Applied Physiology, Nutrition, and Metabolism
Egg oil from Portunus trituberculatus improves insulin resistance through activation of insulin signaling in mice
Journal: Applied Physiology, Nutrition, and Metabolism
Manuscript ID apnm-2018-0718.R7
Manuscript Type: Article
Date Submitted by the 20-Feb-2019 Author:
Complete List of Authors: Hu, Shiwei; Zhejiang Ocean University Wang, Jingfeng; Ocean University of China Yan, Xiaojun; Zhejiang Ocean University Li, Shijie; Zhejiang Ocean University Jiang, Wei;Draft Zhejiang Ocean University Liu, Yu; Zhejiang Ocean University
Egg oil, Portunus trituberculatus, constituents, insulin resistance < Keyword: insulin resistance, insulin signaling, obesity < obesity
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Egg oil from Portunus trituberculatus alleviates insulin
2 resistance through activation of insulin signaling in mice
3 Shiwei Hua, Jingfeng Wangb, Xiaojun Yana*, Huicheng Yangc, Shijie Lia, Wei Jianga,
4 Yu Liua
5
6 aInnovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhoushan, 316022, China.
7 bCollege of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province
8 266003, China.
9 cZhejiang Marine Development Research Institute, Zhoushan 316021, China
Running title: Pt-egg oil improves insulin resistance
Corresponding author at: Institute of InnovationDraft & Application, Zhejiang Ocean University, Zhoushan
316002, Chian. Fax: +86 0580 2262063; Tel: +86 0580 2262589; E-mail: [email protected],
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11 Abstract
12 Marine bioactive lipids have been utilized to overcome insulin resistance. However, oil from
13 swimming crab has never been studied. Here, we analyzed the constituents of egg oil from Portunus
14 trituberculatus (Pt-egg oil) and investigated its protective effects against insulin resistance in mice on a
15 high-fat diet. The results showed that Pt-egg oil contained 52.05% phospholipids, 8.61% free fatty
16 acids (especially eicosapentaenoic acid and docosahexaenoic acid), 32.38% triglyceride, 4.79% total
17 cholesterol, and ditissimus astaxanthin. Animal experiments showed that Pt-egg oil significantly
18 mitigated insulin resistance and was associated with reductions in blood glucose, insulin, glucose
19 tolerance, insulin tolerance, serum lipids, and hepatic glycogen. Pt-egg oil activated the 20 phosphatidylinositol 3-hydroxy kinase (PI3K)/proteinDraft kinase B (Akt)/glucose transporter 4 (Glut4) 21 pathway in skeletal muscle both at the transcriptional level and at the translational level. Pt-egg oil also
22 promoted hepatic glycogen synthesis through activation of the PI3K/Akt/glycogen synthase kinase-3
23 beta (GSK3β) pathway. These indicate that Pt-egg oil can be used as an alternative to marine bioactive
24 lipids, to improve insulin resistance.
25 Keywords: Egg oil; Portunus trituberculatus; constituents; insulin resistance; insulin signaling; obesity
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27 Introduction
28 Insulin resistance underlies the development of several metabolic disorders, such as
29 obesity, cardiovascular disease, type 2 diabetes mellitus, and certain cancers (Czech
30 2017). Insulin resistance occurs when the normal circulating concentration of insulin
31 fails to balance body glucose homeostasis in its target tissues-mainly the liver, skeletal
32 muscle, and adipose tissues (Macdonald 2016). Insulin-dependent glucose disposal
33 primarily takes place in the skeletal muscle: approximately 75%, including glucose
34 translocation (Saltiel and Kahn 2001). The liver precedes resistance in these
35 peripheral tissues, and defects in hepatic glycogen synthesis result from insufficient 36 insulin, which finally causes theDraft disruption of glucose-glycogen homeostasis and 37 hyperinsulinemia (Prada et al. 2018). Therefore, numerous studies on the impaired
38 glucose homeostasis in insulin resistant individuals have been performed in the
39 skeletal muscle and liver (Bai et al. 2015; Wang et al. 2016; Li et al. 2015).
40 Insulin-stimulated glucose disposal is mainly mediated through phosphatidylinositol
41 3-hydroxy kinase (PI3K)/ protein kinase B (Akt) signal transduction (Nandipati et al.
42 2017). Activated Akt protein promotes translocation of glucose transporter 4 (Glut4)
43 from the cytoplasm to the cytomembrane, which provides a tunnel for glucose to enter
44 into cells (Zhang et al. 2017). The phosphorylated Akt also triggers the inactivation of
45 glycogen synthase kinase-3 beta (GSK3β) and subsequent activation of glycogen
46 synthase (GS), finally accelerating glycogen synthesis (Rong Guo et al. 2016).
47 The swimming crab, Portunus trituberculatus, is widely distributed in the coastal
48 waters of China, Japan, Korea, and other East Asian countries (Lv et al. 2017a). This
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49 crustacean species has become one of the most important economic marine products
50 based on its high nutritive value and high production: more than 100,000 tons in
51 China in 2015 (Lv et al. 2017b). Recent studies on Portunus trituberculatus have
52 focused on its gene sequence analysis or aquaculture (Pan et al. 2016; Ng'ambi et al.
53 2016). Few studies have involved its processing or utilization. Swimming crab eggs
54 contain abundant bioactive lipids, and the biological functions of these lipids have not
55 been revealed, including the effects on insulin resistance. However, inspirations can
56 be taken from the favorable activities of fish oil and shrimp oil (de Castro et al. 2015;
57 Nair et al. 2017). Here, we first separated egg oil from Portunus trituberculatus 58 (Pt-egg oil) and described its composition.Draft The effects of Pt-egg oil on alleviation of 59 insulin resistance were also investigated, especially the molecular mechanism of
60 insulin signal cascades. These may provide some theoretical basis for the utilization of
61 Pt-egg oil as a potent functional ingredient against insulin resistance.
62 Materals and methods
63 Materials and regents
64 Portunus trituberculatus eggs were obtained from Tongqu Aquatic Food Company
65 (Zhoushan, Zhejiang, China). Triglyceride (TG), total cholesterol (TC), free fatty
66 acids (FFAs), glucose, glycosylated haemoglobin (HbA1c), glycogen, and BSA kits
67 were purchased from Biosino (Beijing, China). Insulin ELISA kit was form Invitrogen
68 (Carlsbad, CA, USA). Antibody proteins used in this study were all Cell Signaling
69 products (Beverly, MA, USA). The primers of genes for PCR were synthesized by
70 ShanGon (Shanghai, China).
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71 Preparation of Pt-egg oil
72 Pt-egg oil was extracted from dry Portunus trituberculatus eggs with 95% ethanol
73 (1:6 m/V) for 10 h. After centrifugation, the supernatant was disposed by vacuum
74 concentration to obtain crude lipids. Pt-egg oil was gained through a series of process,
75 including re-concentration, blowing with nitrogen, and dehydration with Na2SO4.
76 Pt-egg oil was stored at -20℃ under nitrogen.
77 Composition of Pt-egg oil
78 TG, TC, and FFAs concentrations in Pt-egg oil were detected using commercial
79 kits. Phospholipid and astaxanthin contents were determined according to the methods 80 described by Xie et al. (2017). Draft 81 Free fatty acids composition analysis in Pt-egg oil
82 FFAs composition in Pt-egg oil was analyzed based on methods described in the
83 literature (Yin et al. 2016). Briefly, esterified Pt-egg oil was produced using 2 M
84 KOH in methanol at 80℃ for 1 h. After cooling to 25℃, further transesterification
85 was performed using 2 M H2SO4 under the same conditions. The upper organic layer
86 was diluted with n-hexane after centrifugation for analysis. The esterifiable Pt-egg oil
87 was analyzed using a gas chromatographic system (7820A, Agilent, Santa Clara, CA,
88 USA) equipped with a hydrogen flame ionization detector (FID) and a FFAP-fused
89 silica capillary column (30 m × 0.53 mm, 1μm). The conditions were as follows: oven
90 temperature, 50 to 100℃ (10℃/min, 1 min), 100 to 150℃ (5℃/min, 5min), and 150
91 to 200℃ (20℃/min); nitrogen (carrier gas), 1 mL/min; FID and injector temperature,
92 250℃; injection volume, 2.0 μL. Fatty acids in the samples were identified by
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93 comparing the retention times of the sample peaks with those of a mixture of fatty
94 acid methyl ester standards. The FFAs contents were expressed as the weight
95 percentage (% w/w) of the total FFAs detected with chain lengths of 4-22 carbon
96 atoms.
97 Animal experiments
98 Male C57BL/6J mice (licensed ID SCXK2016-0001), 16-18 g, were purchased
99 from Vital River Laboratory Animal Center (Beijing, China). Animals were housed in
100 normal cages at 23 ± 1 ℃ with a 12:12 h light-dark schedule. All experimental
101 protocols used in this study were approved by the ethical committee for experimental 102 animal care at Zhejiang Ocean University.Draft Animals were randomly assigned to four 103 groups (n=15 per group, 5 mice per cage): control group (fed with normal chow diet:
104 70% carbohydrate, 20% protein, and 10% fat based on weight), high fat diet
105 (HFD)-feeding group (fed with HFD: 29% carbohydrates, 16% protein, and 55% fat
106 based on weight), low Pt-egg oil group (fed with an HFD and intragastric
107 administration of 150 mg/kg of Pt-egg oil), and high Pt-egg oil group (fed with an
108 HFD and 600 mg/kg of Pt-egg oil). Four groups of animals were administered
109 continuously for 16 weeks. At 15 w of feeding, an oral glucose tolerance test (OGTT,
110 eight mice per group) and intraperitoneal insulin tolerance test (IITT, seven mice per
111 group) were conducted to verify the model and the activity of Pt-egg oil. After 1 week
112 of recovery in physical function, the seven mice tested for IITT were used to detect
113 plasma parameters, glycogen contents, and insulin signals at the transcriptional level,
114 while the eight mice tested for OGTT were used to detect insulin stimulation for
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115 phosphorylated protein and plasma membrane Glut4 protein.
116 OGTT and IITT
117 After feeding for 15 weeks, OGTT was performed by detecting blood glucose
118 levels at 0, 0.5, 1, and 2 h after intragastric administration of 2 g/kg glucose to the 5 h
119 fasted mice (eight animals per group). For IITT, the other mice (seven animals per
120 group) were intraperitoneally injected with insulin (0.5 U/kg), and tail vein blood was
121 collected at the indicated time points. Blood glucose levels were measured using a
122 commercial kit. The areas under the curve of the OGTT (AUCOGTT) and IITT
123 (AUCIITT) were both calculated as Formula 1.
124 AUCOGTT/AUCIITT = 0.25 × A Draft+ 0.5 × B +0.75 × C + 0.5 × D (A, B, C, and D 125 represent blood glucose level at 0, 0.5, 1, and 2 h, respectively) (Formula 1)
126 Blood glucose parameters, insulin, TC, and TG concentrations assay
127 After 16 weeks of administration, the mice were sacrificed under anesthesia. Blood
128 was collected from the orbital vein of seven mice given the IITT. The blood was used
129 to determinate fasting blood glucose levels. Serum was obtained from the collected
130 blood after centrifugation at 7,500 rpm for 15 min. Serum HbA1c, TC, and TG levels
131 were assessed by commercial kits. Serum insulin was determined using insulin ELISA
132 assay kits. A homeostasis model assessment of insulin resistance index (HOMA-IR)
133 and quantitative insulin sensitivity check index (QUICKI) were expressed as
134 Formulas 2 and 3, respectively.
135 HOMIA-IR = fasting blood glucose × serum insulin / 22.5 (Formula 2)
136 QUICKI = 1 / [lg (fasting blood glucose) + lg (serum insulin)] (Formula 3)
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137 Hepatic glycogen content detection
138 The liver (80-100 mg) was disrupted in alkali lye and subsequently denatured in
139 boiling water for 30 min. Samples at 96-fold dilution were centrifuged at 7,500 rpm
140 for 30 min. The precipitate was dissolved in 1.5 mL of distilled water, and hepatic
141 glycogen was detected by a commercial kit.
142 Quantitative real time polymerase chain reaction (qRT-PCR) analysis
143 The mRNA expression levels of pivotal insulin signaling genes (IRS1, IRS2, PI3K,
144 Akt, GSK-3β, GS, and Glut4) in skeletal muscle or liver were examined by qRT-PCR
145 according to our previous study (Hu et al. 2014). Briefly, total RNA from skeletal 146 muscle or liver was extractedDraft with TRIzol regent and subsequently 147 reverse-transcribed to cDNA. PCR was tested by amplification of 15 ng cDNA in a 25
148 µL system containing SYBR-Green mix with a quantitative real-time PCR
149 thermocycler (iQ5; Bio-Rad, Hercules, CA, USA). Cycling conditions were
150 referenced form our previous study (Hu et al. 2014). Table S1 shows the primer
151 sequences. Quantitative PCR products were analyzed using the software iCycler iQ5
152 software. β-Actin was used as an internal reference.
153 Insulin stimulation
154 Insulin stimulation can enhance PI3K/Akt signal cascades. In insulin resistant mice,
155 insulin stimulation fails to improve this signaling because of hormone insensitivity,
156 while the inverse is true in normal animals. In order to determine the phosphorylated
157 proteins and plasma membrane Glut4 protein, insulin stimulation was carried out as
158 described (Sim et al. 2007; Hu et al. 2017). Briefly, for phosphorylated proteins,
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159 insulin (40 U/kg) was intraperitoneally injected and the mice were sacrificed to strip
160 hindquarter skeletal muscle and liver 5 min after the injection (four in the eight OGTT
161 mice per group). For plasma membrane Glut4, a similar treatment was performed by
162 replacing a waiting period of 30 min after injection of 0.5 U insulin/kg (remaining
163 four mice in the OGTT). The remaining seven mice were subjected to intraperitoneal
164 injection of normal saline to measure total proteins. The muscle and liver were frozen
165 in liquid nitrogen and stored at -80 °C for subsequent analysis.
166 Plasma membrane preparation
167 The skeletal muscle (3 g) from the mice injected with 0.5 U insulin/kg was 168 homogenized in normal saline. TheDraft homogenate was subjected to triple centrifugation 169 at 1,200, 9,000, and 19,000 × g to gain the crude membrane. The plasma membrane
170 fractions were further separated by sucrose density-gradient centrifugation (25%,
171 32%, and 35%) at 150,000 × g for 16 h. For western blotting experiment, the m-Glut 4
172 protein was collected from the fraction of 25% sucrose solution and centrifuged at
173 190,000 ×g for 1 h.
174 Immunoprecipitation
175 IRSs-associated PI3K was achieved through immunoprecipitation. Briefly, skeletal
176 muscle or liver with intraperitoneal injection of normal saline was lysed in PBS
177 (containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 mM
178 sodium orthovanadate). After centrifugation, the cell lysates (1 mg of total protein)
179 were incubated with anti-IRS1 or anti-IRS2 antibodies for 1 h and precipitated by
180 incubation with protein A-Sepharose for 1 h. The resultant immunocomplexes were
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181 washed four times with the lysis buffer, and PI3K-IRSs was examined by western
182 blotting.
183 Western blot assay
184 Western blotting was performed, as described in our previous study (Hu et al.
185 2017). Briefly, the skeletal muscle or liver (0.1 g) was lysed in IP lysis buffer to
186 dissolve cellular proteins. Equal amounts of proteins were resolved by 10%
187 SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes.
188 After blotting with 5% BSA, the protein was incubated with primary antibodies and
189 subsequently with HRP-IGG secondary antibodies. Members underwent detection by 190 ECL. Phosphorylated proteins andDraft m-Glut4 were normalized by their corresponding 191 proteins, while PI3K in IRSs by immunoprecipitation was normalized by total IRSs.
192 Statistical analysis
193 Results are expressed as mean values and standard deviations. The statistical
194 analysis was performed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). The
195 difference between the control and HFD mice was analyzed using Student’s t-test.
196 Differences among the HFD, low Pt-egg oil group, and high Pt-egg oil group were
197 analyzed using one-way analysis of variance (ANOVA) with post hoc Bonferroni’s
198 multiple comparison tests. A p value of < 0.05 was considered statistically significant.
199 Results
200 Pt-egg oil composition
201 Table 1 shows the composition of Pt-egg oil. Phospholipids (approximately 52%)
202 accounted for the largest section. TG also occupied a large proportion with 32.38%.
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203 Pt-egg oil contained 8.61% FFAs and 4.79% TC. Additionally, astaxanthin content
204 was present at a concentration of 971.79 μg/g in Pt-egg oil.
205 Pt-egg oil free fatty acids compositions
206 As shown in table 2, in the FFAs of Pt-egg oil, unsaturated fatty acids (UFAs)
207 accounted for a greater proportion than saturated fatty acids (SFAs). Polyunsaturated
208 fatty acids (PUFAs) made up 82.75% of UFAs, while monounsaturated fatty acids
209 (MUFAs) only comprised 17.25%. Moreover, in the PUFAs, EPA and DHA were
210 abundant, at approximately 52.73%.
211 Pt-egg oil reduced blood glucose and lipids 212 After 16 weeks of HFD feeding,Draft the mice showed significant increases in body 213 weight gain (p < 0.01, table 3). When administered with Pt-egg oil, body weight gains
214 were markedly decreased in both the low Pt-egg oil (p < 0.05) and the high Pt-egg oil
215 groups (p < 0.01). HFD induced elevations in fasting blood glucose and decreases in
216 HbA1c were significantly decreased by high Pt-egg oil to 26.77% and 32.44%,
217 respectively. Low Pt-egg oil also caused a significant reduction in fasting blood
218 glucose in HFD mice (p < 0.05). As shown in Fig. 1A and B, the data in the OGTT
219 experiment showed that both low and high Pt-egg oil remarkably improved glucose
220 tolerance by lowering AUCOGTT (p < 0.05, p < 0.01). In addition, serum TC and TG
221 were significantly decreased by high Pt-egg oil (p < 0.05, p < 0.01). These data
222 suggest that Pt-egg oil exerts significant anti-hyperglycemic and anti-hyperlipidemic
223 activities.
224 Pt-egg oil increased insulin sensibility
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225 As shown in table 3, high Pt-egg oil treatment induced 21.40% and 41.47%
226 reductions in serum insulin and HOMA-IR score and a 13.78% increase in QUICKI
227 value, respectively. Moreover, low Pt-egg oil also significantly decreased serum
228 insulin and HOMA-IR scores (p < 0.05). In the IITT experiment, the elevation of
229 AUCIITT by HFD was dramatically inhibited in the low and high Pt-egg oil group (p <
230 0.05, p < 0.01, Fig. 1C and D). These results indicate that insulin sensitivity is
231 markedly enhanced by Pt-egg oil.
232 Pt-egg oil promoted hepatic glycogen synthesis
233 As shown in table 3, Pt-egg oil increased hepatic glycogen contents by 63.50% in 234 the low dosage group and 1.72-foldDraft in the high dosage group, respectively. These 235 indicated that Pt-egg oil can promote hepatic glycogen synthesis.
236 Pt-egg oil regulated mRNA expression in insulin signal cascades in the skeletal
237 muscle and liver
238 PI3K/Akt signaling is the main approach to insulin-dependent glucose disposal.
239 PI3K/Akt/Glut4 signal mediates glucose transport, while the PI3K/Akt/GSK3β
240 pathway regulates glycogen synthesis. We further examined the effects of Pt-egg oil
241 on the mRNA expression of this signaling in the liver and skeletal muscle of HFD
242 mice. As shown in Fig. 2, high Pt-egg oil significantly increased IRS1 mRNA in
243 muscle and IRS2 mRNA in liver (p < 0.01). Moreover, low Pt-egg oil also markedly
244 enhanced hepatic IRS2 mRNA (p < 0.05). PI3K mRNA expression was elevated in
245 the liver and muscle of HFD mice when supplemented with low and high Pt-egg oil (p
246 < 0.05, p < 0.01). High Pt-egg oil treatment also remarkably increased the mRNA
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247 expression of Akt in the liver and muscle (p < 0.05, p < 0.01), while there were no
248 changes in the low dosage group. Furthermore, high Pt-egg oil dramatically increased
249 Glut4 mRNA in muscle and GS mRNA in the liver, and decreased GSK3β mRNA in
250 the liver (p < 0.01). Low Pt-egg oil also caused a remarkable elevation in GS mRNA
251 in the liver of HFD mice (p < 0.05). These indicate that Pt-egg oil can promote
252 glucose disposal by insulin through regulation of PI3K/Akt signaling in skeletal
253 muscle and liver.
254 Pt-egg oil activated PI3K/Akt/Glut4 insulin signaling in skeletal muscle
255 Glucose transport from intercellular substances to the cytoplasm is dependent on 256 Glut4 protein translocation fromDraft the cytoplasm to the cytomembrane, which is 257 mediated by a series of activated proteins in insulin signal cascade. We subsequently
258 investigated how Pt-egg oil influenced proteins in skeletal muscle. As shown in Fig. 3,
259 HFD-induced elevation of phosphorylated IRS1 at the serine site was significantly
260 inhibited in both the low and high Pt-egg oil groups (p < 0.05, p < 0.01).
261 Phosphorylated IRS1 at the tyrosine point was remarkably increased by high Pt-egg
262 oil supplementation in HFD mice (p < 0.05). High Pt-egg oil also caused significant
263 increases in IRS1-conjugated PI3K and phosphorylated Akt at the serine site (p < 0.05,
264 p < 0.01). The glucose transporter, Glut4 protein, at the plasma membrane was also
265 enhanced by high Pt-egg oil (p < 0.01). These results indicate that Pt-egg oil exhibits
266 anti-hyperglycemic activities via activation of PI3K/Akt/Glut4 insulin signaling.
267 Pt-egg oil activated the PI3K/Akt/GSK3β/GS pathway in the liver
268 Insulin-mediated glycogen synthesis is affected by the activation of GSK3β and GS.
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269 The Pt-egg oil-influenced PI3K/Akt/GSK3β/GS pathway in the liver is shown in Fig.
270 4. Similarly to the reaction in skeletal muscle, high Pt-egg oil significantly inhibited
271 phosphorylated IRS2 at the serine site, increased phosphorylated IRS2 at the tyrosine
272 point and Akt at the serine point, and increased IRS2-conjugated PI3K (p < 0.01).
273 Moreover, low Pt-egg oil also caused a significant reduction in phosphorylated IRS2
274 at the serine site and increase in phosphorylated Akt at serine point (p < 0.05).
275 Meanwhile, HFD animals showed dramatic decreases in phosphorylated GSK3β at
276 serine site when treated with low and high Pt-egg oil (p < 0.05, p < 0.01). Activated
277 GS protein expression was markedly promoted by low and high Pt-egg oil treatment 278 (p < 0.05, p < 0.01). These data Draftsuggest that Pt-egg oil promotes hepatic glycogen 279 synthesis through activation of the PI3K/Akt/GSK3β/GS pathway.
280 Disscussion
281 In this study, Pt-egg oil was isolated and analyzed for the first time. Pt-egg oil
282 contains abundant phospholipids, FFAs, and astaxanthin. We also demonstrated that
283 treatment with Pt-egg oil for 16 weeks ameliorated insulin resistance, which was
284 associated with the reductions in blood glucose, insulin, glucose intolerance, insulin
285 intolerance, and hepatic glycogen in HFD mice. These results suggest that insulin
286 resistance can be improved by Pt-egg oil because of its active constituents.
287 UFAs are one of the main marine biological lipids, in which EPA and DHA are the
288 main functional lipids. Fish oil contains abundant EPA and DHA (approximately
289 30-40% w/w of total fatty acids), and exhibits significant bioactivities to overcome
290 insulin resistance (Sadeghi et al. 2017). In different species of fish, the ratios of EPA
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291 and DHA were distinguished. Fore example, shark liver oil contains 19.4% EPA and
292 22.1% DHA (Oliver and Shorland 1948), ling liver oil contains 22.9-25.4% EPA and
293 8.9-9.8% DHA (Shorland FB 1939), and Newfoundland oil contains 30.3% EPA and
294 14.1% DHA (Guha et al. 1930). Numerous studies have reported that fish oil can
295 alleviate insulin resistance and maintain glucose homeostasis (Slim et al. 2017;
296 Mikami et al. 2012). Krill oil has also been shown to possess plentiful EPA and DHA
297 (approximately 20-25% of total fatty acids) (Kwantes and Grundmann 2015), and
298 therefore exert improvement in insulin resistance (Ivanova et al. 2015). Skorve et al.
299 analyzed the oil from Northern shrimp caught in the Atlantic area and found 9.9% 300 EPA and 10.3% DHA in the totalDraft fatty acids (Skorve et al. 2015). Our data showed 301 that Pt-egg oil has 27.80% EPA and 5.43% DHA in the total fatty acids, which is
302 similar to fish oil. In difference, neutral lipids such as triacylglycerols predominate in
303 fish oil and krill oil. For example, there are ≥95% triacylglycerols and 1.7% sterols in
304 shrimp oil (Skorve et al. 2015). While in Pt-egg oil, there were 32.38%
305 triacylglycerols and 52.05% phospholipids, which are polar lipids that possess
306 significant effects on insulin resistance (Kachko et al. 2015; Rossmeisl et al. 2014).
307 These are the primary advantage of Pt-egg oil over fish oil and krill oil, and the larger
308 number of phospholipids in Pt-egg oil could enhance absorption. Astaxanthin has
309 been proven to exert alleviation of insulin resistance and activation of insulin
310 signaling (Ni et al. 2015; Bhuvaneswari and Anuradha 2012). Study shows that there
311 is ≥400 μg/g of astaxanthin in shrimp oil (Skorve et al. 2015), but little in fish oil. In
312 our experiments, Pt-egg oil contained abundant astaxanthin (971 μg/g), more than in
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313 shrimp oil (Skorve et al. 2015). When these biological lipids in Pt-egg oil were
314 supplemented to HFD mice, their insulin resistance and glucose homeostasis were
315 significantly improved. Therefore, we believe that these functional lipids in Pt-egg oil
316 such as PUFAs (EPA/DHA), phospholipids, and astaxanthin, directly improve insulin
317 resistance.
318 More than 75% of insulin-disposed glucose occurs in skeletal muscle, which is the
319 foremost approach to glucose disposal (Voss et al. 2017). This procedure is achieved
320 by the translocation of the glucose transporter Glut4 from intracellular sites to the cell
321 surface (Esteves et al. 2017). In this study, Pt-egg oil promoted glucose uptake by 322 increasing membrane Glut4 proteinDraft expression and mRNA level in skeletal muscle. 323 Intracellular glucose is mainly utilized as an energy source to maintain physiological
324 functions, and the redundant glucose is transformed into glycogen and adipose to store
325 energy. HFD caused significantly high blood glucose levels in mice, while treatment
326 with Pt-egg oil dramatically decreased the elevation of blood glucose and glucose
327 tolerance, suggesting that the mice in the Pt-egg oil group utilized more glucose.
328 Moreover, insulin tolerance was also improved by Pt-egg oil supplementation. In
329 molecules, insulin activates IRSs through inhibition of phosphorylation at the serine
330 point and promotion of phosphorylation at the tyrosine site (Hatem-Vaquero et al.
331 2017). Treatment with Pt-egg oil inhibited the elevation in phosphorylated
332 IRSs(Ser307) proteins and the decrease in phosphorylated IRSs(tyr612) proteins,
333 indicating that IRSs were activated by Pt-egg oil supplementation. Activated IRSs
334 polymerize PI3K and trigger the phosphorylation of the p85 regulatory subunit, which
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335 is crucial for the activation of PI3K (Tao et al. 2010; Cook et al. 2010). When PI3K is
336 activated, signal cascades such as Akt are transmitted downstream. Activation of Akt
337 by its phosphorylation promotes Glut4 translocation (Albers et al. 2015). Pt-egg oil
338 increased the protein expression of IRS1-associated PI3K and phosphorylated Akt.
339 These results indicate that Pt-egg oil HFD-induced insulin resistance was inhibited by
340 Pt-egg oil through activation of the PI3K/Akt/Glut4 signaling in skeletal muscle.
341 On the other hand, insulin-mediated glycogen synthesis in the liver is an important
342 mechanism involved in blood glucose homeostasis and metabolism (Bhuvaneswari
343 and Anuradha 2012; Liu et al. 2016). Hepatic glycogen content was significantly 344 increased in the Pt-egg oil group, suggestingDraft that HFD-induced excessive glucose was 345 synthesized to glycogen in the liver when hyperglycemic mice were treated with
346 Pt-egg oil, which also contributed to a reduction in blood glucose. Glycogen synthesis
347 is mediated by GS. GS is activated by deactivation of GSK3β, the upstream protein of
348 GS in insulin signaling, in response to insulin (Yang et al. 2014), and phosphorylated
349 GS directly triggers hepatic glycogen synthesis (Salameh et al. 2006; Gao et al. 2017).
350 Pt-egg oil treatment decreased GSK3β mRNA and phosphorylated protein, while
351 increasing GS in the liver of HFD mice, which supports glycogen synthesis and
352 glucose homeostasis. GS-mediated glycogen synthesis is also triggered by PI3K/Akt
353 signaling (Kang et al. 2017). When treated with Pt-egg oil, insulin resistant mice
354 showed an activated IRS2/PI3K/Akt pathway in the liver. These results indicate that
355 Pt-egg oil mitigates insulin resistance through activation of the IRSs/PI3K/Akt
356 pathway in the liver
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357 Conclusions
358 Pt-egg oil is firstly described as a valuable lipid, containing abundant
359 phospholipids, PUFA and astaxanthin, and plentiful EPA and DHA in particular. This
360 study also suggested that Pt-egg oil significantly alleviats insulin resistance in
361 HFD-fed mice. Activation of the PI3K/Akt/GSK-3β insulin signal cascade was the
362 underlying mechanism for Pt-egg oil-improved hepatic insulin resistance. Activation
363 of the PI3K/Akt/Glut4 pathway in skeletal muscle induced by Pt-egg oil was involved
364 in the alleviation of insulin resistance. This study provides significant implications for
365 the utilization of bioactive lipids from swimming crab as an alternative marine drug to 366 protect insulin resistance. Draft 367 Acknowledgements
368 This study was financially supported by Public Projects of Zhejiang Province
369 (LGN19D060001), Zhoushan Science and Technology project (2016C41012 &
370 2015C21014), and National Natural Science Foundation of China (41806182).
371 Disclosure statement
372 The authors declare that there are no conflicts of interest. The authors alone are
373 responsible for the content and writing of the article.
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509 Figure Captions
510 Figure 1
511 Fig.1 Effects of Pt-egg oil on glucose tolerance and insulin sensitivity in insulin resistant mice fed
512 with HFD. OGTT, 5-h fasting blood at 0, 0.5, 1, and 2 h after intragastrically given 2 g glucose per kg
513 body weight was collected from the caudal vein at 15 weeks for measuring blood glucose level to
514 calculate AUCOGTT. IITT, 5-h fasting blood at the indicated time points after intraperitoneal injection of
515 0.5 U insulin per kg body weight was collected from the caudal vein at 15 weeks for measuring blood
516 glucose level to calculated AUCIITT. A, blood glucose level at indicated time points in OGTT; B,
517 AUCOGTT; C, blood glucose level at indicated time points in IITT; D, AUCIITT. Data are expressed as 518 mean ± SD (n = 8/group for OGTT, n = 7/groupDraft for IITT). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 519 vs HFD.
520 Figure 2
521 Fig.2 Effects of Pt-egg oil on insulin signaling gene mRNA expression in the liver and skeletal muscle
522 of insulin resistant mice. The mRNA expressions were detected by qRT-PCR, and the results were
523 normalized by β-actin. Data are expressed as mean ± SD (n=7/group, without insulin stimulation). ## P
524 < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
525 Figure 3
526 Fig.3 Effects of Pt-egg oil on phosphorylated protein or membrane Glut4 in PI3K/Akt/Glut4 insulin
527 signaling in skeletal muscle of insulin resistant mice. A, protein bands using immunoprecipitation and
528 western blot; B, data of relative expression of phosphorylated protein or membrane Glut4. The results
529 were normalized by their corresponding total protein. Data are expressed as mean ± SD (n = 4/group).
530 ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
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531 Figure 4
532 Fig.4 Effects of Pt-egg oil on phosphorylated protein in PI3K/Akt/GSK3β insulin signaling in the liver
533 of insulin resistant mice. A, protein bands using immunoprecipitation and western blot; B, data of
534 relative expression of phosphorylated protein. The results were normalized by their corresponding total
535 protein. Data are expressed as mean ± SD (n = 4/group). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01
536 vs HFD.
Draft
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Table 1 The composition of Pt-egg oila
Composition Content TG (%) 32.38±1.10 TC (%) 4.79±0.38 FFAs (%) 8.61±1.49 Phospholipids (%) 52.05±2.24 Astaxanthin (μg/g) 971.79±41.73
a Data are presented as mean ± S.D (n=6). TG, triglyceride; TC, total cholesterol; FFAs, free fatty acids.
Draft
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Table 2 Free fatty acids compositions of Pt-egg oila
Fatty acids Proportion (%) Fatty acids Proportion (%) C14:0 3.42±0.13 C20:2 1.84±0.17 C15:0 1.09±0.07 C20:4 14.32±0.86 C16:0 11.19±0.74 C20:5 27.80±0.66 C17:0 1.42±0.25 C22:6 5.43±0.47 C17:1 2.13±0.20 ∑SFA 23.84±0.92 C18:0 5.78±0.61 ∑UFA 76.16±1.57 C18:1 9.10±0.49 ∑MUFA 13.14±0.75 C18:3 0.49±0.05 ∑PUFA 63.02±1.18 C20:0 0.94±0.04 ∑(EPA+DHA) 33.23±0.88 C20:1 1.91±0.16
a Data are presented as mean ± S.D (n=6).Free fatty acid compositions was analyze by gas
chromatography. SFAs, saturated fatty acids; UFAs, unsaturated fatty acids; MUFAs, monounsaturated
fatty acids; PUFAs, polyunsaturated fatty acids. Draft
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Table 3 Effects of Pt-egg oil on blood glucose and insulin parameters, serum TC, TG, and hepatic glycogen levels in HFD micea
Control HFD Low Pt-egg oil High Pt-egg oil Body weight gain (g) 11.46±0.82 23.99±1.26 19.44±0.74* 17.35±0.87** Fasting blood glucose (mmol/L) 8.49±0.73 12.59±0.88## 10.98±0.84* 9.22±0.76** Serum HbA1c (mmol/L) 16.95±3.24 26.88±5.10## 22.47±3.51 18.16±4.04** Serum insulin (mU/L) 9.08±.079 12.43±0.72## 10.96±0.61* 9.77±0.69** HOMA-IR 3.12±0.43 6.92±0.83## 5.33±0.40* 4.05±0.64** QUICKI 0.537±0.012 0.450±0.011## 0.487±0.010 0.512±0.011** Serum TC (mmol/L) 2.54±0.16 4.56±0.37## 4.05±0.14 3.38±0.14* Serum TG (mmol/L) 0.797±0.069 1.345±0.186## 1.069±0.143 0.824±0.085** Hepatic glycogen (mg/g) 9.42±0.84 2.74±0.47## 4.48±0.52* 7.46±0.60**
a Data are presented as mean ± S.D (n=7/group, without insulin stimulation). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD. HFD, highDraft fat diet; TG, triglyceride; TC, total cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance index; QUICKI, quantitative insulin sensitivity check index.
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Draft
Fig.1 Effects of Pt-egg oil on glucose tolerance and insulin sensitivity in insulin resistant mice fed with HFD. OGTT, 5-h fasting blood at 0, 0.5, 1, and 2 h after intragastrically given 2 g glucose per kg body weight was collected from the caudal vein at 15 weeks for measuring blood glucose level to calculate AUCOGTT. IITT, 5- h fasting blood at the indicated time points after intraperitoneal injection of 0.5 U insulin per kg body weight was collected from the caudal vein at 15 weeks for measuring blood glucose level to calculated AUCIITT. A, blood glucose level at indicated time points in OGTT; B, AUCOGTT; C, blood glucose level at indicated time points in IITT; D, AUCIITT. Data are expressed as mean ± SD (n = 8/group for OGTT, n = 7/group for IITT). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
85x62mm (300 x 300 DPI)
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Draft
Fig.2 Effects of Pt-egg oil on insulin signaling gene mRNA expression in the liver and skeletal muscle of insulin resistant mice. The mRNA expressions were detected by qRT-PCR, and the results were normalized by β-actin. Data are expressed as mean ± SD (n=7/group, without insulin stimulation). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
86x99mm (300 x 300 DPI)
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Draft
Fig.3 Effects of Pt-egg oil on phosphorylated protein or membrane Glut4 in PI3K/Akt/Glut4 insulin signaling in skeletal muscle of insulin resistant mice. A, protein bands using immunoprecipitation and western blot; B, data of relative expression of phosphorylated protein or membrane Glut4. The results were normalized by their corresponding total protein. Data are expressed as mean ± SD (n = 4/group). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
90x64mm (300 x 300 DPI)
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Draft
Fig.4 Effects of Pt-egg oil on phosphorylated protein in PI3K/Akt/GSK3β insulin signaling in the liver of insulin resistant mice. A, protein bands using immunoprecipitation and western blot; B, data of relative expression of phosphorylated protein. The results were normalized by their corresponding total protein. Data are expressed as mean ± SD (n = 4/group). ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.
86x68mm (300 x 300 DPI)
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