shows that CE reduces
serum LDL-c to normal level in DIO mice, suggesting that CE may be beneficial
to CVD.Another finding may be noted in this study is that CE treatment improved impaired liver function in DIO mice.
Elevated serum levels of AST and the ratio of AST/ALT usually indicate hepatocyte damage [24], and the most common
presentation is elevated liver enzymes AST and ALT in fatty liver [25]. In DIO mice, serum AST and ALT
were increased notably, but 3 weeks of CE treatment significantly decreased
serum AST level and the ratio of AST/ALT, suggesting that CE may play an
important role in improving liver function.A meta-analysis based on five randomized
clinical trials have shown that the use of cinnamon does not appear to improve
A1C, FBG, or lipid parameters
in patients with type 1 or type 2 diabetes [26]. Our preliminary study also
showed no effect on DIO or db/db mice when cinnamon powder was used without
water extraction. In present study, cinnamon water extract exhibited
hypoglycemic and hypolipidemic effects on both DIO and db/db mice. This is in
agreement with the results reported by Mang et al. demonstrating that the aqueous
cinnamon extract seemed to have a moderate effect in reducing fasting plasma
glucose concentrations in diabetic patients with poor glycaemic control [27]. The water extraction may have
enriched the active components of cinnamon to activate both PPARα and PPARγ
which are beneficial to lipid and glucose metabolism. Thus, preparation of
cinnamon such as water extraction may be considered when designing new clinical
trials.In conclusion,
our study demonstrated that CE had several beneficial effects on type 2
diabetes possibly through the activation of both PPARγ and PPARα resulting in improved
insulin resistance, lowered blood glucose, and serum lipid level without weight
gain and the structure change of the white adipose tissue. In addition, CE improved
the liver function of obese mice. CE may have potential use in management of
obesity-related type 2 diabetes and hyperlipidemia.



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*Source: 581348-2008-12-11.xml*