NO-1886 suppresses diet-induced insulin resistance and cholesterol accumulation through STAT5-dependent upregulation of IGF1 and CYP7A1

in Journal of Endocrinology
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  • 1 Key Laboratory for Atherosclerology of Hunan Province, Department of Nutrition and Metabolism, Clinical Research Center for Diabetes, Institute of Cardiovascular Research, Life Science Research Center, University of South China, Hengyang, Hunan 421001, People's Republic of China

Insulin resistance and dyslipidemia are both considered to be risk factors for metabolic syndrome. Low levels of IGF1 are associated with insulin resistance. Elevation of low-density lipoprotein cholesterol (LDL-C) concomitant with depression of high-density lipoprotein cholesterol (HDL-C) increase the risk of obesity and type 2 diabetes mellitus (T2DM). Liver secretes IGF1 and catabolizes cholesterol regulated by the rate-limiting enzyme of bile acid synthesis from cholesterol 7α-hydroxylase (CYP7A1). NO-1886, a chemically synthesized lipoprotein lipase activator, suppresses diet-induced insulin resistance with the improvement of HDL-C. The goal of the present study is to evaluate whether NO-1886 upregulates IGF1 and CYP7A1 to benefit glucose and cholesterol metabolism. By using human hepatoma cell lines (HepG2 cells) as an in vitro model, we found that NO-1886 promoted IGF1 secretion and CYP7A1 expression through the activation of signal transducer and activator of transcription 5 (STAT5). Pretreatment of cells with AG 490, the inhibitor of STAT pathway, completely abolished NO-1886-induced IGF1 secretion and CYP7A1 expression. Studies performed in Chinese Bama minipigs pointed out an augmentation of plasma IGF1 elicited by a single dose administration of NO-1886. Long-term supplementation with NO-1886 recovered hyperinsulinemia and low plasma levels of IGF1 suppressed LDL-C and facilitated reverse cholesterol transport by decreasing hepatic cholesterol accumulation through increasing CYP7A1 expression in high-fat/high-sucrose/high-cholesterol diet minipigs. These findings indicate that NO-1886 upregulates IGF1 secretion and CYP7A1 expression to improve insulin resistance and hepatic cholesterol accumulation, which may represent an alternative therapeutic avenue of NO-1886 for T2DM and metabolic syndrome.

Abstract

Insulin resistance and dyslipidemia are both considered to be risk factors for metabolic syndrome. Low levels of IGF1 are associated with insulin resistance. Elevation of low-density lipoprotein cholesterol (LDL-C) concomitant with depression of high-density lipoprotein cholesterol (HDL-C) increase the risk of obesity and type 2 diabetes mellitus (T2DM). Liver secretes IGF1 and catabolizes cholesterol regulated by the rate-limiting enzyme of bile acid synthesis from cholesterol 7α-hydroxylase (CYP7A1). NO-1886, a chemically synthesized lipoprotein lipase activator, suppresses diet-induced insulin resistance with the improvement of HDL-C. The goal of the present study is to evaluate whether NO-1886 upregulates IGF1 and CYP7A1 to benefit glucose and cholesterol metabolism. By using human hepatoma cell lines (HepG2 cells) as an in vitro model, we found that NO-1886 promoted IGF1 secretion and CYP7A1 expression through the activation of signal transducer and activator of transcription 5 (STAT5). Pretreatment of cells with AG 490, the inhibitor of STAT pathway, completely abolished NO-1886-induced IGF1 secretion and CYP7A1 expression. Studies performed in Chinese Bama minipigs pointed out an augmentation of plasma IGF1 elicited by a single dose administration of NO-1886. Long-term supplementation with NO-1886 recovered hyperinsulinemia and low plasma levels of IGF1 suppressed LDL-C and facilitated reverse cholesterol transport by decreasing hepatic cholesterol accumulation through increasing CYP7A1 expression in high-fat/high-sucrose/high-cholesterol diet minipigs. These findings indicate that NO-1886 upregulates IGF1 secretion and CYP7A1 expression to improve insulin resistance and hepatic cholesterol accumulation, which may represent an alternative therapeutic avenue of NO-1886 for T2DM and metabolic syndrome.

Introduction

The metabolic syndrome (syndrome X) refers to the aggregation of a cluster of risk factors, including insulin resistance/hyperinsulinemia and dyslipidemia (increased low-density lipoprotein cholesterol (LDL-C), and depressed high-density lipoprotein cholesterol (HDL-C)), and hypertension (Moller & Kaufman 2005).

Insulin-like growth factor 1 (IGF1) is a protein hormone similar in molecular structure to insulin. Nonetheless, unlike insulin that is secreted from pancreas, IGF1 is produced primarily from liver in response to GH stimulation. IGF1 has been found to play an important role in embryonic and childhood growth. In adults, IGF1 is crucial to function on glucose homeostasis, lipolysis, proteolysis, and protein oxidation (Froesch et al. 1996). Decreased levels of circulating IGF1 found in diabetic patients have been discovered to be associated with insulin resistance (Moller & Flier 1991, Clark et al. 1997, Takano et al. 2001). IGF1 directly acts on the balance between GH and insulin to the control of glucose homeostasis (Yakar et al. 2001). In addition to the critical role on glucose metabolism, IGF1 interferes with the development of diabetic myopathy (Kajstura et al. 2001). High levels of IGF1 prevent diabetic complications induced by enhanced oxidative stress observed in type 2 diabetes mellitus (T2DM) subjects (Brownlee 2005). It is thus believed that IGF1 participates in physiological glucose homeostasis.

Cholesterol functions in the membrane of cells to modulate fluidity. It is also the raw material for manufacturing steroid hormones. However, elevation of tissue cholesterol accumulation accelerates the formation of atherosclerotic plaques, leading to heart attacks and strokes (Brown & Goldstein 1986). The excessive cholesterol in peripheral tissues obtained from a combination of local synthesis and the uptake from LDL and very LDL (VLDL) is reversely transported to the liver through the process of reverse cholesterol transport (RCT) by HDL particles (Fielding & Fielding 1995). Liver maintains whole body cholesterol homeostasis by regulating the biosynthesis of cholesterol, its uptake from plasma, storage and catabolism to bile acids. Conversion of cholesterol to bile acids in the liver and its subsequent fecal excretion represents a major route for the elimination of cholesterol from the body (Turley & Dietschy 1988). Bile acids can be synthesized via ‘classical pathway’ and ‘alternate pathway’ utilizing different sequence of initial steps. Compared with the alternative pathway, the classical pathway is quite ‘flexible’. Expressional regulation of the rate-limiting enzyme of cholesterol 7α-hydroxylase (cytochrome P-450, family 7, subfamily A, and polypeptide 1 (CYP7A1, EC1.14.13.17)) represents a major node for hepatic cholesterol catabolism (Chawla et al. 2000). Transcription of CYP7A1 is stimulated by dietary cholesterol in rodents (Horton et al. 1995). In mammals, however, dietary cholesterol fails to stimulate hepatic CYP7A1 expression, resulting in the susceptive accumulation of the peripheral cholesterol and atherosclerosis (Chiang et al. 2001, Goodwin et al. 2003). Recently, transgenic strategies have proved the favorable effects on dyslipidemia by increasing the expression of CYP7A1 (Ratliff et al. 2006), whereas, there is still lack of an effective pharmacological drug to activate CYP7A1 in mammals.

The chemically synthesized compound NO-1886 has been known to activate lipoprotein lipase (LPL) by increasing LPL mRNA and activity (Tsutsumi et al. 1993). Recently, NO-1886 has been found to elevate plasma HDL-C levels by upregulating ATP-binding cassette transporter A1 to facilitate cholesterol efflux from peripheral tissues to nascent HDL particles, suppressing fat accumulation and insulin resistance induced by high-carbohydrate, high-fat diet regimen in animal models (Tsutsumi et al. 1997, Zhang et al. 2006).

Although it has been verified that NO-1886 upregulates HDL-C and RCT, what happens to the excessive hepatic cholesterol uptaken from HDL and/or LDL is still awaiting further elucidation. In this study, we investigated the effects of NO-1886 on IGF1 and CYP7A1 expression. Our data indicate that NO-1886 enhances IGF1 secretion and CYP7A1 expression, which ultimately functions to prevent diet-induced insulin resistance and dyslipidemia. These effects are therefore regarded to be involved in the therapeutic mechanisms of NO-1886 on insulin resistance and dyslipidemia.

Materials and Methods

Reagents

DMEM/Hams F-12 (DMEM/F-12) was purchased from Gibco (Invitrogen Corp). The antibody of anti-CYP7A1 (sc-25536) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-β-actin (ab6276) was from Abcam (Cambridge, UK), anti-signal transducer and activator of transcription 5 (STAT5) and anti-phospho-STAT5 (Tyr-694/Tyr-699) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). The Janus kinase 2 (JAK2)/STAT pathway inhibitor AG 490 (PHZ1204) was from BioSource International, Inc. (Camarillo, CA, USA). The enhanced chemiluminescence kit (ECL) was from Amersham Biosciences. Sucrose was from Liuzhou Sugar Co. (Guangxi, China) and lard was from Hengyang Meat Product Co. (Hunan, China). Cholesterol was from Sigma–Aldrich. High sensitive RIA kits for insulin, GH and IGF1 were from Jiuding Bio-Engineering Corporation (Tianjing, China). BCA Protein Assay Kit was from Pierce Biotechnology (Rockford, IL, USA). The compound NO-1886 ([4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]- phosphonic acid diethyl ester, Chemical Abstracts Service 133208-93-2; lot No. C99L925M) was chemically synthesized in the New Drug Synthesis Section of Otsuka Pharmaceutical factory Inc. (Tokushima, Japan). All of the other chemical reagents were of either reagent or molecular biological grade from Bio Basic Inc. (Markham, ON, Canada).

Cell culture

Human hepatoblastoma cells HepG2 were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM/Hams F-12 with 10% (v/v) fetal bovine serum (FBS, Biofluids, Rockville, MD, USA) supplemented with 100 units/ml penicillin, and 100 μg/ml streptomycin (Biofluids). Cells were maintained at 37 °C in 5% CO2. Cells were cultured overnight with serum-free medium (DMEM/F12 medium with 0.1% FBS) before each experiment. In transient transfection assays, constructs (50 μg) of caSTAT5A1*6 expression plasmid (described elsewhere (Onishi et al. 1998)) were electroporated into HepG2 cells. These cells were then cultured for 24 h before experiments.

Animal

Fifteen male Chinese Bama minipigs (Sus scrofa domestica) were purchased from Experimental Animal Supply Center of the Third Military Medical University (Chongqing, China), aged 30±5 days, and weighting 6.3±1.3 kg. The minipigs were housed in five cages covered with sterile glass chips per room in three rooms at constant temperature of 23±2 °C and 55% humidity, the light/darkness cycle was 12 h. Before experiments, animals were given laboratory standard chow to acclimate for 2 weeks. The animals were observed daily for clinical signs and custody. For the treatment of single dose of NO-1886, after overnight fasting, six male minipigs were randomly divided into two groups according to their body weight (BW); 1 g/kg BW NO-1886 (suspended in 5% Arabic gum) was orally administered within 10 min. Blood was obtained without sedation by pricking an ear vein with a lancet and collecting drops in a hematocrit tube at each hour for 8 h. The plasma samples kept frozen at −80 °C were analyzed within 1 week. For the experiment of long-term treatment of NO-1886, 15 male minipigs were randomly divided into three groups according to their BW. Animals of the first group were given a daily standard chow (control diet, CD); the second group were fed a daily high-fat/high-sucrose/high-cholesterol diet (HFSCD, normal pig diet supplemented with 10% lard, 37% sucrose, and 2% cholesterol); the third group were fed a daily HFSCD supplemented with NO-1886 diet (100 mg/kg BW/day, HFSCD1886), the components were specifically described previously (Zhang et al. 2006). Animals were treated for 5 months; blood samples for plasma lipids, glucose, insulin and GH inspection were obtained without sedation by pricking an ear vein with a lancet and collecting drops in a hematocrit tube at the end of each month after overnight fasting. At the end of treatment period of 5 months, animals were killed by phlebotomy under anesthesia with i.v. injection of sodium pentobarbital (60 mg/kg). Liver tissues were dissected and stocked in liquid nitrogen until western blot analyses and cholesterol quantification within 7 days. All experimental work was conducted in accordance with the humane guidelines for ethical and sensitive care under the Act of Animal Protective Department of Human Province and conformed to the guideline of the UFAW handbook on the care and management of laboratory animals.

RNA preparation and real-time quantitative RT-PCR

Total RNA isolated from cells was reverse transcribed using TaKaRa PrimeScript RT reagent kits (TaKaRa, Kyoto, Japan). Quantitative real-time PCR was performed with the LightCycler system (Roche Diagnostics) using TaKaRa SYBR Premix Ex Taq (TaKaRa) and the gene-specific primers. After the reaction, each PCR product was verified for its single amplification by melting curve analysis. The expression levels of CYP7A1 (forward: 5′-CCG ATG GAT GGA AAT ACC AC-3′; reverse: 5′-GGC AGC GGT CTT TGA GTT AG-3′) were normalized to the expression of 18S rRNA (forward: 5′-AAA CGG CTA CCA CAT CCA AG-3′; reverse: 5′-GGC CTC GAA AGA GTC CTG TA-3′) in each sample and were given arbitrary units. Analyses were performed in triplicate.

Western blot

After treatment as described in the figure legends, cells were rinsed three times with ice-cold PBS (NaCl, 137 mmol/l; KCI, 2.7 mmol/l; Na2HPO4, 10 mmol/l; KH2PO4, 2 mmol/l, pH 7.4) and lysed in RIPA buffer (50 mmol/l Tris, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.5% sodium deoxycholate (w/v), 1% Nonidet P-40 (v/v), 0.1% SDS (w/v), 1 μmol/l aprotinin, 10 μmol/l leupeptin, 0.1 μmol/l phenylmethylsulfonyl fluoride, 20 mmol/l sodium fluoride, 20 mmol/l β-glycerolphosphate and 1 mmol/l sodium orthovanadate, pH 7.4). Cell lysates were obtained by centrifugation at 15 000 g and 4 °C for 20 min. Equal amounts of proteins were subjected to SDS-PAGE (Laemmli 1970). Proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). Immunoblotting was performed with an ECL PLUS system according to the manufacturer's instructions. ECL images of immunoblots were scanned with the White/u.v. transilluminator (Upland, CA, USA) and quantified with Labworks image analysis software from UVP Inc. (Upland, CA, USA).

Measurement of IGF1, GH, insulin, glucose, LDL-C, HDL-C, and total cholesterol

For the experiment using HepG2 cells, concentrations of IGF1 and GH were measured in triplicate as described previously (Osada et al. 1996). Briefly, HepG2 cells that had 80% confluent state in 6-well plates were used. The cells were washed with PBS and incubated in 2 ml of DMEM/F-12 medium containing 1% FBS for 2, 4, 8, 16, and 24 h with NO-1886 treatment. Medium samples for IGF1 determination were obtained and stored at −80 °C until measurement by a commercial RIA kit. Data were normalized by protein assay. For the animal experiment, plasma samples stored at −80 °C were used to detect the IGF1 and GH concentration by RIA kit; plasma glucose, LDL-C, HDL-C and total cholesterol were directly measured by commercial kits within 3 h after performance of sampling.

Determination of total hepatic cholesterol content

Quantification of tissue cholesterol was carried out using the method of cholesterol oxidase–peroxidase after ethyl acetate extraction. Briefly, samples of ∼1 g saponified for 1 h were extracted in triplicate with two volumes of ethyl acetate. After accurately taking 1/10 volume to evaporate ethyl acetate under vacuum, the residue was dissolved in 2 ml glacial acetic acid. The well-suspended solution was applied to determine cholesterol content by colorimetric assay with commercial kit.

Statistical analysis

Each experiment was performed in triplicate and representative data are shown. All of the other results were expressed as means±s.e.m. Values of P were calculated using ANOVA followed with a Scheffe's post hoc test, and values of P<0.05 were considered significant.

Results

NO-1886 increased CYP7A1 expression through activation of STAT5 in HepG2 cells

The CYP7A1 expression is restricted to the liver, and the HepG2 cells have been used as a model for studying the regulation of human CYP7A1 (Pandak et al. 1996). We investigated the dose response and time course of activation of CYP7A1 expression with NO-1886 treatment ranging from 0.01 to 3 μg/ml for 4 h in HepG2 cells. As shown in Fig. 1A, treatment of cells with NO-1886 improved CYP7A1 expression up to 0.3 μg/ml and then reached a plateau at 3 μg/ml. Figure 1B shows the time course of 0.3 μg/ml NO-1886 treatment on CYP7A1 expression in HepG2 cells. The increase in CYP7A1 expression started to be detected from 2nd hour reaching a maximum at 6th hour and gradually decreasing thereafter.

Figure 1
Figure 1

NO-1886 increased CYP7A1 expression through activation of STAT5 in HepG2 cells. After overnight serum starvation, HepG2 cells were left untreated or treated with: (A) NO-1886 under the dose ranging from 0.01 to 3 μg/ml for 4 h; (B and C) NO-1886 at 0.3 μg/ml for 2–24 h; (D) pretreatment with AG490 for 30 min, then cells were treated with 0.3 μg/ml NO-1886 for another 2 h. Cell lysates were subjected to SDS-PAGE and western blot by anti-CYP7A1 antibodies, anti-STAT5 (pTyr694/699) antibodies, anti-STAT5 antibodies and anti-β-actin antibodies. (E) Cells treated as in (D) were applied to determine CYP7A1 gene transcription by a real-time quantitative PCR described in Materials and Methods. (F) Cells electroporated with FLAG-tagged constitutive active STAT5 (pME-caSTAT5-FLAG) or control plasmid (control) were seeded into 6-well plates as described in Materials and Methods followed by the treatment and detection of CYP7A1, FLAG and β-actin as shown in figure by western blot. A representative image from three independent experiments is shown. Results from all the three experiments were also quantified, and the fold increase is shown. Data are means±s.e.m. *P<0.05 and P<0.01 versus control group; NS, no statistical significance.

Citation: Journal of Endocrinology 204, 1; 10.1677/JOE-09-0278

The expression of hepatic CYP7A1 is regulated by various factors (Lu et al. 2000). Among them, we were especially interested in STAT5, a member of the STAT family of transcriptional factors. In response to cytokines, STAT5 is phosphorylated at the site of tyr694/699 by the receptor-associated kinases, then dimerizes and translocates to the nucleus, where they act as transcription activator (Vidal et al. 2007). We hypothesized that NO-1886 might activate CYP7A1 expression through STAT5 activation. As shown in Fig. 1C, similar to CYP7A1 expression, STAT5 tyrosine phosphorylation was significantly upregulated with NO-1886 treatment from 2 to 6 h, and then recovered to the base line gradually. In order to prove whether the STAT5 was involved into the regulation of CYP7A1 by NO-1886, the specific JAK2/STAT inhibitor tyrphostin AG 490 was applied to block the activation of STAT5 (Meydan et al. 1996). Pretreatment of AG 490 completely abrogated NO-1886-induced transcription and expression of CYP7A1 and STAT5 phosphorylation (Fig. 1D and E). In constitutive active STAT5 transfected cells, however, AG 490 failed to inhibit CYP7A1 expression, suggesting the involvement of STAT5 on AG 490 inhibition of CYP7A1 (Fig. 1F).

NO-1886 increased IGF1 secretion in STAT5-dependent manner in HepG2 cells

It was noted that one of the classical targets downstream of STAT5 is IGF1 in GH signaling (Davey et al. 2001, Chia et al. 2006). STAT5 activation motivated us to investigate whether or not IGF1 secretion are also activated (Woelfle et al. 2003, Woelfle & Rotwein 2004).

For proving the hypothesis, we detected the alteration of IGF1 secretion after treating HepG2 cells with NO-1886. As shown in Fig. 2A, treatment of NO-1886 significantly elevated the concentration of secreted IGF1 which reached a peak at the 8th hour, at a time when STAT5 phosphorylation starting to elapse (Fig. 1C), implicating a further downstream event of STAT5 activation. Pretreatment of AG 490 (10 μg/ml), however, completely abolished IGF1 secretion (Fig. 2A). NO-1886 did not interfere with intracellular concentration of IGF1 (Fig. 2B). However, preventing STAT activation by AG 490 significantly upregulated its intracellular accumulation of IGF1 (Fig. 2B). This may probably be due to the lack of feedback inhibition of IGF1 by secreted IGF1, a condition which has been reported previously in myoblast (Frost et al. 2002). These data suggested that STAT5 activation by NO-1886 is responsible for the facilitation of IGF1 secretion, and there might exist other undiscovered mechanisms for IGF1 production.

Figure 2
Figure 2

NO-1886 increased IGF1 secretion in STAT5-dependent manner in HepG2 cells. After overnight serum starvation, under the pretreatment of vehicle of DMSO or AG 490 for 30 min (designated as the start time of ‘0’ shown in figure), HepG2 cells were left untreated or treated with 0.3 μg/ml NO-1886 for 2–24 h. At the end of each time, 1 ml medium were stored at −80 °C until RIA, after subtracting the contents of control group of vehicle, the data were regarded as the alteration of IGF1 secretion (A). Cells were washed with ice-cold PBS and total protein was extracted into RIPA buffer, samples were stored at −80 °C until RIA and protein assay, the control group subtracted data were designated as the changes in intracellular IGF1 (B) Results were controlled by total protein assay (n=3 samples per group were analyzed). Data are means±s.e.m. aP<0.05; bP<0.01 versus starting time point value of 0 (data were analyzed by Student t-test). cP<0.05; dP<0.01 versus the group of NO-1886+AG 490 on the independent time points.

Citation: Journal of Endocrinology 204, 1; 10.1677/JOE-09-0278

NO-1886 elevated plasma IGF1 in minipigs

As NO-1886 activated CYP7A1 expression and IGF1 secretion in HepG2 cells, it might provide beneficial effects on dyslipidemia and insulin resistance respectively. Thus, in order to create a physiological relevance to the role of NO-1886, the in vivo analyses were executed in minipigs, the optimum humanoid diabetes model animals (Xi et al. 2004, Bellinger et al. 2006).

We first checked the effects of single dose distribution of NO-1886 (1 g/kg) on plasma IGF1 and GH protein levels in minipigs. As Fig. 3A shows, plasma IGF1 levels were upregulated from the 2nd hour and then climbed to a peak at 4 h. Comparing with the data in HepG2 cells (Fig. 2), we found an earlier responsiveness of IGF1 to NO-1886 stimulation. This could be due to the different sensitivity between liver and hepatoma cell line and/or the contribution from other IGF1 secreting cell types, such as skeletal muscle cells (Frost et al. 2002). Further investigation found that the upregulation of IGF1 proceeded without similar modification in GH levels (Fig. 3B). Although GH is the main regulator of IGF1 secretion, treatment of NO-1886 induces an important activation of STAT5 and this phenomenon may solely be the mechanism by which IGF1 secretion is promoted rather than through the promotion of GH secretion. However, we could still detect the ‘predicted’ feedback inhibition by the elevation of IGF1 between 2 and 3 h in NO-1886 treatment group (Fig. 3B).

Figure 3
Figure 3

NO-1886 elevated plasma IGF1 in minipigs. After overnight fast, six male minipigs divided into two groups were orally administered with 1 g/kg BW NO-1886 (suspended in 5% Arabic gum). Plasma obtained at the end of each hour for 8 h were analyzed to determine plasma IGF1 (A) and GH (B) concentration by RIA. Data are means±s.e.m. *P<0.05 versus vehicle of Arabic gum control group.

Citation: Journal of Endocrinology 204, 1; 10.1677/JOE-09-0278

Long-term distribution of NO-1886 prevented HFSCD-induced hyperglycemia and insulin resistance by raising IGF1 levels

Treatment of NO-1886 enhanced IGF1 secretion in both the HepG2 cells and minipigs and promoted CYP7A1 expression in HepG2 cells. Therefore, whether NO-1886 prevents diabetogenic diet-induced insulin resistance and dyslipidemia was investigated. As shown in Fig. 4A, from the 3rd month, HFSCD decreased fasting plasma IGF1 concentration compared with CD group. However, long-term supplementation of NO-1886 promoted the elevation of fasting plasma IGF1 without interfering with fasting GH levels (Fig. 4B). At the same time, treatment of NO-1886 prevented the HFSCD-induced impairment of insulin sensitivity and recovered fasting insulin and glucose (Fig. 4C and D). In combination with previous in vivo studies on the role of IGF1 on insulin sensitivity, the present work suggests that improvement of IGF1 by NO-1886 prevents the progression of HFSCD-induced hyperglycemia and insulin desensitization.

Figure 4
Figure 4

Long-term distribution of NO-1886 prevented HFSCD-induced hyperglycemia and insulin resistance by raising IGF1 levels. Fifteen male minipigs were randomly divided into three groups (CD, HFSCD, and HFSCD1886) and treated as described in the Materials and Methods section. Blood samples for plasma IGF1 (A), GH (B), glucose (C), and insulin (D) quantification were obtained at the end of each month after overnight fasting. Data are means±s.e.m. *P<0.05 versus CD group and #P<0.05 versus HFSCD group.

Citation: Journal of Endocrinology 204, 1; 10.1677/JOE-09-0278

NO-1886 increased plasma HDL-C and decreased HFSCD-induced elevation of LDL-C, hepatic accumulation of cholesterol through upregulation of CYP7A1 in minipigs

Although enhancement of HDL-C and RCT from peripheral tissues has been reported previously as mechanisms for preventing diabetic angiopathy by NO-1886, the excessive load of cholesterol is toxic to hepatocytes and cells need to catabolize cholesterol for clearance (Zhang et al. 2006). Transgenic studies found that overexpression of CYP7A1 increases hepatic LDL receptor expression and decreased plasma LDL-C (Spady et al. 1995, Pandak et al. 2001). Therefore, for providing the evidence of NO-1886 on cholesterol metabolism, animals treated for 5 months were used to measure LDL-C, HDL-C and total cholesterol in plasma, and the CYP7A1 expression and total cholesterol content in liver. As shown in Fig. 5A, treatment of NO-1886 suppressed HFSCD-induced elevation of LDL-C to about 44% on average. Owing to the elevation of HDL-C (Fig. 5B), total plasma cholesterol concentration was not apparently interfered by NO-1886 (Fig. 5C). Animals treated for 5 months were killed, and the liver tissues were used for CYP7A1 detection and cholesterol quantification. As shown in Fig. 5D and E, HFSCD (cholesterol) failed to induce hepatic CYP7A1 expression in minipigs, leading to a severe accumulation of cholesterol. Treatment with NO-1886, however, significantly enhanced the protein levels of CYP7A1 and cholesterol clearance in minipigs (Fig. 5D and E).

Figure 5
Figure 5

NO-1886 increased plasma HDL-C and decreased HFSCD-induced elevation of LDL-C, hepatic accumulation of cholesterol through upregulation of CYP7A1 in minipigs. Blood samples taken from 15 male minipigs, treated as described in Materials and Methods section, were used to determine their LDL-C (A), HDL-C (B), and total cholesterol (C) concentration by using commercial kits. At the end of treatment for 5 months, animals were killed, and liver tissues were dissected and stocked in liquid nitrogen until the detection of CYP7A1 expression (D) and hepatic cholesterol content (E). Data are means±s.e.m. *P<0.05, P<0.01 versus vehicle of CD group; #P<0.05 versus HFSCD group; NS, no statistical significance.

Citation: Journal of Endocrinology 204, 1; 10.1677/JOE-09-0278

Discussion

In humans, cholesterol homeostasis is maintained by a delicate balance between cholesterol input and output pathways. The development of new mechanisms of action for the metabolic dyslipidemia has focused attention on the cholesterol catabolism (Repa & Mangelsdorf 2000). In liver, plasma HDL-C is transported into hepatocytes through scavenger receptor class B type I in the plasma membrane by a way of passive diffusion (Trigatti et al. 2000). Thus, to keep the concentration gradient of cholesterol, the hepatocytes need to catabolize cholesterol for its depletion, a process which is controlled by CYP7A1 (Myant & Mitropoulos 1977, Javitt 1994, Chiang 1998, Lu et al. 2000). In the present study, compared with CD group, HFSCD maximally induced an elevation of HDL-C by ∼2.3 folds in plasma and ∼6 folds of cholesterol in liver; whereas supplementation of NO-1886 elevates plasma HDL-C by 3.7 folds and only ∼2.5 folds of hepatic cholesterol (Fig. 5). Inferentially, according to Fick's law of passive diffusion that the flux rate from high concentration to low concentration is proportional to the concentration gradient, treatment of NO-1886 consequently upregluates cholesterol influx rate by:

article image
Activation of CYP7A1 by NO-1886 may efficiently facilitate RCT.

As reported previously, GH enhances CPY7A1 activity and bile acids synthesis to a powerful magnitude by upregulating CYP7A1 expression. Although specific mechanism is still unclear, the role of STAT5 and peroxisome proliferators-activated receptor α (PPARA) might be implicated, activation of STAT5 negatively regulates the suppressive effects of PPARα on CYP7A1 (Zhou & Waxman 1999, Patel et al. 2000). Similar to GH, NO-1886 improved hepatic cholesterol clearance by raising CYP7A1 expression. Recently, NO-1886-induced LPL activation has been shown to rectify diet-induced hepatic steatosis in mice at the same dose used in the present study (Yu et al. 2007). This study also provided the evidence that NO-1886 increased PPARA expression. Thus, further experiments need to be conducted to investigate the role of PPARA on NO-1886-induced CYP7A1 activation.

Insulin resistance, characterized by reduced responsiveness to normal concentration of insulin, is a common feature of many of the patients with T2DM and metabolic syndrome (DeFronzo & Ferrannini 1991, Eckel et al. 2005). Low levels of circulating IGF1 are a risk factor for the desensitization of insulin action (Massa et al. 1993, Yakar et al. 2001). Although GH is important for IGF1 secretion, hypersecretion of GH is related with GH signal desensitization and insulin desensitization (Moller & Flier 1991, Clark et al. 1997, Takano et al. 2001, Miquet et al. 2004). Our present investigation discovered that NO-1886 upregulated hepatic IGF1 secretion without interfering with plasma GH, resulting in the improvement of diet-induced insulin resistance.

NO-1886 has been known as an activator of LPL which increased LPL mRNA and LPL activity (maximum at 3 μg/ml) (Tsutsumi et al. 1993). However, for the activation of CYP7A1 expression, the effective concentration was observed to be at 0.3 μg/ml. These findings imply that, unrelated with the mechanism of LPL activation, there may exist some other activities of the drug responsible for the current observation. On the other hand, although NO-1886 has been proved to prevent diabetic dyslipidemia and atherosclerosis in animal experiments (Yin et al. 2003, 2004a,b, Niho et al. 2005, Zhang et al. 2006), the (patho)physiological role of LPL itself is still under controversy. For example, highly expressed LPL in endothelial cells is regarded as a risk factor for angiopathy and atherosclerosis (Goldberg 1996), which highlights the potential role of NO-1886 as a STAT5 activator to upregulate CYP7A1 and IGF1. This study is consistent with the previous observations regarding the beneficial outcomes of CYP7A1 and IGF1 upregulation.

In summary, NO-1886 promoted hepatic CYP7A1 and IGF1 expression via activation of STAT5. This study discovered a new therapeutic target of NO-1886 on diet-induced insulin resistance and dyslipidemia.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The research was supported in part from Project 973 of China G2000056905 and the National Natural Sciences Foundation of China (project 30370675 and 30470720).

Acknowledgements

We thank Dr Toshio Kitamura at Tokyo University for providing the constitutive active mutant of caSTAT5A1*6 plasmid.

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    • Search Google Scholar
    • Export Citation
  • Clark RG, Thomas GB, Mortensen DL, Won WB, Ma YH, Tomlinson EE, Fairhall KM & Robinson IC 1997 Growth hormone secretagogues stimulate the hypothalamic–pituitary–adrenal axis and are diabetogenic in the Zucker diabetic fatty rat. Endocrinology 138 43164323.

    • Search Google Scholar
    • Export Citation
  • Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ & Grattan DR 2001 STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology 142 38363841.

    • Search Google Scholar
    • Export Citation
  • DeFronzo RA & Ferrannini E 1991 Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14 173194.

    • Search Google Scholar
    • Export Citation
  • Eckel RH, Grundy SM & Zimmet PZ 2005 The metabolic syndrome. Lancet 365 14151428.

  • Fielding CJ & Fielding PE 1995 Molecular physiology of reverse cholesterol transport. Journal of Lipid Research 36 211228.

  • Froesch ER, Hussain MA, Schmid C & Zapf J 1996 Insulin-like growth factor I: physiology, metabolic effects and clinical uses. Diabetes/Metabolism Reviews 12 195215.

    • Search Google Scholar
    • Export Citation
  • Frost RA, Nystrom GJ & Lang CH 2002 Regulation of IGF-I mRNA and signal transducers and activators of transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 myoblasts. Endocrinology 143 492503.

    • Search Google Scholar
    • Export Citation
  • Goldberg IJ 1996 Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. Journal of Lipid Research 37 693707.

    • Search Google Scholar
    • Export Citation
  • Goodwin B, Watson MA, Kim H, Miao J, Kemper JK & Kliewer SA 2003 Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Molecular Endocrinology 17 386394.

    • Search Google Scholar
    • Export Citation
  • Horton JD, Cuthbert JA & Spady DK 1995 Regulation of hepatic 7 alpha-hydroxylase expression and response to dietary cholesterol in the rat and hamster. Journal of Biological Chemistry 270 53815387.

    • Search Google Scholar
    • Export Citation
  • Javitt NB 1994 Bile acid synthesis from cholesterol: regulatory and auxiliary pathways. FASEB Journal 8 13081311.

  • Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A & Anversa P 2001 IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 50 14141424.

    • Search Google Scholar
    • Export Citation
  • Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680685.

  • Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J & Mangelsdorf DJ 2000 Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Molecular Cell 6 507515.

    • Search Google Scholar
    • Export Citation
  • Massa G, Dooms L, Bouillon R & Vanderschueren-Lodeweyckx M 1993 Serum levels of growth hormone-binding protein and insulin-like growth factor I in children and adolescents with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 36 239243.

    • Search Google Scholar
    • Export Citation
  • Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A & Gazit A 1996 Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379 645648.

    • Search Google Scholar
    • Export Citation
  • Miquet JG, Sotelo AI, Bartke A & Turyn D 2004 Suppression of growth hormone (GH) Janus tyrosine kinase 2/signal transducer and activator of transcription 5 signaling pathway in transgenic mice overexpressing bovine GH. Endocrinology 145 28242832.

    • Search Google Scholar
    • Export Citation
  • Moller DE & Flier JS 1991 Insulin resistance – mechanisms, syndromes, and implications. New England Journal of Medicine 325 938948.

  • Moller DE & Kaufman KD 2005 Metabolic syndrome: a clinical and molecular perspective. Annual Review of Medicine 56 4562.

  • Myant NB & Mitropoulos KA 1977 Cholesterol 7 alpha-hydroxylase. Journal of Lipid Research 18 135153.

  • Niho N, Mutoh M, Takahashi M, Tsutsumi K, Sugimura T & Wakabayashi K 2005 Concurrent suppression of hyperlipidemia and intestinal polyp formation by NO-1886, increasing lipoprotein lipase activity in Min mice. PNAS 102 29702974.

    • Search Google Scholar
    • Export Citation
  • Onishi M, Nosaka T, Misawa K, Mui AL, Gorman D, McMahon M, Miyajima A & Kitamura T 1998 Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. Molecular and Cellular Biology 18 38713879.

    • Search Google Scholar
    • Export Citation
  • Osada R, Ohshima H, Ishihara H, Yudoh K, Sakai K, Matsui H & Tsuji H 1996 Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. Journal of Orthopaedic Research 14 690699.

    • Search Google Scholar
    • Export Citation
  • Pandak WM, Stravitz RT, Lucas V, Heuman DM & Chiang JY 1996 Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level. American Journal of Physiology 270 G401G410.

    • Search Google Scholar
    • Export Citation
  • Pandak WM, Schwarz C, Hylemon PB, Mallonee D, Valerie K, Heuman DM, Fisher RA, Redford K & Vlahcevic ZR 2001 Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis. American Journal of Physiology. Gastrointestinal and Liver Physiology 281 G878G889.

    • Search Google Scholar
    • Export Citation
  • Patel DD, Knight BL, Soutar AK, Gibbons GF & Wade DP 2000 The effect of peroxisome-proliferator-activated receptor-alpha on the activity of the cholesterol 7 alpha-hydroxylase gene. Biochemical Journal 351 747753.

    • Search Google Scholar
    • Export Citation
  • Ratliff EP, Gutierrez A & Davis RA 2006 Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia. Journal of Lipid Research 47 15131520.

    • Search Google Scholar
    • Export Citation
  • Repa JJ & Mangelsdorf DJ 2000 The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annual Review of Cell and Developmental Biology 16 459481.

    • Search Google Scholar
    • Export Citation
  • Spady DK, Cuthbert JA, Willard MN & Meidell RS 1995 Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol. Journal of Clinical Investigation 96 700709.

    • Search Google Scholar
    • Export Citation
  • Takano A, Usui I, Haruta T, Kawahara J, Uno T, Iwata M & Kobayashi M 2001 Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Molecular and Cellular Biology 21 50505062.

    • Search Google Scholar
    • Export Citation
  • Trigatti B, Rigotti A & Krieger M 2000 The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Current Opinion in Lipidology 11 123131.

    • Search Google Scholar
    • Export Citation
  • Tsutsumi K, Inoue Y, Shima A, Iwasaki K, Kawamura M & Murase T 1993 The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis. Journal of Clinical Investigation 92 411417.

    • Search Google Scholar
    • Export Citation
  • Tsutsumi K, Inoue Y, Hagi A & Murase T 1997 The novel compound NO-1886 elevates plasma high-density lipoprotein cholesterol levels in hamsters and rabbits by increasing lipoprotein lipase without any effect on cholesteryl ester transfer protein activity. Metabolism 46 257260.

    • Search Google Scholar
    • Export Citation
  • Turley SD & Dietschy JM 1988 The Liver: Biology and Pathobiology, New York: Raven Press.

  • Vidal OM, Merino R, Rico-Bautista E, Fernandez-Perez L, Chia DJ, Woelfle J, Ono M, Lenhard B, Norstedt G & Rotwein P 2007 In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver. Molecular Endocrinology 21 293311.

    • Search Google Scholar
    • Export Citation
  • Woelfle J & Rotwein P 2004 In vivo regulation of growth hormone-stimulated gene transcription by STAT5b. American Journal of Physiology. Endocrinology and Metabolism 286 E393E401.

    • Search Google Scholar
    • Export Citation
  • Woelfle J, Billiard J & Rotwein P 2003 Acute control of insulin-like growth factor-I gene transcription by growth hormone through Stat5b. Journal of Biological Chemistry 278 2269622702.

    • Search Google Scholar
    • Export Citation
  • Xi S, Yin W, Wang Z, Kusunoki M, Lian X, Koike T, Fan J & Zhang Q 2004 A minipig model of high-fat/high-sucrose diet-induced diabetes and atherosclerosis. International Journal of Experimental Pathology 85 223231.

    • Search Google Scholar
    • Export Citation
  • Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD, Mejia W & Le Roith D 2001 Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50 11101118.

    • Search Google Scholar
    • Export Citation
  • Yin W, Yuan Z, Tsutsumi K, Xie Y, Zhang Q, Wang Z, Fu G, Long G & Yang Y 2003 A lipoprotein lipase-promoting agent, NO-1886, improves glucose and lipid metabolism in high fat, high sucrose-fed New Zealand white rabbits. International Journal of Experimental Diabetes Research 4 2734.

    • Search Google Scholar
    • Export Citation
  • Yin W, Liao D, Kusunoki M, Xi S, Tsutsumi K, Wang Z, Lian X, Koike T, Fan J & Yang Y 2004a NO-1886 decreases ectopic lipid deposition and protects pancreatic beta cells in diet-induced diabetic swine. Journal of Endocrinology 180 399408.

    • Search Google Scholar
    • Export Citation
  • Yin W, Liao D, Wang Z, Xi S, Tsutsumi K, Koike T, Fan J, Yi G, Zhang Q & Yuan Z 2004b NO-1886 inhibits size of adipocytes, suppresses plasma levels of tumor necrosis factor-alpha and free fatty acids, improves glucose metabolism in high-fat/high-sucrose-fed miniature pigs. Pharmacological Research 49 199206.

    • Search Google Scholar
    • Export Citation
  • Yu J, Chu ES, Hui AY, Cheung KF, Chan HL, Leung WK, Farrell GC & Sung JJ 2007 Lipoprotein lipase activator ameliorates the severity of dietary steatohepatitis. Biochemical and Biophysical Research Communications 356 5359.

    • Search Google Scholar
    • Export Citation
  • Zhang C, Yin W, Liao D, Huang L, Tang C, Tsutsumi K, Wang Z, Liu Y, Li Q & Hou H 2006 NO-1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs. Journal of Lipid Research 47 20552063.

    • Search Google Scholar
    • Export Citation
  • Zhou YC & Waxman DJ 1999 STAT5b down-regulates peroxisome proliferator-activated receptor alpha transcription by inhibition of ligand-independent activation function region-1 trans-activation domain. Journal of Biological Chemistry 274 2987429882.

    • Search Google Scholar
    • Export Citation

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      Society for Endocrinology

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    NO-1886 increased CYP7A1 expression through activation of STAT5 in HepG2 cells. After overnight serum starvation, HepG2 cells were left untreated or treated with: (A) NO-1886 under the dose ranging from 0.01 to 3 μg/ml for 4 h; (B and C) NO-1886 at 0.3 μg/ml for 2–24 h; (D) pretreatment with AG490 for 30 min, then cells were treated with 0.3 μg/ml NO-1886 for another 2 h. Cell lysates were subjected to SDS-PAGE and western blot by anti-CYP7A1 antibodies, anti-STAT5 (pTyr694/699) antibodies, anti-STAT5 antibodies and anti-β-actin antibodies. (E) Cells treated as in (D) were applied to determine CYP7A1 gene transcription by a real-time quantitative PCR described in Materials and Methods. (F) Cells electroporated with FLAG-tagged constitutive active STAT5 (pME-caSTAT5-FLAG) or control plasmid (control) were seeded into 6-well plates as described in Materials and Methods followed by the treatment and detection of CYP7A1, FLAG and β-actin as shown in figure by western blot. A representative image from three independent experiments is shown. Results from all the three experiments were also quantified, and the fold increase is shown. Data are means±s.e.m. *P<0.05 and P<0.01 versus control group; NS, no statistical significance.

  • View in gallery

    NO-1886 increased IGF1 secretion in STAT5-dependent manner in HepG2 cells. After overnight serum starvation, under the pretreatment of vehicle of DMSO or AG 490 for 30 min (designated as the start time of ‘0’ shown in figure), HepG2 cells were left untreated or treated with 0.3 μg/ml NO-1886 for 2–24 h. At the end of each time, 1 ml medium were stored at −80 °C until RIA, after subtracting the contents of control group of vehicle, the data were regarded as the alteration of IGF1 secretion (A). Cells were washed with ice-cold PBS and total protein was extracted into RIPA buffer, samples were stored at −80 °C until RIA and protein assay, the control group subtracted data were designated as the changes in intracellular IGF1 (B) Results were controlled by total protein assay (n=3 samples per group were analyzed). Data are means±s.e.m. aP<0.05; bP<0.01 versus starting time point value of 0 (data were analyzed by Student t-test). cP<0.05; dP<0.01 versus the group of NO-1886+AG 490 on the independent time points.

  • View in gallery

    NO-1886 elevated plasma IGF1 in minipigs. After overnight fast, six male minipigs divided into two groups were orally administered with 1 g/kg BW NO-1886 (suspended in 5% Arabic gum). Plasma obtained at the end of each hour for 8 h were analyzed to determine plasma IGF1 (A) and GH (B) concentration by RIA. Data are means±s.e.m. *P<0.05 versus vehicle of Arabic gum control group.

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    Long-term distribution of NO-1886 prevented HFSCD-induced hyperglycemia and insulin resistance by raising IGF1 levels. Fifteen male minipigs were randomly divided into three groups (CD, HFSCD, and HFSCD1886) and treated as described in the Materials and Methods section. Blood samples for plasma IGF1 (A), GH (B), glucose (C), and insulin (D) quantification were obtained at the end of each month after overnight fasting. Data are means±s.e.m. *P<0.05 versus CD group and #P<0.05 versus HFSCD group.

  • View in gallery

    NO-1886 increased plasma HDL-C and decreased HFSCD-induced elevation of LDL-C, hepatic accumulation of cholesterol through upregulation of CYP7A1 in minipigs. Blood samples taken from 15 male minipigs, treated as described in Materials and Methods section, were used to determine their LDL-C (A), HDL-C (B), and total cholesterol (C) concentration by using commercial kits. At the end of treatment for 5 months, animals were killed, and liver tissues were dissected and stocked in liquid nitrogen until the detection of CYP7A1 expression (D) and hepatic cholesterol content (E). Data are means±s.e.m. *P<0.05, P<0.01 versus vehicle of CD group; #P<0.05 versus HFSCD group; NS, no statistical significance.

  • Bellinger DA, Merricks EP & Nichols TC 2006 Swine models of type 2 diabetes mellitus: insulin resistance, glucose tolerance, and cardiovascular complications. ILAR Journal/National Research Council, Institute of Laboratory Animal Resources 47 243258.

    • Search Google Scholar
    • Export Citation
  • Brown MS & Goldstein JL 1986 A receptor-mediated pathway for cholesterol homeostasis. Science 232 3447.

  • Brownlee M 2005 The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54 16151625.

  • Chawla A, Saez E & Evans RM 2000 Don't know much bile-ology. Cell 103 14.

  • Chia DJ, Ono M, Woelfle J, Schlesinger-Massart M, Jiang H & Rotwein P 2006 Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription. Journal of Biological Chemistry 281 31903197.

    • Search Google Scholar
    • Export Citation
  • Chiang JY 1998 Regulation of bile acid synthesis. Frontiers in Bioscience 3 d176d193.

  • Chiang JY, Kimmel R & Stroup D 2001 Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha). Gene 262 257265.

    • Search Google Scholar
    • Export Citation
  • Clark RG, Thomas GB, Mortensen DL, Won WB, Ma YH, Tomlinson EE, Fairhall KM & Robinson IC 1997 Growth hormone secretagogues stimulate the hypothalamic–pituitary–adrenal axis and are diabetogenic in the Zucker diabetic fatty rat. Endocrinology 138 43164323.

    • Search Google Scholar
    • Export Citation
  • Davey HW, Xie T, McLachlan MJ, Wilkins RJ, Waxman DJ & Grattan DR 2001 STAT5b is required for GH-induced liver IGF-I gene expression. Endocrinology 142 38363841.

    • Search Google Scholar
    • Export Citation
  • DeFronzo RA & Ferrannini E 1991 Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14 173194.

    • Search Google Scholar
    • Export Citation
  • Eckel RH, Grundy SM & Zimmet PZ 2005 The metabolic syndrome. Lancet 365 14151428.

  • Fielding CJ & Fielding PE 1995 Molecular physiology of reverse cholesterol transport. Journal of Lipid Research 36 211228.

  • Froesch ER, Hussain MA, Schmid C & Zapf J 1996 Insulin-like growth factor I: physiology, metabolic effects and clinical uses. Diabetes/Metabolism Reviews 12 195215.

    • Search Google Scholar
    • Export Citation
  • Frost RA, Nystrom GJ & Lang CH 2002 Regulation of IGF-I mRNA and signal transducers and activators of transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 myoblasts. Endocrinology 143 492503.

    • Search Google Scholar
    • Export Citation
  • Goldberg IJ 1996 Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. Journal of Lipid Research 37 693707.

    • Search Google Scholar
    • Export Citation
  • Goodwin B, Watson MA, Kim H, Miao J, Kemper JK & Kliewer SA 2003 Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Molecular Endocrinology 17 386394.

    • Search Google Scholar
    • Export Citation
  • Horton JD, Cuthbert JA & Spady DK 1995 Regulation of hepatic 7 alpha-hydroxylase expression and response to dietary cholesterol in the rat and hamster. Journal of Biological Chemistry 270 53815387.

    • Search Google Scholar
    • Export Citation
  • Javitt NB 1994 Bile acid synthesis from cholesterol: regulatory and auxiliary pathways. FASEB Journal 8 13081311.

  • Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A & Anversa P 2001 IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 50 14141424.

    • Search Google Scholar
    • Export Citation
  • Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680685.

  • Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J & Mangelsdorf DJ 2000 Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Molecular Cell 6 507515.

    • Search Google Scholar
    • Export Citation
  • Massa G, Dooms L, Bouillon R & Vanderschueren-Lodeweyckx M 1993 Serum levels of growth hormone-binding protein and insulin-like growth factor I in children and adolescents with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 36 239243.

    • Search Google Scholar
    • Export Citation
  • Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A & Gazit A 1996 Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379 645648.

    • Search Google Scholar
    • Export Citation
  • Miquet JG, Sotelo AI, Bartke A & Turyn D 2004 Suppression of growth hormone (GH) Janus tyrosine kinase 2/signal transducer and activator of transcription 5 signaling pathway in transgenic mice overexpressing bovine GH. Endocrinology 145 28242832.

    • Search Google Scholar
    • Export Citation
  • Moller DE & Flier JS 1991 Insulin resistance – mechanisms, syndromes, and implications. New England Journal of Medicine 325 938948.

  • Moller DE & Kaufman KD 2005 Metabolic syndrome: a clinical and molecular perspective. Annual Review of Medicine 56 4562.

  • Myant NB & Mitropoulos KA 1977 Cholesterol 7 alpha-hydroxylase. Journal of Lipid Research 18 135153.

  • Niho N, Mutoh M, Takahashi M, Tsutsumi K, Sugimura T & Wakabayashi K 2005 Concurrent suppression of hyperlipidemia and intestinal polyp formation by NO-1886, increasing lipoprotein lipase activity in Min mice. PNAS 102 29702974.

    • Search Google Scholar
    • Export Citation
  • Onishi M, Nosaka T, Misawa K, Mui AL, Gorman D, McMahon M, Miyajima A & Kitamura T 1998 Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. Molecular and Cellular Biology 18 38713879.

    • Search Google Scholar
    • Export Citation
  • Osada R, Ohshima H, Ishihara H, Yudoh K, Sakai K, Matsui H & Tsuji H 1996 Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs. Journal of Orthopaedic Research 14 690699.

    • Search Google Scholar
    • Export Citation
  • Pandak WM, Stravitz RT, Lucas V, Heuman DM & Chiang JY 1996 Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level. American Journal of Physiology 270 G401G410.

    • Search Google Scholar
    • Export Citation
  • Pandak WM, Schwarz C, Hylemon PB, Mallonee D, Valerie K, Heuman DM, Fisher RA, Redford K & Vlahcevic ZR 2001 Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis. American Journal of Physiology. Gastrointestinal and Liver Physiology 281 G878G889.

    • Search Google Scholar
    • Export Citation
  • Patel DD, Knight BL, Soutar AK, Gibbons GF & Wade DP 2000 The effect of peroxisome-proliferator-activated receptor-alpha on the activity of the cholesterol 7 alpha-hydroxylase gene. Biochemical Journal 351 747753.

    • Search Google Scholar
    • Export Citation
  • Ratliff EP, Gutierrez A & Davis RA 2006 Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia. Journal of Lipid Research 47 15131520.

    • Search Google Scholar
    • Export Citation
  • Repa JJ & Mangelsdorf DJ 2000 The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annual Review of Cell and Developmental Biology 16 459481.

    • Search Google Scholar
    • Export Citation
  • Spady DK, Cuthbert JA, Willard MN & Meidell RS 1995 Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol. Journal of Clinical Investigation 96 700709.

    • Search Google Scholar
    • Export Citation
  • Takano A, Usui I, Haruta T, Kawahara J, Uno T, Iwata M & Kobayashi M 2001 Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Molecular and Cellular Biology 21 50505062.

    • Search Google Scholar
    • Export Citation
  • Trigatti B, Rigotti A & Krieger M 2000 The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Current Opinion in Lipidology 11 123131.

    • Search Google Scholar
    • Export Citation
  • Tsutsumi K, Inoue Y, Shima A, Iwasaki K, Kawamura M & Murase T 1993 The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis. Journal of Clinical Investigation 92 411417.

    • Search Google Scholar
    • Export Citation
  • Tsutsumi K, Inoue Y, Hagi A & Murase T 1997 The novel compound NO-1886 elevates plasma high-density lipoprotein cholesterol levels in hamsters and rabbits by increasing lipoprotein lipase without any effect on cholesteryl ester transfer protein activity. Metabolism 46 257260.

    • Search Google Scholar
    • Export Citation
  • Turley SD & Dietschy JM 1988 The Liver: Biology and Pathobiology, New York: Raven Press.

  • Vidal OM, Merino R, Rico-Bautista E, Fernandez-Perez L, Chia DJ, Woelfle J, Ono M, Lenhard B, Norstedt G & Rotwein P 2007 In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver. Molecular Endocrinology 21 293311.

    • Search Google Scholar
    • Export Citation
  • Woelfle J & Rotwein P 2004 In vivo regulation of growth hormone-stimulated gene transcription by STAT5b. American Journal of Physiology. Endocrinology and Metabolism 286 E393E401.

    • Search Google Scholar
    • Export Citation
  • Woelfle J, Billiard J & Rotwein P 2003 Acute control of insulin-like growth factor-I gene transcription by growth hormone through Stat5b. Journal of Biological Chemistry 278 2269622702.

    • Search Google Scholar
    • Export Citation
  • Xi S, Yin W, Wang Z, Kusunoki M, Lian X, Koike T, Fan J & Zhang Q 2004 A minipig model of high-fat/high-sucrose diet-induced diabetes and atherosclerosis. International Journal of Experimental Pathology 85 223231.

    • Search Google Scholar
    • Export Citation
  • Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD, Mejia W & Le Roith D 2001 Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50 11101118.

    • Search Google Scholar
    • Export Citation
  • Yin W, Yuan Z, Tsutsumi K, Xie Y, Zhang Q, Wang Z, Fu G, Long G & Yang Y 2003 A lipoprotein lipase-promoting agent, NO-1886, improves glucose and lipid metabolism in high fat, high sucrose-fed New Zealand white rabbits. International Journal of Experimental Diabetes Research 4 2734.

    • Search Google Scholar
    • Export Citation
  • Yin W, Liao D, Kusunoki M, Xi S, Tsutsumi K, Wang Z, Lian X, Koike T, Fan J & Yang Y 2004a NO-1886 decreases ectopic lipid deposition and protects pancreatic beta cells in diet-induced diabetic swine. Journal of Endocrinology 180 399408.

    • Search Google Scholar
    • Export Citation
  • Yin W, Liao D, Wang Z, Xi S, Tsutsumi K, Koike T, Fan J, Yi G, Zhang Q & Yuan Z 2004b NO-1886 inhibits size of adipocytes, suppresses plasma levels of tumor necrosis factor-alpha and free fatty acids, improves glucose metabolism in high-fat/high-sucrose-fed miniature pigs. Pharmacological Research 49 199206.

    • Search Google Scholar
    • Export Citation
  • Yu J, Chu ES, Hui AY, Cheung KF, Chan HL, Leung WK, Farrell GC & Sung JJ 2007 Lipoprotein lipase activator ameliorates the severity of dietary steatohepatitis. Biochemical and Biophysical Research Communications 356 5359.

    • Search Google Scholar
    • Export Citation
  • Zhang C, Yin W, Liao D, Huang L, Tang C, Tsutsumi K, Wang Z, Liu Y, Li Q & Hou H 2006 NO-1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs. Journal of Lipid Research 47 20552063.

    • Search Google Scholar
    • Export Citation
  • Zhou YC & Waxman DJ 1999 STAT5b down-regulates peroxisome proliferator-activated receptor alpha transcription by inhibition of ligand-independent activation function region-1 trans-activation domain. Journal of Biological Chemistry 274 2987429882.

    • Search Google Scholar
    • Export Citation