JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2015, 1–12 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.3416

FULL COMMUNICATION

Anti-Diabetic Effect of Aster sphathulifolius in C57BL/KsJ-db/db Mice Xingfu Yin,1 Yuhua Huang,1 Da-Woon Jung,1 Hee Chul Chung,2 Se Young Choung,3 Jae-Hoon Shim,1 and Il-Jun Kang1 1

Department of Food Science and Nutrition, Hallym University, Gwangwon, Korea. 2 Newtree Co., Ltd., Gyeonggi, Korea. 3 Department of Preventive Pharmacy and Toxicology, College of Pharmacy, Kyung Hee University, Seoul, Korea. ABSTRACT In this study, we investigated the anti-diabetic effect of Aster sphathulifolius (AS) extract in C57BL/KsJ-db/db mice. The db/db mice were orally administered with AS 50% ethanol extract at concentrations of 50, 100, and 200 mg/kg/day (db/db-AS50, db/db-AS100, and db/db-AS200, respectively) for 10 weeks. Food and water intake, fasting blood glucose concentrations, blood glycosylated hemoglobin levels, and plasma insulin levels were significantly lower in the db/db-AS200 group than in the vehicle-treated db/db group; whereas glucose tolerance was significantly improved in the db/db-AS200 group. Moreover, AS dose dependently increased both insulin receptor substrate 1 and glucose transporter type 4 expression in skeletal muscle, significantly increased glucokinase expression, and decreased glucose 6-phosphatase and phosphoenolpyruvate carboxykinase expressions in the liver. The expressions of transcription factors, such as sterol-regulatory elementbinding protein, peroxisome proliferator-activated receptor c, and adipocyte protein 2, were upregulated in adipose tissue. Furthermore, immunohistochemical analysis showed that AS upregulated insulin production by increasing pancreatic b-cell mass. In summary, AS extract normalized hyperglycemia by multiple mechanisms: inhibition of glyconeogenesis, acceleration of glucose metabolism and lipid metabolism, and increase of glucose uptake. Using in vivo assays, this study has shown the potential of AS as a medicinal food and suggests the efficacy of AS for the use of prevention of diabetes.

KEYWORDS:  Aster sphathulifolius  blood glucose  C57BL/KsJ-db/db mice  diabetes mellitus  insulin resistance

The first step by which insulin increases energy utilization and storage involves the glucose transporter in skeletal muscle. Skeletal muscle is a major contributor to the dysregulation of glucose homeostasis in T2DM, since skeletal muscle accounts for 85% of whole-body insulin-stimulated glucose uptake.5 Glucose transport in skeletal muscle is mainly facilitated by the insulin-responsive glucose transporter type 4 (GLUT4). In the basal state, GLUT4 is stored in a transporter-enriched intracellular pool. Definitive evidence that GLUT4 is the primary mediator of both basal and insulin-stimulated glucose transport in muscle comes from muscle-specific GLUT4 knockout mice.6 When insulin activates its cell surface receptor, a signal is generated, which results in the stimulation of exocytosis of the intracellular GLUT4 vesicles to the plasma membrane and facilitates the uptake of the glucose from the blood stream.7 This process is initiated on binding of insulin to the transmembrane receptor and is mediated through an intracellular molecular signaling cascade, including the consecutive phosphorylation of several cytosolic proteins, such as insulin receptor substrate (IRS), phosphatidylinositol 3-kinase (PI3K), and protein kinase B.8,9 Another major site that regulates glucose metabolism in T2DM is the liver, which plays a central role in this process by balancing the uptake and storage of glucose via

INTRODUCTION

T

ype 2 diabetes mellitus (T2DM) is a variety of metabolic diseases characterized by insulin resistance in muscle, liver, and adipose tissue. A lack of appropriate compensation by the b-cells leads to relative insulin deficiency.1,2 The functional disturbance of pancreatic b-cells causes decreases in glucose utilization in muscle, liver, and adipose tissue, and an increase in hepatic glucose production by hyperglycemia.3 Insulin plays a key role in glucose homeostasis at some sites, especially increasing the rate of glucose uptake, primarily into striated muscle and adipose tissue, and reducing hepatic glucose output (via decreased gluconeogenesis and glycogenolysis), and it regulates the maintenance of normoglycemia and normallipidaemia.4 Both impaired insulin secretion and insulin resistance, which are the two main mechanisms, contribute to the pathogenesis of the disease to varying degrees on the background of a still unidentified genetic predisposition.

Manuscript received 16 December 2014. Revision accepted 10 March 2015 Address correspondence to: Il-Jun Kang, PhD, Department of Food Science and Nutrition, Hallym University, Chuncheon, Gwangwon 200-702, Republic of Korea, E-mail: ijkang@ hallym.ac.kr

1

2

YIN ET AL.

glycogenesis and the release of glucose via glycogenolysis and gluconeogenesis. Hepatic glucokinase (GK), as one of the key glucose-regulating enzymes in the catabolism of glucose, can phosphorylate glucose and it catalyzes the conversion of glucose into glucose-6-phosphate in the liver. GK-knockout mice exhibit mild hyperglycemia, while the overexpression of GK leads to a relatively lower blood glucose concentration in the diabetic models.10 Glucose-6phosphatase (G-6-Pase) is a key enzyme involved in systemic glucose homeostasis. It catalyzes the last biochemical reaction of both gluconeogenesis and glycogenolysis.11 Phosphoenolpyruvate carboxykinase (PEPCK) is a central enzyme in carbohydrate metabolism, and it catalyzes the conversion of oxaloacetate to phosophoenol pyruvate, helping regulate the blood glucose level.12 Diabetes is often correlated with obesity, leading to a long-standing hypothesis that insulin resistance is a consequence of overnutrition and elevated dietary fatty acids.13 Sterol-regulatory element-binding protein-1c (SREBP1c) was initially discovered as a key player of adipocyte differentiation.14 In addition, SREBP-1c plays a critical role in lipogenesis.15 It works in tandem with peroxisome proliferator-activated receptor c (PPAR-c), which is a direct target of SREBP-1c in adipocytes and contains a sterol response element in its promoter.16 It has been proposed that along with an upregulation of PPAR expression, SREBP-1c is also able to induce PPAR activity by the production of an endogenous ligand, leading to the stimulation of both adipogenesis and lipogenesis.17 Insulin, a key postprandial hormone, stimulates the expression and activity of SREBP1c to accommodate anabolic processes, such as fatty acid synthesis, on feeding.18,19 PPAR-c acts as a transcription factor regulating the expression of several genes involved in metabolic pathways, including energy expenditure.20 Insulin is the primary hormone involved in glucose homeostasis and the stimulation of glucose transport. Circulating insulin rapidly reaches its target tissues, mainly muscle, liver, and adipose tissue, where it interacts with its receptor. Therefore, enhancing glucose uptake in skeletal muscle and liver is a primary therapeutic strategy for treatment of metabolic syndrome and T2DM. Currently, there are six classes of oral antidiabetic drugs, such as metformin, sulfonylureas, meglitinides, thiazolidinediones, dipeptidyl peptidase IV inhibitors, and a-glucosidase inhitors.21 Although these drugs are widely used medications for the treatment of T2DM, most oral therapeutic agents for T2DM have side effects. Currently, there is increasing interest in natural products that have minor side effects as compared with the synthetic drugs. Aster sphathulifolius (AS) Maxim is a perennial herb distributed along the eastern and southern coasts of South Korea, and young leaves of AS have been traditionally used as edible vegetables in Korea.22,23 Won et al. reported that AS has antiviral efficacy against influenza A/PR/8/34 infection.24 In addition, Lee et al. reported that three new diterpene glycosides and eight known substances were isolated from methanol extracts of aerial parts of AS and their cytotoxicity against five human tumor cell lines was evaluated.25 There are

limited reports on the effects of AS against diabetes and diabetic complications in animal models. This study evaluated the possible efficacy of AS extract for altering blood glucose concentration, insulin secretion, insulin resistance, and its effects on the gene expression of genes that regulate glucose metabolism in diabetic C57BL/KsJ-db/db mice. MATERIALS AND METHODS Materials Dried Aster sphathulifolius (AS) collected from Ulleung Island, Korea, was identified by Dr. Il-Jun Kang, professor in the Department of Food Science and Nutrition, Hallym University (Korea). Dry plant materials were extracted with 50% ethanol for 3 h by refluxing and then filtered (3MM CHR; Whatman, Maidstone, United Kingdom). After filtration, various ethanolic extracts were concentrated under reduced pressure, by using a rotary vacuum evaporator (Bu¨chi, Flawil, Switzerland) and lyophilized (IlShin BioBase, Seoul, Korea) to obtain powder. The extraction yield of extract from AS was 26.7%. Reagents Mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Millipore Corporation, Billerica, MA, USA), assay kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol (CHOL), glucose (GLUC), high-density lipoprotein (HDL-Chol), low-density lipoprotein (LDL-Chol), and triglyceride (TG) were purchased from Jung-Ang Experimental Animal Incorporated (Seoul, Korea). Immunoblotting was performed using the following antibody: Insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Animals C57BL/KsJ-db/db male diabetic mice (db/db) and their non-diabetic heterozygote littermates (db/m + ) were purchased from Jung-Ang Experimental Animal Incorporated (Seoul, Korea) at 5 weeks of age and maintained under standard laboratory conditions of 24C – 5C and 55% – 5% relative humidity with a 12-h light/12-h dark cycle. The animals were allowed ad libitum access to water and an AIN-76A Rodent Purified diet ( Jung-Ang Experimental Animal Incorporated, Seoul, Korea) during a 2-week acclimation period. Animal experiments After a 2-week acclimation, animals were randomly divided into diabetic control group (vehicle-treated db/db group; n = 12) and non-diabetic control group (vehicletreated db/m + group; n = 12), while another three groups of db/db mice were orally treated with AS extract at concentrations of 50, 100, and 200 mg/kg/day (db/db-AS50, db/dbAS100, and db/db-AS200; n = 12, respectively). The 50% ethanol extract of AS was dissolved in distilled water and orally administered once a day for 10 weeks. This animal

ANTI-DIABETIC EFFECT OF ASTER SPHATHULIFOLIUS

study was conducted in accordance with guidelines and approval of the Institutional Animal Care and Use Committees (IACUC) of Hallym University (Approval No.: Hallym 2013-103). Determination of food intake, water intake, and body weight All the groups were fed a standard AIN-76A Purified Rodent diet ( Jung-Ang Experimental Animal Incorporated) and had free access to water. The food and water intakes of animals were measured semiweekly, and body weight was monitored weekly. Food and water consumption were determined by subtracting the left-over amount from the total amount provided. Determination of fasting blood glucose During the 10-week experiment, the blood glucose concentration was monitored weekly. Fasting blood glucose levels were measured after the animals had fasted for 12 h. Blood glucose levels were determined in blood samples from the tail vein using a glucose analyzer (Roche Diagnostics Incorporated, Indianapolis, IN, USA). To get the precise data, the measurement of blood glucose was always carried out at the same time period after a 12 h fast. Determination of hemoglobin A1c levels The mice were fasted for 12 h, and the blood samples, via the retro-orbital venous plexus, using heparinized capillary tubes were collected into an EDTA-coated tube. We evaluated the performance of the TOSOH glycohemoglobin analyzer (TOSOH Corporation, Tokyo, Japan). Serum biochemistry analysis At the end of the experimental period, the mice were sacrificed after a 12 h fast. Blood samples were obtained via the retro-orbital venous plexus into a tube, and serum samples were separated from the blood. AST, ALT, CHOL, GLUC, HDL-Chol, LDL-Chol, and TG were determined using an automatic analyzer (Thermo Electron Corporation, Vantaa, Finland). Enzyme-linked immunosorbent assay The blood samples were placed on ice and were centrifuged at 800 ·g for 15 min at 4C. The plasma was stored at - 70C until assay. The level of insulin in the serum from the mice was determined using ELISA assay procedure, and reagents were included in the mouse insulin ELISA kit (Millipore Corporation, Billerica, MA, USA). Ten microliters of assay buffer, 10 lL of sample, and 80 lL of biotinylated detection antibody were added to the wells of a microtiter plate coated by a pre-titered amount of antiinsulin-coated antibodies, and they were then incubated for 2 h at room temperature. After washing away unbound biotinylated antibodies, 100 lL of horse radish peroxidase conjugate streptavidin was added to each well and incubated

3

for 30 min at room temperature. The wells were again washed, 100 lL substrate solution was added to each well for 15 min at room temperature, and color developed in proportion to the amount of target protein bound. The stop solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. Intraperitoneal glucose tolerance test At the end of treatment, an intraperitoneal glucose tolerance test (IPGTT) was performed to assess glucose tolerance of db/db mice. On the test day, the animals were fasted for 12 h and then given an intraperitoneal injection of glucose (2 g/kg body weight). Blood glucose levels were measured in tail blood samples at 0, 15, 30, 60, 90, and 120 min after the glucose treatment using a glucometer (Roche Diagnostics Incorporated). Insulin tolerance test To estimate insulin sensitivity, an insulin tolerance test (ITT) was performed. The test consisted of an insulin solution (0.75 U/kg of body weight) that was administered subcutaneously in the dorsal region. After 30 min, they were intraperitoneally injected with 2 g glucose/kg body weight. Blood glucose levels were measured in tail blood samples at 0, 30, 60, 90, and 120 min after the glucose treatment using a glucometer (Roche Diagnostics Incorporated). Immunohistochemical staining for insulin The pancreatic tissue samples were fixed in 4% formaldehyde, dehydrated in a graded series of ethanol concentrations, and embedded. For immunohistochemistry analysis, the rehydrated sections were treated with 3% H2O2 in methanol for 1 h to quench endogenous peroxidase, and then washed with 0.01 M sodium phosphate buffer for 10 min. For detection of insulin, slides were incubated for 3 h at room temperature with anti-insulin antibody (Diluted 1:500; Santa Cruz Biotechnology Incorporated). Incubation with primary antibody followed rinsing with PBST for 10 min; slides were then treated with a biotinylated link (Universal DakoCytomation, Glostrup, Denmark), which was detected by a reaction with horseradish peroxidase for 30 min. After washing with phosphate-buffered saline containing 0.05% Tween 20 PBST, slides were incubated with 3, 30 -diaminobenzidine tetrahydrochloride (Universal DakoCytomation) and immediately rinsed with distilled water after color development. Counterstaining was performed with Mayer’s Hematoxylin (Universal DakoCytomation). Slides were mounted and were then observed under a light microscope (Carl Zeiss, Go¨ttingen, Germany). RNA isolation For the isolation of tissue RNA, mice were sacrificed; muscle, liver, and adipose muscle tissue were removed under aseptic conditions and immediately frozen at - 70C. Muscle, liver, and adipose tissues were homogenized in easy-blue reagent (Easy-Blue total RNA extraction kit;

4

YIN ET AL.

iNtRON, Gyeonggi, Korea) using Mixer. Total RNA was extracted according to the manufacturer’s protocol. RNA concentrations were measured spectrophotometrically (Thermo Scientific, Waltham, MA, USA) using the absorbance at 260 nm and 280 nm in samples with an appropriate dilution. Real-time polymerase chain reaction analysis The real-time polymerase chain reaction (RT-PCR) analysis was performed using cDNA synthesized from 1 lg total RNA of each sample by using a transcriptor first-strand cDNA synthesis kit as described in the manufacturer’s protocol (Roche Applied Science, Penzberg, Germany). The PCR amplifications were quantified using a LightCycler 480 SYBR Green 1 Master Mix (Roche Applied Science, Mannheim, Germany), and the results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The oligonucleotide primers are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/jmf). The reaction mixtures comprised 5 lL cDNA template, 15 lL SYBR Green Supermix dye (Bio-Rad, Vienna, Austria), including a 10 pmol final concentration of each primer. The RT-PCR program started with 5 min at 95C, followed by 45 cycles of 95C, 59C, and 72C, at 30 s each, with the plate reading step at 59C. The thermal profile for the melt-curve analysis was determined by incubation at 65C for 1 min followed by a gradual increase of temperature to 95C, during which changes in fluorescence were monitored. No template controls were included in duplicate to control for the presence of primer-dimers. The results were obtained using LightCycler Relative Quantifications Software (Roche Applied Science, Basel, Germany). Statistical analysis The results were expressed as the mean – standard deviation (n = 3) of triplicate experiments. An analysis of variance and Duncan’s multiple-range tests were used to determine the significance of differences means, and P < .05 was considered statistically significant.

RESULTS Changes in body weight and food and water intakes At the start of the study, body weight in the db/db group was significantly heavier than that of their lean littermates (vehicletreated db/m + ) at baseline (36.28 – 1.52 vs. 25.37 – 0.54 g for vehicle-treated db/db and vehicle-treated db/m + mice, respectively). After the 10-week period, the average body weight of the db/db-AS200 group was slightly reduced compared with that of the vehicle-treated db/db group (Table 1), but the body weights of the db/db mice treated with AS at concentrations of 50, 100, and 200 mg/kg/day were not statistically different from those of the vehicle-treated db/db group. During the 10week period, food and water intake in the vehicle-treated db/db group was significantly higher than that in the vehicle-treated db/m + group; the vehicle-treated db/db group and db/dbAS50 group were similar throughout the study. The db/dbAS100 and db/db-AS200 groups were significantly lower than the vehicle-treated db/db group. Analysis of fasting blood glucose levels As shown in Figure 1, the initial blood glucose levels were not significantly different among the db/db group. The vehicle-treated db/db group showed a significantly higher level of blood glucose, compared with the vehicle-treated db/m + group. The AS extract-treated group maintained lower blood glucose levels compared with the non-treated group in a dose-dependent manner. However, the blood glucose levels of the db/db-AS200 group were significantly decreased compared with the vehicle-treated db/db group from the 6th week to the end of the study. IPGTT and ITT Changes in blood glucose were shown in Figure 2. Fasting blood glucose level of the vehicle-treated db/db group was significantly higher than that of the vehicle-treated db/m + group. Data from the IPGTT experiment indicated that administration of AS extract induces a slight decrease in blood glucose levels that was observed at 15 min. Glucose values in the db/db-AS200 group were markedly reduced to significant

Table 1. Food and Water Intake, Body Weight of C57BL/KsJ db/db Mice Fed Aster sphathulifolius Extract for 10 Weeks Group Parameters Body weight (g) Initial Final Food intake (g/day) Water intake (mL/day)

db/m +

db/db

db/db-AS50

db/db-AS100

db/db-AS200

25.37 – 0.54b 33.34 – 0.30b 2.65 – 0.14c 3.05 – 0.13c

36.28 – 1.52a 48.72 – 1.05a 4.57 – 0.32a 12.73 – 0.39a

36.52 – 0.50a 46.95 – 1.92a 4.24 – 0.19ab 12.64 – 0.79a

36.58 – 0.44a 46.17 – 1.15a 4.00 – 0.21b 9.30 – 0.75b

36.63 – 0.42a 46.03 – 0.96a 3.83 – 0.18b 8.76 – 0.51b

The values are expressed as mean – SD (n = 12). Values with different superscript letters are significantly different (P < .05). The food intake, water intake of animals was measured semiweekly, and body weight was monitored weekly for 10 weeks. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/dbAS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day. AS, Aster sphathulifolius; SD, standard deviation.

ANTI-DIABETIC EFFECT OF ASTER SPHATHULIFOLIUS

5

FIG. 1. Weekly changes in the fasting blood glucose levels of C57BL/KsJ db/db mice administered with Aster sphathulifolius (AS) extract. During the 10-week experiment, the blood glucose concentration was monitored weekly. The values are expressed as mean – SD (n = 12). *P < .05 vs. to db/db control group based on Tukey’s test. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/ db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day. SD, standard deviation.

values after 15, 30, and 60 min, and these levels returned to almost normal levels after 120 min. Moreover, the significant change in blood glucose level was not detected in the samplelow group and the sample-high group. Because of impaired insulin sensitivity, the blood glucose concentrations increased in the db/db group after 60 min. The blood glucose levels increased in the db/db-AS50 group and the db/db-AS100 group after 90 min, and blood glucose levels of these groups were higher than initial blood glucose levels (Fig. 3). On the other hand, the blood glucose levels of the db/db-AS200 group also increased after 90 min and then declined to the initial blood glucose level in 120 min. These observations strongly suggest that AS improves glucose intolerance and slightly ameliorates insulin sensitivity in the db/db-AS200 group.

Effect of AS extract on plasma parameters After a 10-week administration of AS extracts in C57BL/ KsJ-db/db mice, serum biochemical levels were determined. The results of plasma parameters were summarized in Table 2. As shown in Table 2, the db/db-AS200 group showed significantly decreased CHOL, GLUC, and LDL-Chol, compared with the vehicle-treated db/db group. Effect of AS extract on hemoglobin A1c levels At the end of the experimental period, after 10 weeks, we investigated the percentage of glycosylated hemoglobin A1c (HbA1c) in db/db mice after different concentrations of treatment. Figure 4 shows the results of the HbA1c levels

FIG. 2. Effect of AS extract treatment in glucose tolerance test on C57BL/KsJ- db/db mice at 10 weeks. After a 12 h fast, all mice were intraperitoneally injected with glucose (2 g/kg body weight). The blood glucose concentration was measured at the indicated times and presented as a percent of glucose injection at time zero. The values are expressed as mean – SD (n = 12). *P < .05 vs. to db/db control group based on Tukey’s test. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

6

YIN ET AL.

FIG. 3. Effect of AS extract treatment in insulin tolerance test on C57BL/KsJ- db/db mice at 10 weeks. After a 12 h fast, male mice were intraperitoneally injected with glucose (2 g/kg body weight). The blood glucose concentrations were measured and presented as a percent of glucose injection at time zero. The values are expressed as mean – SD (n = 12). *P < .05 vs. to db/db control group based on Tukey’s test. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/ kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

performed at the 10th week of the treatments. The vehicletreated db/db group showed a significantly higher level of HbA1c, compared with the vehicle-treated db/m + group. The AS-treated group maintained lower HbA1c levels compared with the vehicle-treated db/db group in a dosedependent manner. The HbA1c levels of the db/db-AS200 group was significantly lower (P < .05) than that of the vehicle-treated db/db group. Effect of AS extract on serum insulin concentration levels The changes in plasma insulin levels are shown in Figure 5. In db/db mice, the plasma insulin level of the db/db-AS200 group was significantly lower than that of the vehicle-treated db/db group (P < .05). In db/db mice, the group demonstrated the highest blood insulin level (vehicletreated db/db, 2.605 ng/mL), followed by the sample-low

group (db/db-AS50, 2.175 ng/mL), and the sample-high (db/db-AS200, 1.558 ng/mL). Both the sample-low and the sample-high supplement significantly ameliorated the blood insulin level compared with the vehicle-treated db/db group. Insulin resistance of T2DM can lead to the development of hyperinsulinemia, which is caused by excessive insulin production in the pancreas to compensate for insulin resistance. In db/db mice, the AS supplement decreased the plasma insulin level. This result means that AS may have alleviated hyperinsulinemia by reducing insulin resistance. Expression of genes related to glucose homeostasis in muscle We principally focused on the insulin receptor, IRS-1, which is involved in GLUT4 translocation under insulin

Table 2. Serum Biochemical Values in the Mice Treated Orally with Aster sphathulifolius Extract for 10 Weeks Group Parameters ALT (U/L) AST (U/L) CHOL (mg/dL) GLUC (mg/dL) HDL-Chol (mg/dL) LDL-Chol (mg/dL) TG (mg/dL)

db/m +

db/db

db/db-AS50

db/db-AS100

db/db-AS200

33.51 – 4.85b 95.39 – 8.77b 136.07 – 4.66b 221.11 – 17.09c 113.35 – 4.50a 11.381 – 1.83b 53.34 – 3.32a

555.25 – 50.60a 414.12 – 29.94a 247.91 – 38.56a 623.76 – 40.28a 132.37 – 17.61a 31.98 – 5.49a 68.85 – 7.81a

451.89 – 79.51a 431.82 – 81.37a 248.23 – 36.17a 547.48 – 76.49ab 145.75 – 16.87a 29.21 – 6.82a 73.63 – 9.63a

485.78 – 99.42a 407.75 – 69.86a 245.24 – 9.35a 587.70 – 58.32ab 155.29 – 11.24a 31.48 – 2.30a 70.26 – 4.25a

384.07 – 42.14a 326.29 – 37.42a 209.52 – 22.76ab 422.10 – 75.80b 147.26 – 9.13a 22.01 – 2.15ab 68.73 – 11.74a

The values are expressed as mean – SD (n = 12). Values with different superscript letters are significantly different (P < .05). db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHOL, cholesterol; GLUC, glucose; HDL-Chol, high-density lipoprotein; LDL-Chol, lowdensity lipoprotein; TG, triglyceride.

ANTI-DIABETIC EFFECT OF ASTER SPHATHULIFOLIUS

7

Expression of genes related to glucose homeostasis in liver We measured the expression of genes that were known to be involved in glucose metabolism such as G-6-Pase, PEPCK, and GK in the liver by RT-PCR (Fig. 7). In the RTPCR study, AS significantly reduced the expression of the gene encoding the regulatory enzyme of gluconeogenesis (G-6-Pase and PEPCK) in the liver of the db/db-AS200 group (P < .05). In contrast, AS significantly increased GK expression in the db/db-AS200 group, compared with the vehicle-treated db/db group (P < .05). Expression of genes related to glucose homeostasis in adipose tissue

FIG. 4. Effect of AS extract treatment on HbA1c levels in C57BL/ KsJ-db/db mice at 10 weeks. The values are expressed as mean – SD (n = 12). Values with different lowercase letters are significantly different (P < .05). HbA1c is expressed as percentage of total hemoglobin. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day. HbA1c, hemoglobin A1c.

stimulation. In the RT-PCR study, AS treatment increased both IRS-1 and GLUT4 gene expression level dose dependently (Fig. 6). The db/db-AS200 group showed significantly higher gene expression levels than the vehicle-treated db/db group (P < .05).

The expression of genes involved in lipogenesis and boxidation in the epididymal fat tissue was examined. The gene expression of transcription factors (SREBP-1c and PPAR-c) and their target molecules (adipocyte protein 2 [aP2]) were analyzed using RT-PCR. As shown in Figure 8, the expression of transcription factors and aP2 was upregulated in the db/db group in a dose-dependent manner, compared with the vehicle-treated db/db group. Pancreas immunohistochemistry The pancreatic tissues were immunohistochemically stained, using an antibody to insulin, and examined by light microscopy (Fig. 9). Tissue from the vehicle-treated db/m + group exhibited a higher staining intensity than that from the vehicle-treated db/db group. The level of insulin in pancreatic b-cells of the db/db-AS200 group was significantly higher than that of the db/db group, whereas the levels in the db/db-AS50 and db/db-AS100 groups were not significantly different. These results suggest that AS may improve pancreatic b-cells against destruction in a dose-dependent way. DISCUSSION

FIG. 5. Effect of AS extract treatment on serum insulin levels in C57BL/KsJ- db/db mice at 10 weeks. The values are expressed as mean – SD (n = 12). Values with different lowercase letters are significantly different (P < .05). db/m + , C57BL/KsJ-db/m + mice; db/ db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

In this study, we observed the effects of AS on glucose metabolism in the C57BL/KsJ-db/db mice. The mice were orally administrated with AS (50, 100, and 200 mg/kg/day, respectively) for 10 weeks. During the 10-week period, though AS supplementation did not affect the body weight gain in db/db mice, food intake and water intake of the db/ db-AS100 and the db/db-AS200 group were significantly lower than those of the vehicle-treated db/db group. The typical symptoms of diabetes are polydipsia, polyuria, and polyphagia. The intake of AS extract reduced those symptoms, especially polydipsia and polyphagia, resulting in decreased water and food intake. Chronic hyperglycemia plays a key role in the pathogenesis of T2DM and diabetic complications. Therefore, it is important for preventing or reversing diabetes to find ways to control blood glucose levels.26 The evidence from this study suggested that AS supplementation can decrease serum glucose levels in C57BL/KsJ-db/db mice. Prolonged poor glycemic control often leads to a decline in insulin secretion and the pathogenesis of glucose toxicity. HbA1c, which is formed in a nonenzymatic glycation pathway by hemoglobin’s exposure to

FIG. 6. Effect of AS on gene expression levels of genes associated with glucose metabolism-regulating enzymes: insulin receptor substrate 1 (IRS-1), glucose transporter type 4 (GLUT4) in muscle. The values are expressed as mean – SD (n = 12). Values with different lowercase letters are significantly different (P < .05). db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/ KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/dbAS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/ kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

FIG. 7. Effect of AS on the gene expression levels of glucose metabolism-regulating enzymes glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6-Pase) in the liver. The values are expressed as mean – SD (n = 12). Values with different lowercase letters are significantly different (P < .05). db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/ KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/ KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

8

ANTI-DIABETIC EFFECT OF ASTER SPHATHULIFOLIUS

9

FIG. 8. Effect of AS on the gene expression levels of glucose metabolism-regulating enzymes, sterol-regulatory element-binding protein (SREBP)-1c, peroxisome proliferatoractivated receptor c (PPAR-c), and adipocyte protein (aP2) in adipose tissue. The values are expressed as mean – SD (n = 12). Values with different lowercase letters are significantly different (P < .05). db/m + , C57BL/KsJ-db/ m + mice; db/db, C57BL/KsJ-db/db mice; db/ db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/ KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/ kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day.

plasma glucose,27 is a marker for average blood glucose levels over the previous months before the measurement.28 In this study, we demonstrated that long-term supplementation with AS significantly lowered the HbA1c level in C57BL/KsJ-db/db mice, thereby decreasing the risk of longterm vascular complications of diabetes by controlling blood glucose in diabetic mice.

Insulin produced by b-cells in the pancreas promotes glucose uptake, glycogenesis, and lipogenesis in skeletal muscle, liver, and adipose tissue.29–31 Therefore, insulin is the most important factor in the regulation of plasma glucose homeostasis. Hyperinsulinemia is characterized as a condition where excess insulin is circulating in the blood, resulting from reduced sensitivity to insulin and disturbances in insulin

FIG. 9. Immunochemical examination of the pancreas of C57BL/KsJ-db/db mice administered with AS extract for 10 weeks. Stained tissues are indicated by arrows. Original magnification, · 400. db/m + , C57BL/KsJ-db/m + mice; db/db, C57BL/KsJ-db/db mice; db/db-AS50, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 50 mg/kg/day; db/db-AS100, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 100 mg/kg/day; db/db-AS200, C57BL/KsJ-db/db mice were orally treated with AS ethanol extract at concentrations of 200 mg/kg/day. Color images available online at www.liebertpub.com/jmf

10

YIN ET AL.

release.32 Insulin resistance is the inability of the body cells to utilize insulin; classically, this refers to impaired sensitivity to insulin-mediated glucose disposal.33 Hyperinsulinemia and insulin resistance can lead to the development of a number of degenerative diseases and potentially life-threatening metabolic consequences.34,35 As shown in IPGTT, which measures the ability to metabolize circulating blood glucose, AS improved impaired glucose tolerance. In addition, AS alleviated hyperinsulinemia by reducing insulin resistance. Taken together, these results demonstrated that db/db mice treated with AS for 10 weeks were less insulin resistant and had a mild improvement in glucose homeostasis compared with their untreated controls. To further determine the effects of AS extract on insulin sensitivity, we performed ITT in C57BL/KsJ-db/db mice. Results demonstrated that AS can improve insulin sensitivity. The underlying defect must be expressed in skeletal muscle, which accounts for 70–80% of insulin-stimulated glucose uptake in vivo.8 IRS-1, which is the first intracellular protein to be phosphorylated, plays a key role via insulin signaling in skeletal muscle.36 Insulin stimulates glucose uptake by increasing the translocation of glucose transport molecules, mainly GLUT4, which is primarily found in skeletal muscle, from intracellular vesicles to the cell surface.37 Overexpression of GLUT4 in the muscle of genetically db/db mice alleviates insulin resistance and improves glycemic control by elevating both basal and insulinstimulated glucose transport.38 The insulin resistance of T2DM is characterized by defects at many levels, with decreases in the concentration and phosphorylation of IRS-1 and -2, PI3K activity, glucose transporter translocation, and the activity of intracellular enzymes in the muscle.39 Treatment with AS significantly increased the expression of IRS-1 and GLUT4, suggesting that AS may stimulate glucose uptake in skeletal muscles through activation of IRS-1 and GLUT4. Insulin resistance profoundly contributes to the pathophysiology of T2DM and it reduces glucose utilization and increases glucose production from the liver, thus leading to hyperglycemia.40 The liver plays a key role in glucose homeostasis increasing or decreasing glucose output and uptake during fasting and feeding.41 In the liver, GK is the glucose-sensing enzyme, which only binds to and phosphorylates glucose,42 and is an appealing target for hepatic glucose metabolism. In addition, PEPCK and G-6-Pase each catalyze the rate-limiting step and the final step of gluconeogenesis, respectively.43 The expressions of hepatic genes in liver have been shown to be associated with the pathogenesis of diabetes.44 In our study, the hypoglycemic action of AS in db/db mice was related with a significant decrease in the expression of the gene encoding the regulatory enzymes of gluconeogenesis and glycogenolysis, such as G-6-Pase and PEPCK in the liver. In contrast, AS extract significantly increased GK expression in the db/db-AS200 group, compared with the vehicle-treated db/db group. Accordingly, AS treatment appears to improve glucose metabolism through an increase in expression of GK and a decrease in G-6-Pase and PEPCK gene expression. Abnormalities of lipogenesis in insulin-sensitive tissues are an important component of T2DM. Lipogenesis is the metabolic pathway allowing the

conversion of an excess of carbohydrates into fatty acids, which are ultimately esterified with glycerol 3-phosphate to form TGs in adipose tissue, which is the primary site of energy storage.45 SREBP-1c is a key transcription factor that regulates lipid homeostasis by controlling the expression of a range of enzymes for endogenous cholesterol, fatty acid, and triacylglycerol and phospholipid synthesis.46 It works in tandem with the adipogenic transcription factor (PPAR-c). PPAR-c is the main adipogenic factor that triggers adipocyte differentiation; it is highly expressed in the liver, muscle, kidney, and heart, where it stimulates the b-oxidative degradation of fatty acids.47 The aP2 is unregulated in response to PPAR-c agonist exposure; it is a carrier protein for fatty acids that is primarily expressed in adipocytes and is also called fatty acid binding protein 4.48 AS has traditionally been used to treat diabetes in oriental medicine, probably due to the presence of bio active compounds in AS extracts.22,23 In our study, the expression of transcription factors (SREBP-1c, PPAR-c) and their target molecules (aP2) was upregulated in the AS-treated db/db group in a dose-dependent manner, compared with the vehicle-treated db/db group. These findings suggest that AS increased activation of insulin by upregulating SREBP-1c, PPAR-c, and aP2 expression in adipose tissue, promoted fatty acid synthesis, and inhibited fatty acid oxidation. Immunohistological assay of the pancreas revealed that the db/db-AS200 group increased the b-cell staining intensity, resulting from improved pancreatic b-cells against destruction in a dose-dependent way. In conclusion, AS treatment was effective for normalizing hyperglycemia, glucose tolerance, the level of HbA1c, glucose metabolism, and lipid metabolism through gene expression in insulin-sensitive tissues. In addition, immunohistochemical analysis showed that AS upregulated insulin production by increasing pancreatic b-cell mass. These findings provide in vivo evidence that AS has therapeutic efficacy as an anti-diabetic agent.

ACKNOWLEDGMENTS This work was supported by the Technology Innovation Program (industrial strategic technology development program, 10045275, development of functional food product for improving metabolic syndrome using natural resources and extending global market) funded by the Ministry of Trade, Industry & Energy (MOTIE) and by the Hallym University Research Fund (HRF-201407-013). AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. DeFronzo RA: Pathogenesis of type 2 (non-insulin dependent) diabetes mellitus: A balanced overview. Diabetologia 1992;35: 389–397. 2. Yki-Jarvinen H: Pathogenesis of non-insulin-dependent diabetes mellitus. Lancet 1994;343:91–95.

ANTI-DIABETIC EFFECT OF ASTER SPHATHULIFOLIUS 3. Ferre T, Pujol A, Riu E, Bosch F, Valera A: Correction of diabetic alterations by glucokinase. Proc Natl Acad Sci USA 1996;93:7225–7230. 4. Reaven GM: Role of insulin resistance in the pathophysiology of non-insulin dependent diabetes mellitus. Diabetes Metab Rev 1993;9:5S–12S. 5. Bjornholm M, Zierath JR: Insulin signal transduction in human skeletal muscle: Identifying the defects in Type II diabetes. Biochem Soc Trans 2005;33:354–357. 6. Zisman A, Peroni OD, Abel ED, et al.: Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat Med 2000;6:924–928. 7. Saltiel AR, Kahn CR: Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001;414:799–806. 8. DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP: The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 1981;30:1000–1007. 9. Carvalho E, Kotani K, Peroni OD, Kahn BB: Adipose-specific overexpression of GLUT4 reverses insulin resistance and diabetes in mice lacking GLUT4 selectively in muscle. Am J Physiol Endocrinol Metab 2005;289:E551–E561. 10. Postic C, Shiota M, Niswender KD, et al.: Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 1999;274:305–315. 11. Chatelain F, Pegorier JP, Minassian C, et al.: Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine, and kidney: in vivo and in vitro studies in cultured fetal hepatocytes. Diabetes 1998;47:882–889. 12. O’Brien RM, Granner DK: PEPCK gene as model of inhibitory effects of insulin on gene transcription. Diabetes Care 1990;13: 327–339. 13. Muoio DM, Newgard CB: Mechanisms of disease: Molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nat Rev Mol Cell Biol 2008;9:193–205. 14. Tontonoz P, Kim JB, Graves RA, Spiegelman BM: ADD1: A novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol 1993; 13:4753–4759. 15. Horton JD, Shimomura I: Sterol regulatory element-binding proteins: Activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 1999;10:143–150. 16. Fajas L, Schoonjans K, Gelman L, et al.: Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: Implications for adipocyte differentiation and metabolism. Mol Cell Biol 1999;19:5495–5503. 17. Kim JB, Wright HM, Wright M, Spiegelman BM: ADD1/ SREBP1 activates PPARgamma through the production of endogenous ligand. Proc Natl Acad Sci USA 1998;95:4333–4337. 18. Kim JB, Sarraf P, Wright M, et al.: Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest 1998;101:1–9. 19. Flier JS, Hollenberg AN: ADD-1 provides major new insight into the mechanism of insulin action. Proc Natl Acad Sci USA 1999; 96:14191–14192. 20. Puigserver P, Spiegelman BM: Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): Transcriptional coactivator and metabolic regulator. Endocr Rev 2003;24:78–90.

11

21. Luna B, Feinglos MN: Oral agents in the management of type 2 diabetes mellitus. Am Fam Physician 2001;63:1747–1756. 22. Kim TJ: Wild Flowers of Korea. Kugilmedia, Seoul, Korea, 1996, p. 232. 23. Lee CB: Illustrated Flora of Korea. Hyangmoonsa, Seoul, Korea, 1999, p. 740. 24. Won JN, Lee SY, Song DS, Poo H: Antiviral activity of the plant extracts from Thuja orientalis, Aster sphathulifolius, and Pinus thunbergii against influenza virus A/PR/8/34. J Microbiol Biotechnol 2013;23:125–130. 25. Lee SO, Choi SZ, Choi SU, et al.: Labdane diterpenes from Aster sphathulifolius and their cytotoxic effects on human cancer cell lines. J Nat Prod 2005;68:1471–1474. 26. Klein R: Hyperglycemia and microvascular and macrovascular disease in diabetes. Diabetes Care 1995;18:258–268. 27. Larsen ML, Horder M, Mogensen EF: Effect of long-term monitoring of glycosylated hemoglobin levels in insulin-dependent diabetes mellitus. N Engl J Med 1990;323:1021–1025. 28. Stratton IM, Adler AI, Neil HA, et al.: Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): Prospective observational study. BMJ 2000;321:405–412. 29. Hansen ME, Tippetts TS, Anderson MC, et al.: Insulin increases ceramide synthesis in skeletal muscle. J Diabetes Res 2014;2014: 765–784. 30. Coomans CP, Geerling JJ, Guigas B, et al.: Circulating insulin stimulates fatty acid retention in white adipose tissue via KATP channel activation in the central nervous system only in insulinsensitive mice. J Lipid Res 2011;52:1712–1722. 31. Hiriart M, Sanchez-Soto C, Diaz-Garcia CM, et al.: Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes. Front Endocrinol (Lausanne) 2014;5:95. 32. Odeleye OE, de Courten M, Pettitt DJ, Ravussin E: Fasting hyperinsulinemia is a predictor of increased body weight gain and obesity in Pima Indian children. Diabetes 1997;46:1341–1345. 33. Liu J, Wu YY, Huang XM, et al.: Ageing and type 2 diabetes in an elderly chinese population: The role of insulin resistance and beta cell dysfunction. Eur Rev Med Pharmacol Sci 2014;18:1790–1797. 34. Campos G, Ryder E, Diez-Ewald M, et al.: Prevalence of obesity and hyperinsulinemia: Its association with serum lipid and lipoprotein concentrations in healthy individuals from Maracaibo, Venezuela. Invest Clin 2003;44:5–19. 35. Hansen J, Rinnov A, Krogh-Madsen R, et al.: Plasma follistatin is elevated in patients with type 2 diabetes: Relationship to hyperglycemia, hyperinsulinemia, and systemic low-grade inflammation. Diabetes Metab Res Rev 2013;29:463–472. 36. Van Obberghen E, Baron V, Delahaye L, et al.: Surfing the insulin signaling web. Eur J Clin Invest 2001;31:966–977. 37. James DE, Strube M, Mueckler M: Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 1989;338:83–87. 38. Garvey WT, Birnbaum MJ: Cellular insulin action and insulin resistance. Baillieres Clin Endocrinol Metab 1993;7:785–873. 39. Pessin JE, Saltiel AR: Signaling pathways in insulin action: Molecular targets of insulin resistance. J Clin Invest 2000;106: 165–169. 40. McGarry JD: Disordered metabolism in diabetes: Have we underemphasized the fat component? J Cell Biochem 1994;55:29–38. 41. Pirgon O, Bilgin H, Cekmez F, Kurku H, Dundar BN: Association between insulin resistance and oxidative stress parameters in

12

YIN ET AL.

obese adolescents with non-alcoholic fatty liver disease. J Clin Res Pediatr Endocrinol 2013;5:33–39. 42. Matschinsky FM: Regulation of pancreatic beta-cell glucokinase: From basics to therapeutics. Diabetes 2002;51:S394–S404. 43. Seoane J, Barbera A, Telemaque-Potts S, Newgard CB, Guinovart JJ: Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats. J Biol Chem 1999;274:31833–31838. 44. Misawa E, Tanaka M, Nomaguchi K, et al.: Oral ingestion of aloe vera phytosterols alters hepatic gene expression profiles and ameliorates obesity-associated metabolic disorders in zucker diabetic fatty rats. J Agric Food Chem 2012;60:2799–2806.

45. Acheson KJ, Schutz Y, Bessard T, Ravussin E, Jequier E, Flatt JP: Nutritional influences on lipogenesis and thermogenesis after a carbohydrate meal. Am J Physiol 1984;246:E62–E70. 46. Ferre P, Foufelle F: Hepatic steatosis: A role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 2010;12:83–92. 47. Ferre P: The biology of peroxisome proliferator-activated receptors: Relationship with lipid metabolism and insulin sensitivity. Diabetes 2004;53:S43–S50. 48. Baxa CA, Sha RS, Buelt MK, et al.: Human adipocyte lipidbinding protein: Purification of the protein and cloning of its complementary DNA. Biochemistry 1989;28:8683–8690.