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Article

Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet

by
Ju Ri Ham
1,†,
Ra-Yeong Choi
2,†,
Yongjin Lee
3 and
Mi-Kyung Lee
1,*
1
Department of Food and Nutrition, Sunchon National University, Suncheon 57922, Korea
2
Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Korea
3
Department of Pharmacy, Sunchon National University, Suncheon 57922, Korea
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Int. J. Mol. Sci. 2021, 22(7), 3615; https://doi.org/10.3390/ijms22073615
Submission received: 25 February 2021 / Revised: 27 March 2021 / Accepted: 29 March 2021 / Published: 31 March 2021
(This article belongs to the Special Issue Molecular Link between Steatosis and Obesity)
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Abstract

:
Mealworms (Tenebrio molitor larva) are an edible insect and a protein-rich food; however, research on mealworms as a substitute protein is insufficient. In this study, mealworm fermentation extract (TMP) was assessed as a replacement for soy protein (SP) in a control diet (CON) or a high-fat diet (HFD) of mice for 12 weeks. TMP substitution reduced body weight, body weight gain, body fat mass (perirenal and mesenteric), fat size, glucose intolerance, and insulin resistance compared to the HFD-SP group. TMP alleviated hepatic steatosis (lipid contents and lipid droplets) in high-fat-fed mice and down-regulated the PPARγ, CD36, and DGAT2 gene levels. Proteomic analysis showed that a HFD for 12 weeks up-regulated 20 proteins and down-regulated 17 proteins in mice fed SP. On the other hand, TMP reversed the protein profiles. TMP significantly down-regulated KHK, GLO1, ATP5H, SOD, and DDAH1 and up-regulated DLD, Mup1, CPS1, Ces3b, PDI, and HYOU1 compared to the HFD-SP group. These proteins are involved in the glucose, lipid, and amino acid metabolism, as well as in oxidative stress and endoplasmic reticulum stress. Thus, substituting SP for TMP helped improve HFD-induced obesity, steatosis, and insulin resistance in mice. These results suggest that TMP is a potential substitute for commonly used protein sources.

1. Introduction

The epidemic of obesity has increased globally and is consistently related to the rising prevalence of non-alcoholic fatty liver disease (NAFLD). NAFLD is distinguished by more than 5% fat in the liver and progresses to steatohepatitis, which can lead to fibrosis and ultimately to cirrhosis and hepatocellular carcinoma [1]. Many studies have indicated that the prevalence of NAFLD in obese individuals was higher than in lean individuals in the Asia-Pacific region, Netherlands, and the USA [2,3,4,5,6].
Edible insects have been utilized as alternative protein sources. Besides, their market size has increased continuously [7]. Previous studies have showed the hepatoprotective influences of insect extracts, such as Oxya chinensis sinuosa, Gryllus bimaculatus, and Protaetia brevitarsis seulensis, in high-fat diet (HFD)-induced NAFLD animals [8]. Among edible insects, Tenebrio molitor larvae are known as mealworms, which have a high quantity and quality of amino acid and protein [9]. Therefore, they are considered a highly sustainable protein source and alternative to soy protein (SP) or livestock meat [9]. Gessner et al. [10] reported that the isonitrogenous replacement of casein with mealworms exhibited lipid-lowering efficacy in the plasma and liver by inhibiting hepatic triglyceride (TG) and cholesterol synthesis in hyperlipidemic obese Zucker rats. Sim et al. [11] reported that fermented mealworm powder improved NAFLD in orotic acid-induced steatosis rats. A recent study reported that defatted mealworm fermentation extract (TMP) attenuated alcoholic hepatic steatosis in rats by regulating lipid synthesis, inflammation, and glutathione contents and changed the hepatic metabolites and gut microbiota [12]. TMP has a high percentage of free amino acids and essential amino acids compared to non-fermented mealworm extract.
Previous meta-analysis showed that plant protein intake was associated with a decreased risk of obesity, dyslipidemia, and diabetes, whereas animal proteins were associated with an increase [13,14]. Nutritional intervention research in animals and human beings suggests that SP reduces fat mass, body weight, and plasma cholesterol and TG levels [15]. Therefore, this study examined the effects of TMP as a substitute protein for SP in mice with obesity-induced hepatosteatogenesis and compared its underlying mechanisms.

2. Results

2.1. TMP Reduced the Body Weight and Fat Size

As expected, feeding a HFD for 12 weeks increased the body fat mass, fat size, and body weight in mice significantly compared with the control diet (CON)-fed mice (Figure 1). The fat size, body weight gain, and body weight showed a significant interaction between fat and TMP. HFD containing TMP (HFD-TMP) substitution effectively lessened the fat size, body weight gain, and body weight compared to the HFD containing soy protein (HFD-SP) group by 46%, 26%, and 13%, respectively (Figure 1). TMP did not affect food intakes (data not shown).
HFD-TMP significantly reduced mesenteric and perirenal weights of white adipose tissue (WAT) compared with HFD-SP. The weight of subcutaneous WAT was similar to the groups (Figure 1).

2.2. TMP Affected the Insulin Resistance Induced by High-Fat Diet

The blood glucose level did not differ between groups, whereas the serum insulin concentration and homeostasis model assessment of insulin resistance (HOMA-IR) showed only a TMP effect. Although there was no statistical significance, HFD-TMP lowered the insulin level compared to the HFD-SP, which showed a significant decrease in the HOMA-IR in the HFD-TMP group (Figure 2). The glucose tolerance test and serum leptin level also showed only a TMP effect. That is, HFD-TMP decreased glucose intolerance and leptin level significantly compared to the HFD-SP (Figure 2).

2.3. TMP Attenuated Hepatic Steatosis Induced by High-Fat Diet

Oil Red O and hematoxylin and eosin (H&E) staining indicated that the hepatic lipid droplets were higher in the HFD-SP group than in the CON-SP group. Trichrome staining also showed slight fibrosis progressing in the HFD groups. On the other hand, TMP reversed the histological changes (Figure 3). There were no significant differences in hepatic TG content between CON-SP and HFD-SP groups; however, HFD-TMP decreased the TG level significantly compared to the HFD-SP, by 23%. The hepatic free fatty acid (FFA) concentration was significantly lowered by TMP substitution in both the CON and HFD, while cholesterol level had no significant effect on TMP or fat, respectively (Figure 3).
TMP affected PPARγ and CD36 genes, whereas fat affected PPARγ and ChREBP genes in the liver. DGAT2 gene expression showed the significant interaction between TMP and fat. Therefore, HFD-TMP significantly down-regulated the lipogenic transcription factor PPARγ and TG synthesis gene DGAT2 expression compared to the HFD-SP.

2.4. TMP Modified Hepatic Changes of Proteomic Profiles

To determine the effect of TMP on hepatic protein changes, proteomic analysis was carried out. When compared to the CON-SP, 20 proteins (ALDOB, ADH, KHK, FBP1, PC, GLO1, hsc71, HSPA9, BHMT, PHB, MAT1A, BCKDHA, hutU, MGCS2, ATP5H, SOD, DDAH1, GSTM2, ALDH1A1, and IMMT) were up-regulated and 17 proteins (ACO1, DLD, Mup1, CPS1, Ces3b, Ces3a, PDI, HSPA5, HYOU1, PNP, NME1, GSTP1, DDT, Tf, FGA, ALDH6A1, and ALDH12A1) were down-regulated in the HFD-SP group; and HFD-TMP significantly down-regulated KHK, GLO1, ATP5H, SOD, and DDAH1 and up-regulated DLD, Mup1, CPS1, Ces3b, PDI, and HYOU1 compared to the HFD-SP (Figure 4 and Figure 5). These proteins are related to the lipid metabolism, fructose/glucose metabolism, amino acid metabolism, and endoplasmic reticulum (ER) and oxidative stress (Table 1 and Table 2). When comparing CON-SP and CON-TMP, the pattern of changes was similar to the change between HFD-SP and HFD-TMP, but the change was less than that of the HFD (data not shown).

2.5. TMP Has a Similar Effect on the Serum Lipid Contents and Inflammation Markers to SP

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, TG level, high-density lipoprotein (HDL)-cholesterol/total cholesterol ratio (HTR), atherogenic index (AI) in the serum, and fecal FFA level showed only a fat effect. The fecal TG and cholesterol concentration revealed an interaction between TMP and fat. TMP did not change the parameters significantly (Table 3).

3. Discussion

A recent study reported that defatted TMP (200 mg/kg body weight, oral supplementation) prevented alcoholic hepatic steatosis in rats by regulating de novo TG and cholesterol synthesis [12]. It had a higher amino acid content than the non-fermented extract. Moreover, its total and essential free amino acid levels were approximately 170 and 112 times higher than those of SP used in this study, respectively (data not shown). Therefore, this study examined the role of TMP as an alternative protein by comparing it with SP. SP is a beneficial plant protein for various metabolic diseases; for example, obesity, diabetes, and hyperlipidemia.
HFD-TMP fed for 12 weeks helped suppress the body weight gain, fat size, and visceral fat mass (perirenal and mesenteric WAT) in mice. Hepatic TG and FFA contents in HFD-SP were not significantly different compared to the CON-SP, but SP did not suppress the high-fat-induced lipid droplets, as evidenced by H&E and Oil Red O staining. However, HFD-TMP decreased hepatic lipid accumulation (lipid contents and lipid droplets) as compared to the HFD-SP group, which might be mediated by the down-regulation of PPARγ (lipid synthesis transcription factor) and its downstream genes, such as CD36 (fatty acid uptake) and DGAT2 (TG synthesis).
Proteomic analysis was accomplished the understanding of the underlying mechanism of the anti-steatotic effects of TMP. The proteins whose expression was increased or decreased by the HFD-SP were also affected by HFD-TMP. The expressed proteins were involved mainly in fructolysis/gluconeogenesis, energy expenditure, amino acid metabolism, and stress response.
The fructolysis/gluconeogenesis-controlling proteins, such as ALDOB, KHK, and FBP1, were up-regulated in the HFD-SP group compared with the CON-SP group, which is consistent with a previous result [16]. Fructose is metabolized mainly via ketohexokinase (KHK), which phosphorylates fructose to fructose-1-phosphate in the liver. ALDOB then splits fructose-1-phosphate into dihydroxyacetone phosphate (DHAP) and glyceraldehyde (GA). Fructose-derived triose-phosphate can be used for gluconeogenesis or de novo lipogenesis [17]. A previous study announced that the suppression of KHK in the liver resulted in a reduction of the enzymes involved in fatty acid synthesis [18]. The results showed that TMP substitution down-regulated KHK and FBP1 expression compared to the HFD-SP group; the change in ALDOB expression showed a lower trend. FBP1 is a gluconeogenesis enzyme that is promoted by obesity and high fat intake [19,20]. Therefore, TMP might suppress hepatic lipogenesis and HOMA-IR by regulating the fructose/glucose-related metabolism, such as KHK and FBP1, in HFD-fed mice.
Interestingly, an HFD-SP suppressed DLD and ACO1 protein expression, whereas it induced GLO1 protein expression compared to the CON-SP group. The decreases in DLD and ACO1 protein expression mean a decrease in the tricarboxyl acid (TCA) cycle, which can lead to an increase in methylglyoxal production from triose-phosphate. Methylglyoxal is the most potent glycating agent. Its accumulation in the liver could be an early incident in decreased insulin sensitivity due to binding to the major players of the insulin-signaling pathway [21]. GLO1 is involved in the detoxification of methylglyoxal that is mainly produced by removing phosphate from glyceraldehyde 3-phosphate (GA3P) and DHAP [21]. A previous proteomics study presented that the GLO1 protein in the liver of ob/ob mice was decreased threefold compared to the lean controls [22]. In the current study, GLO1 protein expression was up-regulated under the condition of HFD consumption, but TMP significantly suppressed GLO1, indicating lower triose-phosphate production in the HFD-TMP group. The above-mentioned reduction of DLD and ACO1 by the HFD may be associated with the up-regulation of ATP5H. ATP5H, a subunit of ATP synthase, produces ATP [23], which is involved in the adaptive response of SP on a HFD. However, this activated ATP5H is related to the production of reactive oxygen species (ROS). In this study, SOD and DDAH1 were up-regulated by the HFD. DDAH1 plays a major role in the degradation of asymmetrical dimethylarginine, which is an inhibitor of nitric oxide synthesis [24]. On the other hand, TMP reversed the changes in ATP5H, SOD, and DDAH1 induced by the HFD.
TMP dramatically up-regulated Mup1 expression that had been decreased by the HFD. Mup1 is a secreted protein produced predominantly in the liver, which increases energy expenditure and physical activity and improves the glucose intolerance and hyperinsulinemia [25]. Zhou et al. [26] suggested that hepatic Mup1 reduction contributes to hyperglycemia, insulin resistance, and glucose intolerance in diabetes. Baur et al. [27] reported that resveratrol increased mitochondrial biogenesis and energy expenditure in high-fat-induced obese mice, which was mediated by up-regulating hepatic Mup1 expression. CPS1 protein expression in the HFD-TMP was also up-regulated significantly compared to the HFD-SP. CPS1 is required for ammonia elimination and amino acid catabolism, whose gene and protein expression was reduced by the HFD [27]. De Chiara et al. [28] showed that hepatosteatosis was associated with decreased CPS1 expression in a dose-dependent manner [28]. NAFLD progression is related to the mitochondrial dysfunction and metabolites conversion of the urea cycle [29]. Previous studies reported that NAFLD patients [29] and a diet-induced non-alcoholic steatohepatitis (NASH) model [30] are deficient in ureagenesis. In this study, TMP up-regulated CPS1 accompanied by a decrease in fat in the liver.
A dysfunctional metabolism is located at the center of the pathogenesis for NAFLD and involves mitochondrial dysfunction, lipid dysmetabolism, and oxidative stress. Oxidative stress is believed to be a key etiology for NAFLD [31]. The ER provides an environment for disulfide formation and protein folding. Each disulfide bond generated during oxidative protein folding creates a single ROS. It is estimated that secretory cells produce 3–6 million disulfide bonds per minute. In this way, protein folding in the ER is linked to the production of ROS and oxidative stress [32,33].
On the contrary, cellular oxidative stress can interfere with ER homeostasis and induce ER stress [34,35,36]. In the current study, a HFD-SP caused the down-regulation of PDI, HSPA5, and HYOU1 compared to the CON-SP group, but HFD-TMP up-regulated HYOU1 and PDI significantly relative to the HFD-SP. HYOU1, an ER chaperone protein, increases in various tissues under hypoxic conditions but is highly expressed in the pancreatic β cells and liver, even in normoxia [37,38]. High expression of HYOU1 is considered to protect ER stress and can lower insulin resistance in mice [39,40]. Wang et al. [41] reported that the amelioration of lipid-induced ER stress in the liver by Sirt1/AMPK pathway activation is associated with the induction of HYOU1. Moreover, protein disulfide-isomerase (PDI) is required for optimal insulin production to maintain glucose homeostasis in metabolic diseases [42]. The results of the present study suggest that the selective up-regulation of protein chaperones by TMP alternative supplementation in the liver should improve insulin action and hepatic steatosis.
Finally, Ces3b hydrolyzes long-chain fatty acids and thioesters that would play a role in the lipid metabolism. Ces3b helps provide substrates for the assembly of very-low-density lipoprotein (VLDL) in the liver. Previous in vitro and in vivo studies showed that the suppression of Ces3b lowered VLDL secretion [43]. Blocking VLDL assembly and secretion can lead to severe lipid accumulation in the liver [44]. This situation was avoided in TG hydrolase-deficient mice because of the compensatory effect of the decreased non-esterified fatty acid flux from the adipose tissue to the liver and the elevated fatty acid oxidation [45,46]. Elevated TGH expression was observed in patients with steatosis and NASH [43]. This study showed that down-regulation of Ces3b in the HFD-TMP group might be related to lower hepatic lipid concentration.
In conclusion, the substitution of SP with TMP alleviated HFD-induced obesity, hepatosteatosis, and insulin resistance, which might be mediated by regulating the protein expression related to energy expenditure, lipid synthesis from glucose, and ER stress in the liver of mice. These results propose that TMP is possibly better than SP under HFD feeding conditions. Moreover, TMP may potentially be a substitute for a commonly used protein source.

4. Materials and Methods

4.1. TMP Preparation

Preparation of TMP was as described in a previous study [12]. Defatted and freeze-dried powder was fermented for 72 h using a Saccharomyces cerevisiae strain (KCTC 17299, Korean Cell Line Bank, Seoul, Korea). Fermented mealworm powder was added to replace the peptone in the yeast–peptone dextrose medium. After that, 1 L yeast/defatted mealworm fermented broth were with 70% fermented alcohol (1L). Extracts were concentrated and freeze-dried.

4.2. Animal Experiments

Male C57BL/6N mice (4-week-old, n = 40) were obtained (Orient Bio Inc., Seoul, Korea) and housed under controlled light (light 8 a.m.–8 p.m., dark 8 p.m.–8 a.m.), temperature (22 ± 2 °C), and humidity (50 ± 5%) conditions. Mice had free access to food and water. Experiments were performed in accordance with the Sunchon National University Institutional Animal Care and Use Committee ethical guidelines (approved under no. SCNU_IACUC-2020–08).
After 7 days of adaptation, the mice were divided randomly into the four groups (n = 10 per group): control diet containing SP (CON-SP), control diet containing TMP (CON-TMP), high-fat diet containing SP (HFD-SP), and high-fat diet containing TMP (HFD-TMP). All groups received an AIN-76 standard diet with 20% protein. SP (Shandong Yuxin Bio-Tech Co., Ltd., Shandong, China) and TMP groups had 20% SP or 20% TMP added into the control and high-fat diet, respectively, for 12 weeks. Composition of the diets is presented in Table 4. Food intake and body weight were monitored three times and once per week, respectively. At the end of the experimental period, mice were fasted overnight and anesthetized. The organs (serum, liver, and WAT) were collected.

4.3. Oral Glucose Tolerance Test

After 12 weeks of treatment, the mice were fasted for 6 h, and then administered glucose (1 g/kg body weight). Blood samples were collected from the tail vein at four time-points: 0, 30, 60, and 120 min after oral glucose load. The fasting blood glucose concentration was monitored using a G-doctor (AllMedicus, Co., Ltd., Anyang, Korea).

4.4. Serum Parameters

The serum insulin (M1104, Morinaga Institute of Biological Science, Inc., Yokohama, Japan), tumor necrosis factor-α (TNF-α; BMS607HS, Invitrogen, Carlsbad, CA, USA), interleukin-6 (IL-6; BMS603HS, Invitrogen), adiponectin (DY1119, R&D Systems, Minneapolis, MN, USA), and leptin levels (DY498, R&D Systems) were determined using an enzyme-linked immunosorbent assays (ELISA) kit. The serum AST and ALT levels were measured using a Fuji Dri-Chem 3500i (Fujifilm, Tokyo, Japan). The serum glucose level was determined using the enzymatic method (Asan Pharmaceutical Co., Ltd., Seoul, Korea). The HOMA-IR was calculated using the following formula: insulin (µIU/mL) × glucose (mmol/L) / 22.5.

4.5. Lipid Profile Evaluation

The serum TG, cholesterol, HDL cholesterol (Asan Pharmaceutical Co., Ltd.), and FFA (Shinyang Diagnostics, Seoul, Korea) levels were measured using reagents according to the manufacturers’ instructions. The hepatic and fecal lipid contents (TG, FFA, and cholesterol) were monitored after extraction, according to Folch et al. [47].

4.6. Histological Analysis

The liver and epididymal WAT were fixed in 10% neutral buffered formalin and stained by H&E. The liver sections were stained with trichrome and Oil Red O. All images were captured using an optical microscope at 200× magnification.

4.7. RNA Isolation and Quantitative Real-Time PCR Analysis

The total RNA was isolated from the liver tissue using a TRIzol reagent (15596018, Invitrogen) according to the manufacturer’s instructions. The RNA concentration and purity were evaluated using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The complementary DNA (cDNA) was synthesized from the total RNA (1 μg) using a ReverTra Ace qPCR RT master mix (FSQ-201, Toyobo, Osaka, Japan). Real-time PCR was performed using an SYBR green PCR kit (204143, Qiagen, Hilden, Germany) with a specific primer (Table S1) in a CFX96 TouchTM real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were analyzed using the 2−△△CT method [48] and normalized to GAPDH in the same cDNA samples.

4.8. Protein Sample Preparation

The liver tissues were homogenized directly using motor driven homogenizer (PowerGen125, Fisher Scientific, Pittsburgh, PA, USA) in sample lysis solution containing 7 M urea (Sigma-Aldrich, St. Louis, MO, USA), 2 M thiourea (Sigma-Aldrich) containing 4% (w/v) 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate (CHAPS, Sigma-Aldrich), 1% (w/v) dithiothreitol (DTT, Sigma-Aldrich), 2% (v/v) pharmalyte (Amersham Biosciences, Little Chalfont, UK), and 1 mM benzamidine (Sigma-Aldrich). The proteins were extracted for one hour at room temperature with vortexing. After centrifugation at 15,000× g for one hour at 15 °C, the insoluble material was discarded and the soluble fraction was used for two-dimensional gel electrophoresis. The protein concentration was assayed using the Bradford method [49].

4.9. Two-Dimensional Polyacrylamide Gel Electrophoresis (2-DE)

2-DE was conducted as previously described [50]. For 2-DE analysis, immobilized pH gradient (IPG) strips (Genomine Inc., Pohang, Korea) were rehydrated in swelling buffer containing 7 M urea (Sigma-Aldrich), 2 M thiourea (Sigma-Aldrich), 1% (w/v) DTT (Sigma-Aldrich), and 4% (w/v) CHAPS (Sigma-Aldrich). Isoelectric focusing (IEF) was performed at 20 °C. The gels were stained with Coomassie G250, and then destained using deionized water and images were acquired with an image scanner (Bio-Rad Laboratories) as described by Anderson et al. [31].

4.10. Peptide Mass Fingerprinting (PMF)

Peptide mass fingerprinting was performed as previous described [51]. For protein identification by peptide mass fingerprinting, the protein spots were excised, digested with trypsin (Promega, Madison, WI, USA), mixed with α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in 50% acetonitrile (Sigma-Aldrich) with 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich), and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF, Microflex LRF 20, Bruker Daltonics, Billerica, MA, USA), as described by Fernandez et al. [52]. The spectra were collected from 300 shots per spectrum over the m/z range 700–4000 and calibrated by two-point internal calibration using Trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). The peak list was generated using Flex Analysis 3.0. The search program MASCOT, developed by The Matrixscience (http://www.matrixscience.com/, accessed on 8 December 2020), was used for protein identification by peptide mass fingerprinting.

4.11. Statistical Analysis

The data are presented as the means ± standard error (S.E.), and p values < 0.05 were considered significant. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS version 26, SPSS Inc., Chicago, IL, USA). The data were analyzed by two-way ANOVA followed by a Turkey post hoc test to examine the differences between the group means. The proteomic data between the HFD-SP group and HFD-TMP group were compared using a Student’s t-test.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms22073615/s1, Table S1: Primer sequences.

Author Contributions

Conceptualization, J.R.H. and M.-K.L.; Data curation, R.-Y.C., J.R.H., and Y.L.; Formal analysis, J.R.H., R.-Y.C., and M.-K.L.; Investigation, J.R.H., R.-Y.C., and Y.L.; Methodology, J.R.H., R.-Y.C., and M.-K.L.; Supervision, M.-K.L.; Writing—original draft, J.R.H. and M.-K.L.; Writing—review & editing, R.-Y.C. and M.-K.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2020R1A2C1009008).

Institutional Review Board Statement

The study was approved by the Inatitutional Animal Care and Use Committee of Sunchon National University (approval no. SCNU_IACUC-2020-08 and approval date. 06/04/2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Anstee, Q.M.; Reeves, H.L.; Kotsiliti, E.; Govaere, O.; Heikenwalder, M. From NASH to HCC: Current concepts and future challenges. Nat. Rev. Gastroenterol. Hepatol. 2019, 16, 411–428. [Google Scholar] [CrossRef] [PubMed]
  2. Berg, E.H.V.D.; Amini, M.; Schreuder, T.C.M.A.; Dullaart, R.P.F.; Faber, K.N.; Alizadeh, B.Z.; Blokzijl, H. Prevalence and determinants of non-alcoholic fatty liver disease in lifelines: A large Dutch population cohort. PLoS ONE 2017, 12, e0171502. [Google Scholar] [CrossRef]
  3. Ong, J.P.; Elariny, H.; Collantes, R.; Younoszai, A.; Chandhoke, V.; Reines, H.D.; Goodman, Z.; Younossi, Z.M. Predictors of nonalcoholic steatohepatitis and advanced fibrosis in morbidly obese patients. Obes. Surg. 2005, 15, 310–315. [Google Scholar] [CrossRef]
  4. Eguchi, Y.; Nafld, J.; Hyogo, H.; Ono, M.; Mizuta, T.; Ono, N.; Fujimoto, K.; Chayama, K.; Saibara, T. Prevalence and associated metabolic factors of nonalcoholic fatty liver disease in the general population from 2009 to 2010 in Japan: A multicenter large retrospective study. J. Gastroenterol. 2012, 47, 586–595. [Google Scholar] [CrossRef]
  5. Chang, Y.; Jung, H.-S.; Cho, J.; Zhang, Y.; Yun, K.E.; Lazo, M.; Pastor-Barriuso, R.; Ahn, J.; Kim, C.-W.; Rampal, S.; et al. Metabolically healthy obesity and the development of nonalcoholic fatty liver disease. Am. J. Gastroenterol. 2016, 111, 1133–1140. [Google Scholar] [CrossRef]
  6. Pang, Q.; Zhang, J.-Y.; Song, S.-D.; Qu, K.; Xu, X.-S.; Liu, S.-S.; Liu, C. Central obesity and nonalcoholic fatty liver disease risk after adjusting for body mass index. World J. Gastroenterol. 2015, 21, 1650–1662. [Google Scholar] [CrossRef] [PubMed]
  7. Van Thielen, L.; Vermuyten, S.; Storms, B.; Rumpold, B.; Van Campenhout, L. Consumer acceptance of foods containing edible insects in Belgium two years after their introduction to the market. J. Insects Food Feed. 2019, 5, 35–44. [Google Scholar] [CrossRef]
  8. Im, A.-R.; Yang, W.-K.; Park, Y.-C.; Kim, S.H.; Chae, S. Hepatoprotective effects of insect extracts in an animal model of nonalcoholic fatty liver disease. Nutrients 2018, 10, 735. [Google Scholar] [CrossRef] [PubMed]
  9. Hong, J.; Han, T.; Kim, Y. Mealworm (Tenebrio molitor Larvae) as an alternative protein source for monogastric animal: A review. Animals 2020, 10, 2068. [Google Scholar] [CrossRef]
  10. Gessner, D.K.; Schwarz, A.; Meyer, S.; Wen, G.; Most, E.; Zorn, H.; Ringseis, R.; Eder, K. Insect meal as alternative protein source exerts pronounced lipid-lowering effects in hyperlipidemic obese Zucker rats. J. Nutr. 2019, 149, 566–577. [Google Scholar] [CrossRef]
  11. Sim, S.; Jang, S.; Cho, Y.; Ahn, H. Effect of Tenebrio molitor larvae fermented with Saccharomyces cerevisiae M1 (KACC 93023) on non-alcoholic fatty liver-induced rats. J. Life Sci. 2020, 30, 434–442. [Google Scholar]
  12. Choi, R.-Y.; Ham, J.R.; Ryu, H.-S.; Lee, S.S.; Miguel, M.A.; Paik, M.-J.; Ji, M.; Park, K.-W.; Kang, K.-Y.; Lee, H.-I.; et al. Defatted Tenebrio molitor larva fermentation extract modifies steatosis, inflammation and intestinal microflora in chronic alcohol-fed rats. Nutrients 2020, 12, 1426. [Google Scholar] [CrossRef]
  13. Shang, X.; Scott, D.; Hodge, A.M.; English, D.R.; Giles, G.G.; Ebeling, P.R.; Sanders, K.M. Dietary protein intake and risk of type 2 diabetes: Results from the Melbourne collaborative cohort study and a meta-analysis of prospective studies. Am. J. Clin. Nutr. 2016, 104, 1352–1365. [Google Scholar] [CrossRef]
  14. Navas-Carretero, S.; San-Cristobal, R.; Livingstone, K.M.; Celis-Morales, C.; Marsaux, C.F.; Macready, A.L.; Fallaize, R.; O’Donovan, C.B.; Forster, H.; Woolhead, C.; et al. Higher vegetable protein consumption, assessed by an isoenergetic macronutrient exchange model, is associated with a lower presence of overweight and obesity in the web-based Food4me European study. Int. J. Food Sci. Nutr. 2019, 70, 240–253. [Google Scholar] [CrossRef] [Green Version]
  15. De Chiara, F.; Checcllo, C.U.; Azcón, J.R. High protein diet and metabolic plasticity in non-alcoholic fatty liver disease: Myths and truths. Nutrients 2019, 11, 2985. [Google Scholar] [CrossRef] [Green Version]
  16. Soltis, A.R.; Kennedy, N.J.; Xin, X.; Zhou, F.; Ficarro, S.B.; Yap, Y.S.; Matthews, B.J.; Lauffenburger, D.A.; White, F.M.; Marto, J.A. Hepatic dysfunction caused by consumption of a high-fat diet. Cell Rep. 2017, 21, 3317–3328. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Chiu, S.; Mulligan, K.; Schwarz, J.M. Dietary carbohydrates and fatty liver disease: De novo lipogenesis. Curr. Opin. Clin. Nutr. Metab. Care 2018, 21, 277–282. [Google Scholar] [CrossRef] [PubMed]
  18. Softic, S.; Gupta, M.K.; Wang, G.-X.; Fujisaka, S.; O’Neill, B.T.; Rao, T.N.; Willoughby, J.; Harbison, C.; Fitzgerald, K.; Ilkayeva, O.; et al. Divergent effects of glucose and fructose on hepatic lipogenesis and insulin signaling. J. Clin. Investig. 2017, 127, 4059–4074. [Google Scholar] [CrossRef] [Green Version]
  19. Andrikopoulos, S.; Proietto, J. The biochemical basis of increased hepatic glucose production in a mouse model of type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1995, 38, 1389–1396. [Google Scholar] [CrossRef] [PubMed]
  20. Song, S.; Andrikopoulos, S.; Filippis, C.; Thorburn, A.W.; Khan, D.; Proietto, J. Mechanism of fat-induced hepatic gluconeogenesis: Effect of metformin. Am. J. Physiol. Metab. 2001, 281, E275–E282. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  21. Sanchez, J.-C.; Converset, V.; Nolan, A.; Schmid, G.; Wang, S.; Heller, M.; Sennitt, M.V.; Hochstrasser, D.F.; Cawthorne, M.A. Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57Bl/6 lep/lep mice. Mol. Cell. Proteom. 2002, 1, 509–516. [Google Scholar] [CrossRef] [Green Version]
  22. Rabbani, N.; Thornalley, P.J. Glyoxalase in Diabetes, Obesity and Related Disorders, Seminars in Cell & Developmental Biology; Elsevier: Amsterdam, The Netherlands, 2011; pp. 309–317. [Google Scholar]
  23. Xu, C.; Zhang, X.; Yu, C.; Lu, G.; Chen, S.; Xu, L.; Ding, W.; Shi, Q.; Li, Y. Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5β. Proteomics 2009, 9, 409–419. [Google Scholar] [CrossRef]
  24. Ogawa, T.; Kimoto, M.; Sasaoka, K. Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats. Biochem. Biophys. Res. Commun. 1987, 148, 671–677. [Google Scholar] [CrossRef]
  25. Hui, X.; Zhu, W.; Wang, Y.; Lam, K.S.L.; Zhang, J.; Wu, D.; Kraegen, E.W.; Li, Y.; Xu, A. Major urinary protein-1 increases energy expenditure and improves glucose intolerance through enhancing mitochondrial function in skeletal muscle of diabetic mice. J. Biol. Chem. 2009, 284, 14050–14057. [Google Scholar] [CrossRef] [Green Version]
  26. Zhou, Y.; Jiang, L.; Rui, L. Identification of MUP1 as a regulator for glucose and lipid metabolism in mice. J. Biol. Chem. 2009, 284, 11152–11159. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Baur, J.A.; Pearson, K.J.; Price, N.L.; Jamieson, H.A.; Lerin, C.; Kalra, A.; Prabhu, V.V.; Allard, J.S.; Lopez-Lluch, G.; Lewis, K.; et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006, 444, 337–342. [Google Scholar] [CrossRef] [PubMed]
  28. De Chiara, F.; Heebøll, S.; Marrone, G.; Montoliu, C.; Hamilton-Dutoit, S.; Ferrandez, A.; Andreola, F.; Rombouts, K.; Grønbæk, H.; Felipo, V.; et al. Urea cycle dysregulation in non-alcoholic fatty liver disease. J. Hepatol. 2018, 69, 905–915. [Google Scholar] [CrossRef] [Green Version]
  29. Eriksen, P.L.; Vilstrup, H.; Rigbolt, K.; Suppli, M.P.; Sørensen, M.; Heebøll, S.; Veidal, S.S.; Knop, F.K.; Thomsen, K.L. Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion. Liver Int. 2019, 39, 2094–2101. [Google Scholar] [CrossRef]
  30. De Chiara, F.; Thomsen, K.L.; Habtesion, A.; Jones, H.; Davies, N.; Gracia-Sancho, J.; Manicardi, N.; Hall, A.; Andreola, F.; Paish, H.L.; et al. Ammonia scavenging prevents progression of fibrosis in experimental nonalcoholic fatty liver disease. Hepatology 2020, 71, 874–892. [Google Scholar] [CrossRef]
  31. Anderson, N.L.; Esquer-Blasco, R.; Hofmann, J.-P.; Meheus, L.; Raymackers, J.; Steiner, S.; Witzmann, F.; Anderson, N.G.; Anderson, N.L.; Anderson, N.G. An updated two-dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies. Electrophoresis 1995, 16, 1977–1981. [Google Scholar] [CrossRef]
  32. Santos, C.X.; Tanaka, L.Y.; Wosniak, J., Jr.; Laurindo, F.R. Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: Roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid. Redox Signal. 2009, 11, 2409–2427. [Google Scholar] [CrossRef]
  33. Shimizu, Y.; Hendershot, L.M. Oxidative folding: Cellular strategies for dealing with the resultant equimolar production of reactive oxygen species. Antioxid. Redox Signal. 2009, 11, 2317–2331. [Google Scholar] [CrossRef]
  34. Hanada, S.; Harada, M.; Kumemura, H.; Omary, M.B.; Koga, H.; Kawaguchi, T.; Taniguchi, E.; Yoshida, T.; Hisamoto, T.; Yanagimoto, C.; et al. Oxidative stress induces the endoplasmic reticulum stress and facilitates inclusion formation in cultured cells. J. Hepatol. 2007, 47, 93–102. [Google Scholar] [CrossRef] [PubMed]
  35. Xue, X.; Piao, J.-H.; Nakajima, A.; Sakon-Komazawa, S.; Kojima, Y.; Mori, K.; Yagita, H.; Okumura, K.; Harding, H.; Nakano, H. Tumor necrosis factor α (TNFα) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFα. J. Biol. Chem. 2005, 280, 33917–33925. [Google Scholar] [CrossRef] [Green Version]
  36. Yokouchi, M.; Hiramatsu, N.; Hayakawa, K.; Okamura, M.; Du, S.; Kasai, A.; Takano, Y.; Shitamura, A.; Shimada, T.; Yao, J.; et al. Involvement of selective reactive oxygen species upstream of proapoptotic branches of unfolded protein response. J. Biol. Chem. 2008, 283, 4252–4260. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Ikeda, J.; Kaneda, S.; Kuwabara, K.; Ogawa, S.; Kobayashi, T.; Matsumoto, M.; Yura, T.; Yanagi, H. Cloning and expression of cDNA encoding the human 150 kDa oxygen-regulated protein, ORP150. Biochem. Biophys. Res. Commun. 1997, 230, 94–99. [Google Scholar] [CrossRef]
  38. Kobayashi, T.; Ogawa, S.; Yura, T.; Yanagi, H. Abundant expression of 150-kDa oxygen-regulated protein in mouse pancreatic beta cells is correlated with insulin secretion. Biochem. Biophys. Res. Commun. 2000, 267, 831–837. [Google Scholar] [CrossRef]
  39. Ozawa, K.; Miyazaki, M.; Matsuhisa, M.; Takano, K.; Nakatani, Y.; Hatazaki, M.; Tamatani, T.; Yamagata, K.; Miyagawa, J.-I.; Kitao, Y.; et al. The endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes. Diabetes 2005, 54, 657–663. [Google Scholar] [CrossRef] [Green Version]
  40. Sanson, M.; Augé, N.; Vindis, C.; Muller, C.; Bando, Y.; Thiers, J.; Marachet, M.; Zarkovic, K.; Sawa, Y.; Salvayre, R. Oxidized low-density lipoproteins trigger endoplasmic reticulum stress in vascular cells: Prevention by oxygen-regulated protein 150 expression. Circ. Res. 2009, 104, 328–336. [Google Scholar] [CrossRef] [Green Version]
  41. Wang, Y.; Wu, Z.; Li, D.; Wang, D.; Wang, X.; Feng, X.; Xia, M. Involvement of oxygen-regulated protein 150 in AMP-activated protein kinase-mediated alleviation of lipid-induced endoplasmic reticulum stress. J. Biol. Chem. 2011, 286, 11119–11131. [Google Scholar] [CrossRef] [Green Version]
  42. Jang, I.; Pottekat, A.; Poothong, J.; Yong, J.; Lagunas-Acosta, J.; Charbono, A.; Chen, Z.; Scheuner, D.L.; Liu, M.; Itkin-Ansari, P.; et al. PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity. eLife 2019, 8, 44528. [Google Scholar] [CrossRef]
  43. Lian, J.; Wei, E.; Groenendyk, J.; Das, S.K.; Hermansson, M.; Li, L.; Watts, R.; Thiesen, A.; Oudit, G.Y.; Michalak, M.; et al. Ces3/TGH deficiency attenuates steatohepatitis. Sci. Rep. 2016, 6, 25747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Raabe, M.; Véniant, M.M.; Sullivan, M.A.; Zlot, C.H.; Björkegren, J.; Nielsen, L.B.; Wong, J.S.; Hamilton, R.L.; Young, S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J. Clin. Investig. 1999, 103, 1287–1298. [Google Scholar] [CrossRef] [Green Version]
  45. Wei, E.; Ben Ali, Y.; Lyon, J.; Wang, H.; Nelson, R.; Dolinsky, V.W.; Dyck, J.R.; Mitchell, G.; Korbutt, G.S.; Lehner, R. Loss of TGH/Ces3 in mice decreases blood lipids, improves glucose tolerance, and increases energy expenditure. Cell Metab. 2010, 11, 183–193. [Google Scholar] [CrossRef] [Green Version]
  46. Lian, J.; Wei, E.; Wang, S.P.; Quiroga, A.D.; Li, L.; Di Pardo, A.; Van Der Veen, J.; Sipione, S.; Mitchell, G.A.; Lehner, R. Liver specific inactivation of carboxylesterase 3/triacylglycerol hydrolase decreases blood lipids without causing severe steatosis in mice. Hepatology 2012, 56, 2154–2162. [Google Scholar] [CrossRef]
  47. Folch, J.; Lees, M.; Stanley, G.S. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957, 226, 497–509. [Google Scholar] [CrossRef]
  48. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  49. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  50. Bahk, Y.Y.; Kim, S.A.; Kim, J.-S.; Euh, H.-J.; Bai, G.-H.; Cho, S.-N.; Kim, Y.S. Antigens secreted from Mycobacterium tuberculosis: Identification by proteomics approach and test for diagnostic marker. Proteomics 2004, 4, 3299–3307. [Google Scholar] [CrossRef]
  51. Lee, Y.; Hwang, Y.-H.; Kim, K.-J.; Park, A.-K.; Paik, M.-J.; Kim, S.H.; Lee, S.U.; Yee, S.-T.; Son, Y.-J. Proteomic and transcriptomic analysis of lung tissue in OVA-challenged mice. Arch. Pharmacal Res. 2018, 41, 87–100. [Google Scholar] [CrossRef] [Green Version]
  52. Fernandez, J.; Gharahdaghi, F.; Mische, S.M. Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis 1998, 19, 1036–1045. [Google Scholar] [CrossRef] [PubMed]
Ijms 22 03615 g001 550
Figure 1. Body weight, body weight gain, fat mass, and adipocyte size. The values are expressed as the means ± S.E. (n = 10 per group). † vs. CON-SP, †† vs. CON-TMP and ††† vs. HFD-SP by two-way ANOVA followed by a Turkey post hoc test (p < 0.05). * p < 0.05 vs. HFD-SP by Student’s t-test. Histological analysis magnification 200×. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); 2WA TMP × FAT, interaction between TMP and fat diet in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP.
Figure 1. Body weight, body weight gain, fat mass, and adipocyte size. The values are expressed as the means ± S.E. (n = 10 per group). † vs. CON-SP, †† vs. CON-TMP and ††† vs. HFD-SP by two-way ANOVA followed by a Turkey post hoc test (p < 0.05). * p < 0.05 vs. HFD-SP by Student’s t-test. Histological analysis magnification 200×. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); 2WA TMP × FAT, interaction between TMP and fat diet in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP.
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Ijms 22 03615 g002 550
Figure 2. Serum insulin, glucose, and leptin levels; HOMA-IR; and OGTT. The values are expressed as the means ± S.E. (n = 10 per group). * p < 0.05, *** p < 0.001 vs. HFD-SP by Student’s t-test. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP; HOMA-IR, homeostasis model assessment of insulin resistance; OGTT, oral glucose tolerance test.
Figure 2. Serum insulin, glucose, and leptin levels; HOMA-IR; and OGTT. The values are expressed as the means ± S.E. (n = 10 per group). * p < 0.05, *** p < 0.001 vs. HFD-SP by Student’s t-test. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP; HOMA-IR, homeostasis model assessment of insulin resistance; OGTT, oral glucose tolerance test.
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Figure 3. Histology, lipid contents, and lipid metabolism-related gene expression in the liver. The values are expressed as the means ± S.E. (n = 10 per group). † vs. CON-SP and ††† vs. HFD-SP by two-way ANOVA followed by a Turkey post hoc test (p < 0.05). ### p < 0.001 vs. CON-SP, *** p < 0.001 vs. HFD-SP by Student’s t-test. Histological analysis magnification 200×. Yellow arrows indicated the lipid droplets or fibrosis. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); 2WA TMP × FAT, interaction between TMP and fat diet in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP; H&E, hematoxylin and eosin.
Figure 3. Histology, lipid contents, and lipid metabolism-related gene expression in the liver. The values are expressed as the means ± S.E. (n = 10 per group). † vs. CON-SP and ††† vs. HFD-SP by two-way ANOVA followed by a Turkey post hoc test (p < 0.05). ### p < 0.001 vs. CON-SP, *** p < 0.001 vs. HFD-SP by Student’s t-test. Histological analysis magnification 200×. Yellow arrows indicated the lipid droplets or fibrosis. 2WA FAT, fat diet effect in two-way ANOVA (p < 0.05); 2WA TMP, TMP effect in two-way ANOVA (p < 0.05); 2WA TMP × FAT, interaction between TMP and fat diet in two-way ANOVA (p < 0.05); CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP; H&E, hematoxylin and eosin.
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Figure 4. Two-dimensional electrophoresis of proteins in the liver. The 2-D PAGE image from the liver was used as a master gel and reference map. The HFD caused 37 spots to change. The protein spots were identified by MALDI-TOF (arrow) and are marked by their spot numbers. pI, isoelectric point.
Figure 4. Two-dimensional electrophoresis of proteins in the liver. The 2-D PAGE image from the liver was used as a master gel and reference map. The HFD caused 37 spots to change. The protein spots were identified by MALDI-TOF (arrow) and are marked by their spot numbers. pI, isoelectric point.
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Figure 5. Two-dimensional electrophoresis patterns of the proteins in the liver.
Figure 5. Two-dimensional electrophoresis patterns of the proteins in the liver.
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Table
Table 1. Effect of TMP on the proteins up-regulated by the high-fat diet.
Table 1. Effect of TMP on the proteins up-regulated by the high-fat diet.
No.SymbolProtein DescriptionAccessionFold Change Protein RatioProtein Sequence Coverage (%) bMWpIScore c
B/AC/Bp-Value of C/B a
1ALDOBFructose-bisphosphate aldolase BNP_659152.11.90.050.30645248.52148
2ADHAlcohol dehydrogenase 1NP_031435.12.380.170.33532398.44114
3KHKKetohexokinaseNP_032465.21.570.620.01963305.81158
4FBP1Fructose-1,6-bisphosphatase 1NP_062268.11.60.590.05761466.15242
5PCPyruvate carboxylase, mitochondrialNP_000911.21.690.610.40446626.25447
6GLO1Lactoylglutathione lyase (=glyoxalase 1)NP_079650.31.440.430.02972385.24182
7hsc71Heat shock cognate 71 kDaNP_112442.23.230.390.31144465.37224
8HSPA9Stress-70 proteinNP_034611.22.730.260.27737725.81274
9BHMTBetaine—homocysteine S-methyltransferase 1NP_057877.12.780.070.35157568.01160
10PHBProhibitinNP_032857.11.820.220.13853205.57181
11MAT1AS-adenosylmethionine synthase isoform type-1NP_598414.11.60.470.08948535.51183
12BCKDHA2-oxoisovalerate dehydrogenase subunit alphaNP_031559.31.910.630.11748458.15160
13hutUUrocanate hydratase NP_659189.21.470.620.27541447.27290
14HMGCS2Hydroxymethylglutaryl-CoA synthase, mitochondrialNP_032282.21.650.10.28137428.65120
15ATP5HATP synthase subunit d, mitochondrialNP_082138.11.690.680.03565495.52108
16SODSuperoxide dismutaseNP_035564.11.930.420.02242106.02108
17DDAH1N(G),N(G)-dimethylarginine dimethylaminohydrolase 1NP_081269.11.570.180.02235255.6488
18GSTM2Glutathione S-transferase Mu 7NP_080948.21.790.60.54249566.34146
19ALDH1A1Retinal dehydrogenase 1NP_038495.21.460.210.29937637.92123
20IMMTMICOS complex subunit MIC60NP_001240617.11.780.560.63133447.65135
a The p-value means Student’s t-test between the B and C groups. b Protein sequence coverage (%) is defined as the percentage of the whole length of the protein sequence which is covered by matched peptides identified by the MALDI-TOF/MS analysis. c Mascot scores greater than 61 in NCBI p ≤ 0.05 from Mascot search on MALDI-TOF/MS data were considered. A, control diet containing SP (CON-SP); B, high-fat diet containing SP (HFD-SP); C, high-fat diet containing TMP (HFD-TMP).
Table
Table 2. Effects of TMP on the proteins down-regulated by the high-fat diet.
Table 2. Effects of TMP on the proteins down-regulated by the high-fat diet.
No.SymbolProtein DescriptionAccessionFold Change Protein RatioProtein Sequence Coverage (%) bMWpIScore c
B/AC/Bp-Value of C/B a
21ACO1Cytoplasmic aconitate hydrataseNP_031412.20.492.030.14148567.23311
22DLDDihydrolipoyl dehydrogenase, mitochondrial NP_031887.20.435.620.02752457.99172
23Mup1Major urinary protein 1NP_001334083.10.4918.30.03466344.96130
24CPS1Carbamoyl-phosphate synthase ammonia, mitochondrialNP_001074278.10.297.260.01928516.48249
25Ces3bCarboxylesterase 3BNP_001152887.10.343.490.00020285.6584
26Ces3aIsoform 2 of carboxylesterase 3ANP_001158153.10.462.730.06934355.43123
27PDIProtein disulfide-isomeraseNP_035162.10.622.470.04350424.77223
28HSPA5Endoplasmic reticulum chaperone BiPNP_001156906.10.681.310.61141535.07268
29HYOU1Hypoxia up-regulated protein 1NP_067370.30.297.260.01922515.1278
30PNPPurine nucleoside phosphorylaseNP_038660.10.671.410.32141315.7899
31NME1Nucleoside diphosphate kinase ANP_032730.10.591.580.10773336.84131
32GSTP1Glutathione S-transferase P 1NP_038569.10.541.60.23662307.68164
33DDTD-dopachrome decarboxylase NP_034157.10.291.710.42985356.09107
34TfSerotransferrin NP_598738.10.611.430.21733366.94156
35FGAIsoform 2 of fibrinogen alpha chain NP_001104518.10.622.950.27233477.16122
36ALDH6A1Methylmalonate-semialdehyde dehydrogenase (acylating), mitochondrialNP_598803.10.613.790.21725578.2967
37ALDH12A1Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrialNP_780647.30.622.950.12826478.4580
a The p-value means Student’s t-test between the B and C groups. b Protein sequence coverage (%) is defined as the percentage of the whole length of the protein sequence which is covered by matched peptides identified by the MALDI-TOF/MS analysis. c Mascot scores greater than 61 in NCBI p ≤ 0.05 from Mascot search on MALDI-TOF/MS data were considered. A, control diet containing SP (CON-SP); B, high-fat diet containing SP (HFD-SP); C, high-fat diet containing TMP (HFD-TMP).
Table
Table 3. Serum markers and fecal lipid contents.
Table 3. Serum markers and fecal lipid contents.
CON-SPCON-TMPHFD-SPHFD-TMP2WA 1
Serum markers
AST (U/L)49.55 ± 3.3339.33 ± 1.5653.00 ± 5.2942.77 ± 2.67F 2 (p = 0.008)
ALT (U/L)22.36 ± 1.9517.22 ± 3.4833.82 ± 5.5727.80 ± 6.22F (p = 0.018)
TG (mg/dL)97.01 ± 9.7974.84 ± 8.8578.15 ± 7.1570.38 ± 6.51F (p = 0.000)
FFA (μmol/L)0.64 ± 0.040.55 ± 0.040.52 ± 0.040.52 ± 0.04
TC (mg/dL)120.01 ± 5.40112.91 ± 5.51149.41 ± 8.67153.84 ± 6.58
HDL-C (mg/dL)84.64 ± 4.7287.52 ± 4.5692.47 ± 5.3297.03 ± 5.64
HTR (%)70.40 ± 2.5977.53 ± 1.8762.19 ± 2.1763.34 ± 3.25F (p = 0.000)
AI0.44 ± 0.050.30 ± 0.030.63 ± 0.050.62 ± 0.08F (p = 0.000)
TNF-α (pg/mL)9.84 ± 0.719.49 ± 0.8010.68 ± 0.9110.86 ± 0.85
IL-6 (pg/mL)18.07 ± 4.6711.84 ± 1.2117.63 ± 2.6916.51 ± 2.54
Adiponectin (μg/mL)3.63 ± 0.183.55 ± 0.163.60 ± 0.143.36 ± 0.18
Fecal lipid contents
TG (mg/g)67.94 ± 5.66390.60 ± 15.67 †333.32 ± 15.26 †416.33 ± 73.37 †TxF 3 (p = 0.000)
FFA (μmol/g)61.16 ± 4.6364.83 ± 4.18155.48 ± 7.75172.52 ± 17.41F (p = 0.000)
Cholesterol (mg/g)66.39 ± 3.0748.69 ± 1.54 †51.22 ± 2.49 †60.23 ± 3.50TxF (p = 0.013)
The values are expressed as the means ± S.E. (n = 10 per group). † vs. CON-SP by two-way ANOVA followed by a Turkey post hoc test (p < 0.05). 1 2WA, two-way ANOVA, 2 F, fat diet effect in two-way ANOVA (p < 0.05), 3 TxF, interaction between TMP and fat diet in two-way ANOVA (p < 0.05). CON-SP, control diet containing SP; CON-TMP, control diet containing TMP; HFD-SP, high-fat diet containing SP; HFD-TMP, high-fat diet containing TMP.
Table
Table 4. Composition of experimental diets.
Table 4. Composition of experimental diets.
Ingredients (g/kg Diet)CON-SPCON-TMPHFD-SPHFD-TMP
Soy protein200-200-
TMP 1-200-200
DL-methionine3333
Choline bitartrate2222
Corn starch500500340340
Sucrose150150150150
Cellulose50505050
Corn oil50503030
Lard00180180
Mineral mixture35353535
Vitamin mixture10101010
1Tenebrio molitor larva fermentation extract.
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Ham, J.R.; Choi, R.-Y.; Lee, Y.; Lee, M.-K. Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet. Int. J. Mol. Sci. 2021, 22, 3615. https://doi.org/10.3390/ijms22073615

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Ham JR, Choi R-Y, Lee Y, Lee M-K. Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet. International Journal of Molecular Sciences. 2021; 22(7):3615. https://doi.org/10.3390/ijms22073615

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Ham, Ju Ri, Ra-Yeong Choi, Yongjin Lee, and Mi-Kyung Lee. 2021. "Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet" International Journal of Molecular Sciences 22, no. 7: 3615. https://doi.org/10.3390/ijms22073615

APA Style

Ham, J. R., Choi, R. -Y., Lee, Y., & Lee, M. -K. (2021). Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet. International Journal of Molecular Sciences, 22(7), 3615. https://doi.org/10.3390/ijms22073615

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