Glycosaminoglycan from Apostichopus japonicus Improves Glucose Metabolism in the Liver of Insulin Resistant Mice

Holothurian glycosaminoglycan isolated from Apostichopus japonicus (named AHG) can suppress hepatic glucose production in insulin resistant hepatocytes, but its effects on glucose metabolism in vivo are unknown. The present study was conducted to investigate the effects of AHG on hyperglycemia in the liver of insulin resistant mice induced by a high-fat diet (HFD) for 12 weeks. The results demonstrated that AHG supplementation apparently reduced body weight, blood glucose level, and serum insulin content in a dose-dependent manner in HFD-fed mice. The protein levels and gene expression of gluconeogenesis rate-limiting enzymes G6Pase and PEPCK were remarkedly suppressed in the insulin resistant liver. In addition, although the total expression of IRS1, Akt, and AMPK in the insulin resistant liver was not affected by AHG supplementation, the phosphorylation of IRS1, Akt, and AMPK were clearly elevated by AHG treatment. These results suggest that AHG could be a promising natural marine product for the development of an antihyperglycemic agent.


Introduction
Type 2 diabetes mellitus(T2DM) is characterized by long-term persistent hyperglycemia with several complications such as eye injury, renal failure, cardiovascular disease, and nervous system damage [1]. According to a report from the International Diabetes Federation (IDF) published in 2017, the number of people with diabetes has risen to 425 million in the world (2017, IDF, http://diabetesatlas.org) and T2DM makes up about 90% of cases of diabetes [2]. Thus, T2DM has become a chronic disease globally. Insulin resistance is the hallmark of T2DM and describes a condition whereby the ability of insulin to trigger glucose uptake, metabolism, or storage is impaired [3,4]. As the major site of glucose utilization during the post-prandial period and the main human tissue of glucose synthetization, the liver is the vital target organ of insulin resistance, and liver insulin resistance is the main reason for the development of T2DM [5,6]. When insulin resistance develops in the liver, increased hepatic glucose production and decreased glucose utilization contribute to the elevated level of blood glucose [7]. Therefore, an increasing number of studies are examining the liver, and specifically the inappropriate glucose metabolism in insulin resistant individuals.

AHG Decreased Body Weight in Mice Fed with HFD
As shown in Figure 2A, after 12 weeks of HFD treatment, mice exerted two-fold increase in body weight gain compared to mice of LFD group. AHG (20 mg/kg/day) supplementation showed no significant change in body weight gain compared to HFD group, however, treatment of AHG (50 mg/kg/day) and AHG (100 mg/kg/day) alleviated the enhancement by showing 12.1% and 18.9% inhibition in the body weight gain of the HFD group, indicating that AHG affected an HFD-induced insulin resistant model. As a positive control, the body weight gain of mice fed metformin was 24% lower than that of mice fed HFD. To further detect whether the decreasing body weight was caused by the change of energy intake, the food intake was measured. The results showed ( Figure 2B) that food intake was significantly lower in the HFD group compared to the LFD group. However, AHG supplementation showed no effect on food intake in HFD treatment. Metformin showed a markedly suppression on food intake compared to LFD group, which was consistently in agreement with the literature [25]. Effects of AHG supplementation on body weight in insulin resistant mice induced with HFD. C57BL/6J mice were fed with HFD for 12 weeks and treated with low, medium and high doses (20, 50, and 100 mg/kg/day, respectively) of AHG for eight weeks. (A) Body weight gain; (B) food intake per week. Data are showed as mean ± SD, n = 8; *p < 0.5, **p < 0.1, ***p < 0.01 vs. LFD group, # p < 0.5, ## p < 0.1, ### p < 0.01 vs. HFD group.

AHG Improved Glucose Metabolism in Mice Fed with HFD
As shown in Figure 3A, compared to LFD group, an obvious increase in fasting blood glucose levels was observed in HFD group (p < 0.01), however, the increasement was attenuated by AHG in a dose-dependent manner. Also, fasting plasma glucose in H-AHG group was similar to Metformin group, indicating that AHG supplementation (100 mg/kg/day) had a similar hypoglycemic effect as metformin. HFD impaired glucose tolerance sharply, which was attenuated by the supplementation of AHG in a concentration-dependent pattern ( Figure 3C-D). Consistent with this result, insulin injection failed to decline blood glucose in HFD mice, whereas blood glucose decreased normally in H-AHG mice compared to HFD mice, which was reflected in the area under the curve for ITT ( Figure 3E-F). Moreover, no significant difference of blood glucose level was found between H-AHG group and Metformin group in OGTT (p = 0.48) and ITT (p = 0.25) ( Figure 3C-E). The consumption of the HFD mice also caused high level of serum insulin, which was increased four-fold when compared with the basal level of insulin content in LFD mice ( Figure 3B). However, this effect was abolished in the H-AHG group. There was no notable difference in the serum insulin content between the H-AHG and Metformin groups. Overall results confirmed that the treatment of AHG improved insulin resistance induced by HFD in C57BL/6J mice. Data are showed as mean ± SD, n = 8; *p < 0.5, **p < 0.1, ***p < 0.01 vs. LFD group, # p < 0.5, ## p < 0.1, ### p < 0.01 vs. HFD group.

AHG alleviated liver injury in mice fed with HFD
Considering that the liver is the main target tissue of insulin resistance, we next explored whether AHG affected liver tissue in HFD-induced insulin resistance mice. The liver tissue weight, ALT level and AST level were measured. As shown in Figure 4A, a high dose of AHG significantly decreased the liver/body weight ratio in HFD mice (p < 0.001). Additionally, the value of ALT and AST showed the similar trend, indicating that AHG alleviated liver injury caused by HFD ( Figure 4B,C). Gene expression analysis indicated that HFD stimulated inflammatory cytokines transcriptional levels of TNF-α, IL-6 and IL-1β in control mice by 5.7, 4.0, and 11.5-fold, respectively. The elevation of the gene expression of TNF-α, IL-6 and IL-1β was reduced when mice fed with high dose AHG ( Figure 4D). These results indicate that AHG attenuated spontaneous liver injury and inflammation caused by HFD.

AHG Suppressed Hepatic Gluconeogenesis in Fasting Mice Fed A High Fat Diet
Our previous study showed that AHG suppressed hepatic gluconeogenesis in insulin resistant hepatocytes [24]. Next, we determined whether AHG affected hepatic gluconeogenesis in an insulin resistant liver. As shown in Figure 5, the protein level and gene expression of gluconeogenesis rate-limiting enzymes G6Pase and PEPCK were measured. In the livers, HFD significantly increased the protein level of G6Pase and PEPCK by 50% and 20%, respectively, but the increased potential in mice with the administration of high dose AHG was, respectively, 12% and 5%, with a significant reduction compared to HFD mice ( Figure 5A,B). The Q-PCR result showed the same trend. As a positive control, metformin repressed the transcriptional and translational level of G6Pase and PEPCK as reported in the literature [26]. This confirms that AHG suppressed hepatic gluconeogenesis in insulin resistant mice fed with a HFD. Data are showed as mean ± SD, n = 3; (C) Gene expression of G6Pase and PEPCK in livers were quantified by RT-PCR. Data are shown as mean ± SD, n = 4; *p < 0.5, **p < 0.1, ***p < 0.01 vs. LFD group, # p < 0.5, ## p < 0.1, ### p < 0.01 vs. HFD group.

AHG Improved Insulin Signaling Pathway in Liver of Mice Fed with HFD
To explore the effect of AHG on insulin signaling pathway in liver, mice were administered with or without insulin by intraperitoneal injection 2 min before euthanasia. The protein level of major cascades of insulin signaling pathway were assessed by Western Blot. HFD consumption suppressed the basal and insulin-stimulated protein level of p-IRS1(S302) and p-Akt (T308), supplementation of AHG resulted in a dose-dependent increase of IRS1 and Akt protein level in basal and insulin-stimulated state ( Figure 6A,B). Metformin also activated the insulin signaling cascade pathway, which was consistent with the previously reported work [27]. The above results suggest that the observed alterations in glucose metabolism caused by HFD are associated with an impaired IRS1/Akt pathway in liver, and this effect is mitigated by AHG supplementation. showed as mean ± SD, n=3; *p < 0.5, **p < 0.1, ***p < 0.01 vs. insulin untreated HFD group, # p < 0.5, ## p < 0.1, ### p < 0.01 vs. insulin treated HFD group.

AHG Activated AMPK in Liver of Mice Fed a High Fat Diet
AMPK and Akt are the two primary effectors in response to glucose metabolism [28]. AMPK maintains blood glucose levels and reduces hepatic gluconeogenesis gene expression-PEPCK and G6Pase. It is currently unknown that whether AHG activates AMPK in the insulin resistant liver. To address whether AMPK was involved in the effect of AHG on glucose homeostasis in liver, the protein levels of phosphorylated AMPK and an AMPK substrate, acetyl-CoA carboxylase (ACC) were measured. As illustrated in Figure 7, in the liver of insulin resistance mice induced by HFD, AHG elevated the p-AMPK and p-ACC in a dose-dependent manner. Also, there was no difference in activating AMPK between H-AHG and Metformin groups. These results state clearly that the possible role of AHG on repressing hepatic gluconeogenesis via AMPK signaling pathway. Data are showed as mean ± SD, n = 3; * p < 0.5, **p < 0.1, ***p < 0.01 vs. LFD group, # p < 0.5, ## p < 0.1, ### p < 0.01 vs. HFD group.

Discussion
Having high nutritional and medicinal qualities, sea cucumber has been drawn notable attention [9]. There is culminating evidence that polysaccharides from sea cucumber have an effective anti-diabetic activity [16,[29][30][31]. In our previous study, AHG was shown to have a therapeutic effect on hepatic glucose metabolism in vitro, however, there is no report target the effect of AHG on glucose metabolism in vivo. In the current study, we provided evidence that AHG treatment significantly alleviated fasting blood glucose and improved insulin resistance by suppressing gluconeogenesis in HFD-fed insulin resistant mice. Also, AHG supplementation elevated IRS1/Akt activation and AMPK phosphorylation in the liver of insulin resistant mice induced by HFD. All of these data suggest that AHG improved glucose metabolism in vivo.
The liver plays a crucial role in organismic energy metabolism, especially glucose metabolism, and dysfunction of the liver can result in acute metabolic abnormalities [32,33]. Due to the primary site of insulin clearance, liver is associated with the development of insulin resistance. In insulin resistance, the increased gluconeogenesis is the main mechanism involving in elevated blood glucose [34]. In the present study, the effect of AHG on gluconeogenesis was detected. HFD supplementation significantly enlarged the gene expression and protein levels of G6Pase and PEPCK in the liver, and that administration with high dose AHG remarkably inhibited this trend. These results implied that AHG maintained glucose metabolism in the liver via suppressing gluconeogenesis.
Insulin is the crucial hormone regulating gluconeogenesis and insulin represses gluconeogenesis by activating the insulin signaling pathway [35]. Insulin binds to insulin receptor and then activates phosphorylation of insulin receptor substrate (p-IRS), followed by recruitment and phosphorylation of Akt. These are key protein kinases involving in restraining gluconeogenesis related genes G6Pase and PEPCK [36,37]. Therefore, the activation of insulin signaling pathway in liver may be a promising strategy for improving insulin sensitivity. In the current study, HFD impaired the phosphorylation of IRS1 and Akt induced by insulin in the liver. However, AHG supplementation alleviated these alterations. Also, AHG improved insulin tolerance, glucose tolerance and alleviated serum insulin content in HFD group mice, suggesting that AHG improved insulin sensitivity induced by HFD, these provided insight into the anti-insulin resistance actions of AHG.
As an intracellular energy sensor, AMPK is another well-known suppressor of gluconeogenesis in addition to insulin pathway and the suppression of AMPK accompanies insulin resistance [38]. Thus, AMPK is considered as a potential target for diabetes prevention and insulin resistance treatment. So far, cumulative research has reported that various natural products regulated glucose metabolism through AMPK, such as epigallocatechin-3-gallate (EGCG) [39], ginsenoside Rg1 [40], rosmarinic acid [41], and astragalus polysaccharide [42]. To the best of our knowledge, it is barely published to target the polysaccharide isolated from marine organism especially sea cucumber on the effect of glucose metabolism through AMPK. Here, we explored how AHG activated AMPK in an insulin resistant liver induced by HFD. Thus, one of the mechanisms of the effect of AHG on glucose homeostasis in insulin resistance mice might be related to AMPK pathway.
Chronic inflammation plays a vital role in the process of liver injury induced by insulin resistance and associated with disease risk [43]. It is reported that proinflammatory cytokines TNF-α and IL-6 produced by HFD were able to induce insulin resistance [44]. In inflammation, proinflammatory cytokine is reported to activate various serine kinases such as c-Jun N-terminal kinase (JNK) [45], S6 kinase (S6K) [46], double-stranded RNA-dependent protein kinase mammalian [47], and inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ) [48]. These serine kinases can alleviate the activation of IRS1, resulting in the impairment of insulin receptor-mediated signaling and the occurrence of insulin resistance. In our HFD-induced insulin resistant model, AHG attenuated HFD-induced liver inflammation by inhibiting the expression of inflammatory genes. The anti-inflammatory effect of AHG on the liver may be one potential explanation for the beneficial effects of AHG on insulin resistance.
The high molecular weight is thought to be the main factor that suppress the transepithelial transport of glycosaminoglycan. It is reported that a fucosyl chondroitin sulfate chains (Mw=12.00 KDa) isolated from sea cucumber can be detected in plasma and urine after oral administration (50 mg/kg) [49]. Additionally, a glycosaminoglycan from Cucumaria frondosa (Mw=21.53 kDa) can improve glucose metabolism in the liver of the insulin resistant mice after oral administration [30]; a glycosaminoglycan from Acaudina molpadioides (Mw=21.53 kDa) was found to improve glucose metabolism in the liver and skeletal muscle of the insulin resistant mice after oral administration [16,29]. A sulfated polysaccharide from sea cucumber Stichopus japonicus (SCSP, Mw=179.4 kDa) can prevent obesity in association with modification of gut microbiota in HFD-fed mice [31]. These studies indicated that glycosaminoglycans from sea cucumber have the possibility to be absorbed in liver and muscle skeletal of insulin resistant mice. Based on our previously study (data unpublished), AHG is a moderate absorption drug in an endocytosis manner using Caco-2 and M cell models. In Caco-2 and M cell models, the Papp (AP-BL) values of AHG were about 2 ×10 -6 cm/s and 8 ×10 -6 cm/s, respectively. This indicates AHG is absorbed in the intestinal tracts. The pharmacokinetic study of AHG in animals will be of interest for further study.
Cumulative evidence supports a beneficial effect of AHG on glucose homeostasis as we observed in the model of HFD-induced insulin resistant mice. Oral treatment with AHG significantly reduced body weight and fasting blood glucose level. Furthermore, AHG supplementation improved insulin resistance by repressing gluconeogenesis related genes. In addition, the activation of IRS1/Akt and AMPK signaling pathway are the possible mechanisms underlying the effect of AHG on glucose metabolism in insulin resistant liver. In summary, these findings provide evidence that AHG has the potential to become a novel marine natural product to provide a therapy for the prevention and treatment of insulin resistance and type 2 diabetes. To have a better understanding of the effect of AHG on glucose metabolism in vivo, further investigation is necessary to verify the effect of AHG on regulation of glucose homeostasis in other insulin target tissues, such as skeletal muscle and adipose tissues.

Preparation of AHG
AHG was prepared as previously described [23]. Specifically, AHG was extracted from the body wall of the sea cucumber Apostichopus japonicus, purchased from the Nanshan market of Qingdao, China. AHG was prepared as previously described. The body wall of fresh sea cucumber Apostichopus japonicus was grinded into homogenate and extracted with KOH (1 mol/L) at 60 • C for 60 min. After neutralization with cold HCl, diastase vera (EC 3.3.21.4) was added to hydrolyze the protein. The crude polysaccharide was precipitated with 60% ethanol. The crude AHG was further fractionated by a Q Sepharose Fast Flow column (300 mm × 30 mm) with elution by a step-wise gradient of 0.75 and 1.5 M NaCl. The fractions eluted with 1.5 M NaCl were further purified on a Sephadex 25 column (100 × 2.6 cm) with deionized water at a flow rate of 0.3 mL/min. The purified AHG were pooled and lyophilized. The yield of AHG isolated from the fresh sea cucumber Apostichopus japonicus was 0.51% by weight. The average molecular weight of AHG was 98.07 kDa and the purity of AHG was over 99% by using gel filtration chromatography. Monosaccharide composition analysis based on pre-column derivatization reversed-phase HPLC showed that AHG consisted of glucuronic acid, galactosamine and fucose in the molar ratio of 1/1.03/1.16. The sulfate content was 33.20% determined by high performance capillary electrophoresis.

Animals and Animal Care
Male C57BL/6J mice (18-22 g) were obtained from Jinan Pengyue Experimental Animal Breeding Co. Ltd. (License Number: SCXK (lu) 2014-0007) and housed in a controlled condition (25 • C, 50 ± 5 % humidity and 12 h dark-light cycles). All mice were randomly divided into six groups for 12 weeks (8 mice/group): (1) LFD group, given low fat diet (LFD) for 12 weeks; (2) HFD group, given High Fat Diet (HFD) for 12 weeks; (3) L-AHG group, given HFD for 4 weeks, then given HFD simultaneously with oral administration of low dose AHG (20 mg/kg/day) for another 8 weeks; (4) M-AHG group, given HFD for 4 weeks, then given HFD simultaneously with oral administration of medium dose AHG (50 mg/kg/day) for another 8 weeks; (5) H-AHG group, given HFD for 4 weeks, then given HFD simultaneously with high dose oral administration of AHG (100 mg/kg/day) for another 8 weeks; (6) Metformin group, given HFD for 4 weeks, then given HFD simultaneously with oral administration of metformin (200 mg/kg/day) for another 8 weeks. LFD (TP23523) and HFD (60% fat, TP23520) were purchased from Nantong Teluofei Feed Technology Co., Ltd. (Nantong, China). At the end of treatment, mice were fasted for 12 h and intraperitoneally injected with either saline or insulin (20 units/kg). Mice were euthanized 2 min after injection. Blood were collected and centrifuged at 3000 rpm for 15min at 4 • C to obtain serum. Tissues were weighed and flash frozen in liquid nitrogen. The anti-hyperglycemic action of metformin is mainly a consequence of suppressed glucose output owing to inhibition of liver gluconeogenesis [50]. The centre of mechanism of metformin on liver gluconeogenesis is the alteration of the cell energy metabolism. metformin inhibits mitochondrial complex I, resulting a drop in ATP production. the inhibition of ATP stimulates the activation of 5'-AMP-activated protein kinase (AMPK) [51][52][53]. All procedures applied to animals were performed in accordance with the guidelines of the Laboratory Animal Center of Ocean University of China and were approved by Animal Ethics Committee of School of Medicine and Pharmacy, Ocean University of China (Qingdao, China).

Glucose Tolerance Test and Insulin Tolerance Test
For the glucose tolerance test (GTT), mice were fasted for 16 h and administered orally with glucose (2 g/kg). Blood glucose was collected before and at 15, 30, 60 and 120 min. For insulin tolerance test (ITT), mice were injected with insulin (2 units/kg) after 4 h fasting. Blood glucose was collected before and at 15, 30, 45, and 60 min. Blood glucose was measured with a glucometer (Roche, Penzberg, Germany).

Serum Insulin Level Assay
For serum insulin level, blood was collected from mice in fed state. The concentration of serum insulin was measured by Invitrogen insulin mouse ELISA kit (Thermo Fisher, Shanghai, China).

Serum Biochemical Analysis
Serum samples were obtained from blood after 30 min centrifugation at 4 • C. The levels of Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed by ALT and AST assay kits (Nanjing Jiancheng, Nanjing, China).

Western Blot analysis
Proteins from the liver tissues were measured by BCA Protein Assay Kit (Thermo Fisher, Shanghai, China) and separated by SDS-PAGE. After separated, proteins were transferred to PVDF membranes and then membranes were blocked in TBST containing 5% BSA and incubated with indicated antibodies overnight at 4 • C, followed by secondary antibody for 1 hour at room temperature. Finally, the protein bands were detected by using ECL (Nanjing Jiancheng, Nanjing, China) and were quantified by Image J.

Quantitative Real-time PCR
Total RNA was extracted from the liver tissues by using TRIzol reagent (Invitrogen, Carlsbad, USA) and was reversed to cDNA by using iScript cDNA Synthesis Kit (Bio-Rad, USA). Gene expression was measured with SYBR Green Supermix (Bio-Rad, Hercules, California, USA) in Bio-Rad CFX384 system. The mRNA expression was calculated by ∆∆CT methods and Cyclophilin was used as a load control. The primers used for this study were as follows, G6Pase (forward-5 '-TGG TAG  CCC TGT CTT TCT TTG-3

Statistical Analysis
All results were expressed as the mean ± SD. Statistical significance between the two groups was calculated using unpaired two-tailed t test. In Figure 2B, ANOVA tests were used to compare two groups among multiple groups. The value of p < 0.05 was considered statistically significantly. Data were considered no statistically significant at NS.