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Supplementation of non-fermented and fermented goji berry (Lycium barbarum) improves hepatic function and corresponding lipid metabolism via their anti-inflammatory and antioxidant properties in high fat-fed rats

Supplementation of non-fermented and fermented goji berry (Lycium barbarum) improves hepatic... Development of obesity is associated with excessive fat accumulation and oxidative stress along with chronic inflam‑ mation. Goji berries (Lycium barbarum) are high in polyphenolic compounds and have anti‑inflammatory, anti‑ oxidant, and hypolipidemic properties that may alleviate the pathogenesis of obesity and related metabolic complications. Thus, the aim of this study was to investigate potential metabolic benefits of GB supplementation against high fat (HF) diet‑induced obesity and its comorbidities in HF diet ‑fed rats (male Sprague–Dawley, n = 8/group, 6 weeks old). We also sought to examine the potential metabolic benefits of fermented GB (FGB) with L. plantarum CB3 and possi‑ ble distinctions in the degree and/or mechanism of action compared to GB. GB and FGB supplementation suppressed the gene expression of inflammation indices at the local (adipose tissues) and systemic (liver) levels. In addition, GB and FGB supplementation upregulated the gene expression of antioxidant enzymes compared to the HF and/or even low fat (LF) group with more remarkable antioxidant effects by GB supplementation. Also, GB and FGB supplementa‑ tion protected from HF‑induced damages of the liver and dyslipidemia. In conclusion, we demonstrated that GB and FGB supplementation protected from HF‑induced metabolic complications primarily by improving hepatic function and corresponding lipid metabolism via their anti‑inflammatory and antioxidant properties. To our knowledge, this is the first in vivo study confirming metabolic benefits of GB in a fermented form. Thus, these findings support the potential application of both GB and FGB to ameliorate obesity‑associated metabolic abnormalities. Keywords: Dyslipidemia, Fermentation, Goji berry (Lycium barbarum), Hepatic function, Inflammation, Obesity, Oxidative stress Introduction Obesity is primarily characterized by abnormal or excessive fat accumulation and a low-level systemic inflammation [1]. An increase in visceral adipos- ity serves as a strong predictor of local and systemic inflammation with its association with macrophage *Correspondence: suhhj@sunmoon.ac.kr infiltration and subsequent secretion of pro-inflamma- Department of Food Science, Research Center for Food and Bio tory cytokines [2]. The pivotal role of pro-inflamma- Convergence, Sun Moon University, Asan, Chungchengnam‑do 31460, Republic of Korea tory cytokines such as interleukin-1beta (IL-1β) and © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lee et al. Appl Biol Chem (2021) 64:70 Page 2 of 11 tumor necrosis factor alpha (TNFα) has been identi- Materials and methods fied in the development of obesity and related meta- Materials bolic dysregulation [3]. Further, the inflammatory Commercial kits for animal tests were purchased cascade can facilitate the formation of toxic reac- from Elabscience (Houston, Texas, USA). Goji berries tive oxygen species and the subsequent generation (Lycium barbarum) were purchased from local mar- of oxidative stress, which inhibits the expression of kets in Cheongyang Korea. Pectinex Ultra SP-L was antioxidant enzymes and consequently impairs the obtained from Vision Biochem (Seongnam, Korea). antioxidant defense system [3]. Excessive oxidative Lactobacillus plantarum CB3, isolated from kimchi in stress also can impair liver functions, in the regulation our laboratory, was used as a starter culture for fermen- of hepatic lipid metabolism in particular [4]. Previous tation of goji berries. studies have reported that HF-induced hepatic dys- function demonstrated by the elevated level of alkaline Preparation of GB and FGB powder phosphatase (ALP) and aspartate transaminase (AST) For the preparation of GB extract, 10  g of dried GB enzyme activities in circulation was associated with was added to 100  mL of 90  °C water and extracted for the upregulation of adipogenesis and lipogenesis tran- 6  h. Twenty mL of GB extract was taken and used as scription factors, resulting in hepatic fat accumulation a substrate for the production of FGB. Pectinex Ultra and dyslipidemia [5, 6]. SP-L (0.5%, w/v) was added into the GB extract, and Extensive attention has been paid to the role of ber- the mixture was reacted at 55 °C for 6 h with shaking at ries in the prevention of obesity and related meta- 160  rpm and sterilized at 100  °C for 10  min. The steri - bolic disturbances. Goji berries (Lycium barbarum) lized GB suspension was inoculated with 5% (v/v) of L. are high in polyphenolic compounds and have anti- plantarum CB3 culture [1 × 10 colony-forming units inflammatory, anti-oxidant, and hypolipidemic prop- (CFU)/mL] to give a final cell concentration of about erties that may affect disease pathogenesis [7]. Dietary 1 × 10   CFU/mL, and fermented at 37  °C for 72  h supplementation with goji berry (GB) reduced bio- with shaking at 160 rpm. The GB and FGB suspensions markers of oxidative stress and lipid peroxidation as were lyophilized to 88% solids using an EYELA freeze well as inflammatory gene expression in both experi- dryer (FDU-1200, Tokyo Rikakikai Co.) and used for mental and clinical studies [8–10]. In addition, Yang experiments. et  al. reported that GB supplementation ameliorated HF-induced insulin resistance via activation of phos- Analysis of betaine phatidylinositol-3 kinase/protein kinase B/nuclear fac- Quantitative analysis of betaine was performed using tor-E2-related factor 2 (PI3K/AKT/Nrf2) pathway [11]. the method proposed by Lee et al. with some modifica - Despite a growing evidence on metabolic benefits tions [15]. Betaine from GB and FGB samples (lyophi- of GB, there is limited knowledge on the potential lized to 88% solids) was detected using a 1260 Infinity benefits of GB in obesity and related metabolic dis- system (Agilent Technologies, Santa Clara, CA, USA) orders. Thus, the aim of this study was to investigate attached with an evaporative light scattering detector metabolic benefits of GB supplementation against HF (ELSD; Agilent Technologies, Santa Clara, CA, USA) diet-induced obesity and its comorbidities in rats. We and a Discovery C18 column (4.6 × 250  mm, 5  μm, hypothesized that GB supplementation would improve SUPELCO, PA, USA). The mobile phase contained HF-induced obese phenotypes and dysregulation of water and acetonitrile and was used in isocratic elution lipid metabolism in association with anti-inflamma- as at a 95:5 ratio (v/v %), respectively. The flow rate of tory and anti-oxidant mechanisms. Further, consid- the elution was 0.5 mL/min and the evaporator and the ering that fermentation of bioactive prebiotics with nebulizer temperature for the ELSD were 50 and 70 °C, probiotics such as Lactobacillus or Bifidobacterium respectively. has been confirmed to enhance the pharmacological efficacy and metabolic functions of the original non- Measurement of total phenolics and antioxidant activity fermented prebiotics in previous studies [12–14], we The total phenolic content of GB and FGB samples also sought to examine the potential metabolic ben- (lyophilized to 88% solids) was determined by modify- efits of fermented GB (FGB) with L. plantarum CB3 as ing the method of previous studies [16]. To determine probiotics and possible distinctions in the degree and/ total phenolic compound and antioxidant activity, or mechanism of action compared to GB. lyophilized GB and FGB samples were diluted with 80% methanol to a concentration of 5%, respectively. For the calibration curve of phenolic compounds, L ee et al. Appl Biol Chem (2021) 64:70 Page 3 of 11 gallic acid stock solution (5  mg/mL) was prepared Animals and experimental design and diluted with water. Five-hundred microliters Animals were maintained and handled in accordance of each sample extract was mixed with 300  μl of 1  N with protocols approved by the Institutional Ani- Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) mal Care and Use Committee (Sun Moon University; in a tube. After reacting for 3  min at room tempera- SM-2020-02-01). Male Sprague–Dawley rats (n = 8/ ture, and 3  mL of 2% sodium carbonate was added in group; 6  weeks old; Samtako Co., Osan, Korea) were the same tube. After reacting at room temperature for individually housed in a controlled environment at 30  min, the absorbance of the mixture was measured 23 ± 1  °C at 50 ± 5% relative humidity under a 12  h at 720 nm in glass cuvettes using a spectrophotometer light/dark cycle. After acclimation for a week on low- (Libra S22, Biochrom Ltd., Cambridge, UK). The same fat (LF) diet, animals were split into four weight- procedure was performed for diluted gallic acid (as a matched groups and fed either a low-fat (LF; 10% kcal standard). A calibration curve for gallic acid was used as fat), high-fat (HF; 45% kcal as fat), or HF diet sup- to determine the total phenolic contents in the sam- plemented with non-fermented (HF/GB) or fermented ples, and expressed as gallic acid equivalent (GAE). goji berry at 2% (w/w) in diet for 6  weeks (Additional The antioxidant activities of GB and FGB samples file  1: Table S1). Selection of the 2% goji berry concen- were determined using diphenylpicrylhydrazyl (DPPH) tration was based on previously published studies [21– radical scavenging activity and ferric reducing anti- 23]. Body weight and food intake were measured on a oxidant power assay (FRAP). DPPH radical scavenging weekly and daily basis, respectively. Food efficiency activity was performed with a 100  μM DPPH solution ratio (FER) was determined as weight gain (g)/energy proposed by Liang et  al. and Park et  al. [17, 18]. One intake (kcal). After 6 weeks on respective diets, animals and half mL of each methanol extract of GB and FGB were fasted overnight and euthanized by carbon diox- samples was dispensed into a tube and added to 1.5 mL ide inhalation. Blood was placed into a sterile Vacu- of 100  μM DPPH solution. After vortexing and react- tainer plastic tube (BD Vacutainer, Plymouth, UK) and ing for 30  min in the dark, absorbance was measured centrifuged at 1000×g for 10 min at 4 °C for serum col- at 517  nm using a spectrophotometer (Libra S22, bio- lection. The liver and visceral fat pads (retroperitoneal chrom Co.). 80% of methanol (1.5 mL) and DPPH solu- and epididymal) were collected and weighed; an adi- tion (1.5  mL) were collected as a control sample. All posity index was determined. Serum and all the tissues samples were measured in triplicate. DPPH scavenging were snap-frozen and stored − 80 °C until analysis. activity was calculated using the following equation: Antioxidant activity (%) = 1 − [(As − Ab)/Ac] × 100 RNA extraction and quantitative RT‑PCR Total RNA from liver and epididymal fat tissues was where antioxidant activity-DPPH radical scaveng- extracted using the RNeasy Mini Kit (Qiagen, Hilden, ing activity; As, Ab and Ac represent the absorbance of Germany) per the manufacturer’s instructions. cDNAs DPPH with specific samples, blank, and DPPH solutions, were synthesized from 2  μg of purified RNA samples respectively. using TOPscript RT DryMIX (dT18 plus; Enzynomix, The FRAP analysis is based on reduction of ferric- 3+ Daejeon, Korea) following the manufacturer’s protocol. tripyridyltriazine (Fe -TPTZ) complex into ferrous 2+ Real-time PCR was performed with the CFX96 Touch tripyridyltriazine (Fe -TPTZ) by interacting with Real-Time PCR Detection System (Bio-Rad, Hercules, antioxidants in the sample [19]. FRAP assay was per- CA, USA) using ToprealTM qPCR 2 × PreMIX SYBR formed using the modified Benzie and Strain method Green (Enzynomix) for detection. GAPDH was used as [20]. The FRAP reagent was prepared by mixing a housekeeping gene. Genes of interest were analyzed 300 mM sodium acetate buffer (pH3.6), 10 mM TPTZ −ΔΔCT according to the 2 method [24] and compared with solution, and 20  mM F eCl ∙6H O solution in a ratio 3 2 control samples. Primer sequences are provided in Addi- of 10:1:1 (v/v). 50  μL of each of the methanol extract tional file 1: Table S2. of GB and FGB was mixed with 1.5  mL of the FRAP reagent and reacted for 5  min at room temperature in the dark. Then, the absorbance of the mixtures was Biochemical analysis measured at 450  nm. The calibration curve was pre- The levels of serum alkaline phosphatase (ALP) and pared with ferric sulfate and FRAP results for samples aspartate aminotransferase (AST) were measured via col- are expressed in μg/mL. All samples were measured in orimetric assay kits (ALP: E-BC-K091-S and AST: E-BC- triplicate. K236-M, Elabscience, Houston, TX, USA) according to the manufacturer’s guideline. Lee et al. Appl Biol Chem (2021) 64:70 Page 4 of 11 Statistical analysis On the other hand, little difference was observed in Unless stated otherwise (microbiome analysis), statistical cumulative food intake and food efficiency among analysis was performed by using Prism software (Prism groups (Fig. 1B, C). Visceral adiposity was evaluated from 8.4.3; GraphPad Software, La Jolla, CA, USA). Two-factor epididymal and retroperitoneal fat depots (Fig.  1D). HF repeated-measures analysis of variance (ANOVA) was feeding significantly increased fat mass compared to the used to analyze body weight and one-factor ANOVA was LF group (LF vs. HF, p < 0.0001) and little difference was performed to analyze the rest of the parameters. Differ - found among HF-fed. ences between groups were analyzed by using Fisher’s least-significant-difference test. Differences were consid - Supplementation of GB and FGB attenuates HF‑induced ered significant if p < 0.05. Data are presented as means inflammation ± standard error of the mean (SEMs). In adipose tissues, both GB and FGB supplementa- tion significantly down-regulated the gene expression Results of macrophage chemoattactrant protein 1 (MCP-1), as a Betaine, total polyphenol contents and antioxidant marker of macrophage infiltration, and pro-inflammatory activities cytokines, IL-6 and TNFα, compared to the HF group The concentrations of betaine, total polyphenols and (MCP-1: HF vs. GB or FGB, p < 0.01; IL-1β: HF vs. GB antioxidant activity were measured in GB and FGB sam- or FGB, p < 0.05; TNFα: HF vs. GB or FGB, p < 0.05; ples (lyophilized to 88% solids) (Table  1). After lyophili- Fig. 2A–C). zation to 88% solids, the betaine contents of the GB and At the systemic level, HF feeding significantly upregu - FGB samples were measured to be 129.29  mg/mL and lated the gene expression of IL-1β in the liver compared 165.51  mg/mL, respectively, indicating that the betaine to the LF group, which was suppressed by GB (signifi - concentration was increased by about 1.3-fold by fer- cantly) and FGB (partially) supplementation (LF vs. HF, mentation (p < 0.05). p < 0.01; HF vs. GB, p < 0.05; HF vs. FGB, p = 0.075; Total phenolic contents, DPPH radical scavenging abil- Fig. 2D). Further, the gene expression of IL-10 as an anti- ity, and FRAP assay were significantly increased by fer - inflammatory cytokine was upregulated by GB and FGB mentation (p < 0.05). The total phenolic contents of the supplementation by 98% and 97% compared to the HF GB and FGB were 230.76  mg/mL and 280.53  mg/mL, group although this did not reach statistical significance respectively, indicating that the total phenolic contents of (HF vs. GB, p = 0.075; HF vs. FGB, p = 0.076; Fig. 2E). the GB sample were significantly increased by about 1.2- fold that of initial during fermentation. The FRAP assay in the GB and FGB were 273.03  μg/mL and 479.56  μg/ Supplementation of GB and FGB exerts antioxidant effects mL, respectively. This means that the FRAP assay of GB To investigate the antioxidant properties of GB and FGB was increased dramatically to about 1.8-fold after fer- supplementation against the HF challenge, the gene mentation. On the other hand, the DPPH radical scav- expression of antioxidant enzymes was examined. While enging ability of the GB was slightly increased by about HF feeding showed little effect on the gene expression of 3.6% after fermentation. antioxidant enzymes, both GB and FGB supplementation significantly upregulated their gene expression compared Supplementation of GB and FGB improves hyperphagic to the HF and/or even LF group with more remarkable phenotypes antioxidant effects by GB supplementation (CAT: LF or After six weeks on respective diets, all HF fed groups HF vs. HF/GB, p < 0.001; HF/GB vs. HF/FGB, p < 0.05; showed significantly (HF, HF/FGB) or partially (HF/GB) GPX: LF or HF vs. HF/GB, p < 0.0001; HF vs. HF/FGB, higher body weight than the control LF group (LF vs. HF p < 0.01; HF/GB vs. HF/FGB, p < 0.01; GSR: LF or or HF/FGB, p < 0.05; LF vs. HF/GB, p = 0.057; Fig. 1A). HF vs. HF/GB, p < 0.01; SOD1: LF or HF vs. HF/GB, Table 1 Betaine, total phenol contents and antioxidant activities of GB and FGB Sample Betaine (mg/mL) Total polyphenol (mg/mL) DPPH (%) FRAP (μg/mL) b b b b GB 129.29 ± 0.49 230.76 ± 0.59 64.93 ± 0.57 273.03 ± 6.40 a a a a FGB 165.51 ± 1.37 280.52 ± 1.01 68.51 ± 0.68 479.56 ± 6.61 Data are expressed as mean ± SD Different superscript letters in the same column mean significantly different at p < 0.05 by Duncan’s multiple range test GB Freeze-dried goji berry sample (88% solids); FGB Freeze-dried goji berry samples (88% solids) pretreated with Pectinex Ultra SP-L and fermented with L. plantarum CB3 L ee et al. Appl Biol Chem (2021) 64:70 Page 5 of 11 Fig. 1 Supplementation of non‑fermented and fermented goji berry has little effects on hyperphagic phenotypes. Body weight (A), cumulative energy intake (B), feed efficiency [weight gain (g)/energy intake (kcal); (C)], and visceral adiposity (D) in rats fed an LF or HF with or without 1% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. For Fig. 1A, significant # /#/ /## differences are denoted as an asterisk (*) for LF vs. HF, sharp ( ) for LF vs. HF/GB, and dagger (†) for HF vs. HF/FGB at * †p < 0.05 and ** p < 0.01. For Fig. 1B–D, significant differences are denoted as an asterisk (*) at *p < 0.05 and **p < 0.01. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat p < 0.0001; LF vs. HF/FGB, p < 0.001; HF vs. HF/FGB, p (PPARγ) was significantly upregulated by HF feeding < 0.01; Fig. 3). compared to the LF group, which was normalized to the LF level by both GB and FGB supplementation (LF vs. Supplementation of GB and FGB improves hepatic HF, p < 0.0001; HF vs. GB or FGB, p < 0.0001; Fig.  5). functions Similarly, the gene expression of hepatic sterol regula- Hepatic functions were examined by the level of ALP and tor element-binding protein 1c (SREBP1c), as a marker AST in circulation as markers of liver damage (Fig. 4). HF of adipogenesis, was partially upregulated by HF feed- feeding had little effect on the level of serum ALP but the ing compared to the LF group and this was significantly level was significantly decreased by 2% GB supplemen - suppressed by both GB and FGB supplementation (LF tation compared to the HF group (Fig.  4A). The serum vs. HF, p = 0.059; HF vs. GB, p < 0.01; HF vs. FGB, p < ALP level in the HF/GB group was even lower than the 0.001; Fig. 5B). The similar effect of GB and FGB supple - LF group by 27%. Similarly, the serum AST level was sig- mentation on lipid profile was also confirmed at the sys - nificantly reduced by both GB and FGB supplementation temic level; the serum TG level was significantly reduced compared to the HF group (HF vs. GB, p < 0.001; HF vs. by both GB and RGB supplementation compared to the FGB, p < 0.05; Fig. 4B). HF group (HF vs. GB or FGB, p < 0.01; Fig. 5C). Supplementation of GB and FGB improves lipid profiles Discussion In addition to visceral adiposity, lipid profile was fur - In this present study, we investigated the beneficial effects ther examined in the liver and in circulation (Fig.  5). As of GB supplementation on obesity and related metabolic a marker of lipogenesis, the gene expression of hepatic disorders in HF diet-fed rats. We also sought to examine peroxisome proliferator-activated receptor gamma the beneficial effects of fermented GB with L. Plantarum Lee et al. Appl Biol Chem (2021) 64:70 Page 6 of 11 Fig. 2 Supplementation of non‑fermented and fermented goji berry attenuates HF‑induced inflammation. Gene expression of IL ‑6 and TNFα in epididymal fat (A–C) and liver (D, E) tissues of rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05 and **p < 0.01. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); IL interleukin; LF low fat; MCP-1 macrophage chemoattractant protein 1; TNFα tumor necrosis factor alpha CB3, elucidating the possible metabolic benefits result - it functions as a methyl donor and has the effects of ing from the fermentation process. Our hypothesis was improving liver function, cell replication, detoxification, GB and FGB supplementation as a part of HF diet would and kidney protection [25, 26]. improve the HF-induced disturbances in lipid, inflam - In this study, we observed that betaine concentra- matory, and oxidative profiles along with hepatic func - tion of goji berry was significantly increased by enzyme tions, possibly preventing the development of obesity and (Pectinex Ultra SP-L) treatment and L. plantarum CB3 related metabolic abnormalities, and that these effects fermentation, confirming the previous finding that the would be enhanced by fermentation. betaine concentration increases after fermenting goji ber- As major bioactive components of GB, concentrations ries with various lactic acid bacteria [27]. Total phenolic of betaine and total polyphenolic and their correspond- contents, DPPH radical scavenging ability, and FRAP ing antioxidant activities were measured in GB and FGB assay were significantly increased after fermentation with samples. Betaine has similar functions to choline, folic L. plantarum CB3 inoculation, which is corresponds to acid, vitamin B , and the amino acid methionine known the finding of the previous studies [28]. This seems to be as SAMe (S-adenosylmethionine). In the human body, because the enzyme and carboxylic acid produced by L. L ee et al. Appl Biol Chem (2021) 64:70 Page 7 of 11 Fig. 3 Supplementation of non‑fermented and fermented goji berry exerts antioxidant effects. Gene expression of antioxidant enzymes, CAT (A), GPX (B), GSR (C), and SOD1 (D), in the liver of rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. CAT catalase; GPX glutathione peroxidases; GSR glutathione reductase; HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat; SOD1 superoxide dismutase 1 Fig. 4 Supplementation of non‑fermented and fermented goji berry protects hepatic functions; levels of serum ALP (A) and AST (B) in rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05 and ***p < 0.001. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w). ALP alkaline phosphatase; AST aspartate transaminase; HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat plantarum destroy the cellular structure of GB and cause systemic (IL-1β in the liver) levels. In addition, GB and the release of phenolic substances in the fermentation FGB supplementation upregulated the gene expression process [29]. of antioxidant enzymes, catalase (CAT), glutathione per- In vivo, GB and FGB supplementation suppressed oxidases (GPX), glutathione reductase (GSR), superoxide the gene expression of inflammation indices at the dismutase (SOD1) compared to the HF and/or even LF local (MCP-1, IL-1β, TNFα in the adipose tissues) and group with more remarkable antioxidant effects by GB Lee et al. Appl Biol Chem (2021) 64:70 Page 8 of 11 GB and FGB supplementation improved HF-induced metabolic disturbances and these improvements were associated with their anti-inflammatory and anti-oxidant properties. It is widely demonstrated that consumption of a HF diet can lead to increases in body weight, energy intake, and visceral fat mass [30]. In this present study, HF feed- ing for six weeks significantly increased the body weight. While little difference was observed in overall energy intake and feed efficiency between LF and HF fed groups, an increase in adiposity was still observed in HF-fed rats compared to the LF group, confirming the previous finding that total and visceral body fat weights increases proportionally to the level of fat in the diet even when fed isocalorically [31]. It was unexpected that GB and FGB supplementation did not prevent HF-diet induced increases in body weight gain and adiposity. Thus, to bet - ter understand the limited effects of supplementation on these obesity phenotypes, we further investigated the related parameters at the molecular level. The pathogenesis of obesity involves infiltration of macrophages into expanding adipose tissue and the acti- vated macrophages can contribute to local (adipose tis- sue) and systemic (liver) inflammation, which is primarily promoted by the release of pro-inflammatory cytokines [2]. In particular, studies have demonstrated that HF diet- induced obese animals developed chronic inflammation in adipose and hepatic tissues [32–34]. In this present study, the HF group showed the highest expression of MCP-1 gene, which was remarkably down-regulated by GB and FGB supplementation. Correspondingly, GB and FGB supplementation also exerted anti-inflammatory effects against other pro-inflammatory cytokines, IL-1β and TNFα and in favor of IL-10 as an anti-inflammatory cytokine at the local and systemic levels, which corre- sponds to the finding of the previous study [35]. The inflammatory cascade facilitates the formation of toxic reactive oxygen species and the subsequent produc- Fig. 5 Supplementation of non‑fermented and fermented goji berry tion of oxidative stress, which inhibits the expression of improves lipid profiles. Gene expression of PPARγ (A) and SREBP1c antioxidant enzymes and consequently impairs the anti- (B) and serum TG level (C) in rats fed an LF or HF with or without oxidant defense system [3]. Here, we observed that GB 2% non‑fermented or fermented goji berry supplementation diet and/or FGB supplementation remarkably upregulated for 6 weeks. Values are means ± SEMs; n = 8/group. Significant the gene expression of antioxidant enzymes in the liver differences are denoted as an asterisk (*) at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HF high fat; HF HF without goji including CAT, GPX, GSR, and SOD1 compared both the berry; HF/FGB HF with fermented goji berry in diet by 1% (w/w); LF and HF groups, which is consistent to the previous HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low findings of antioxidant effects of GB and FGB in vitro and fat; PPARγ peroxisome proliferator‑activated receptor gamma; SREBP1c in vivo [10, 36, 37] sterol regulator element‑binding protein 1c; TG triglyceride Excessive oxidative stress also can cause tissue damage to the liver [4]. It is known that ALP and AST enzymes are reliable markers of hepatic function and increased supplementation. Also, GB and FGB supplementation levels of circulating ALP and AST indicate hepatic dam- protected from HF-induced damages of the liver and ages [38, 39]. Here, the highest levels of serum ALP and dyslipidemia. Taken together, these data show that both AST were observed in the HF group and their activities L ee et al. Appl Biol Chem (2021) 64:70 Page 9 of 11 were significantly inhibited by GB and/or FGB sup - metabolic effects between non-fermented and fermented plementation. It is important to note that the elevation prebiotics resulting from fermentation with probiotics in these hepatic enzymes have been associated with [45–47]. In these studies, metabolic benefits were more abnormal hepatic lipid metabolism [5]. Further, studies enhanced or different mechanism of action was resulted have previously shown that HF feeding caused ectopic by fermentation compared to the original prebiotics. In fat deposition, which was closely associated with the this regard, it is possible that different pathways might upregulated expression of hepatic PPARγ as a transcrip- have been involved in metabolic benefits observed in the tion factor for adipogenesis [32]. Considered as the mas- HF/FGB group. Thus, further investigation into multiple ter regulator of adipogenesis [40], its overexpression has possible metabolic pathways would identify the potential been known to be sufficient for inducement of hepatic fat mechanism of action by FGB for its metabolic benefits. deposition [41] in concert with SREBP1c as a transcrip- Further, since the gut microbiota has been identified the tion factor of lipogenesis and also a mediator of PPARγ primary target by prebiotics and probiotics for their met- expression [42]. In this study, both GB and FGB signifi - abolic benefits, metagenomic and metabolomic analyses cantly suppressed HF-induced upregulation of PPARγ would provide a better understanding of distinct mecha- and SREBP1c gene expression in the liver. This was also nism of action on metabolic profiles between GB and confirmed at the systemic level. As a well-known indica - FGB. tor of obesity, dyslipidemia is primarily characterized by In conclusion, we demonstrated that GB and FGB sup- the elevation of triglycerides in circulation [6]. In this plementation protected from HF-induced metabolic present study, the serum TG level was increased in the complications primarily by improving hepatic function HF group by 19% compared to the LF group and this ele- and corresponding lipid metabolism via their anti-inflam - vation was significantly suppressed by both GB and FGB matory and antioxidant properties. To our knowledge, supplementation. The beneficial effects of GB supple - this is the first in  vivo study confirming metabolic ben - mentation also has been reported in previous studies via efits of GB in a fermented form. u Th s, these findings inhibiting the expression of hepatic lipogenesis factors support the potential application of both GB and FGB to and/or decreasing the level of triglyceride in circulation, ameliorate obesity-associated metabolic abnormalities. which confirms the findings of this study [43, 44]. Taken together, it can be confirmed that GB and FGB Abbreviations supplementation improved HF-induced metabolic dys- ALP: Alkaline phosphatase; AST: Aspartate transaminase; ANOVA: Analysis of regulation in adipose and liver tissues at the molecular variance; CAT : Catalase; GPX: Glutathione peroxidases; GSR: Glutathione reduc‑ tase; HF: High fat; HF/FGB: HF with fermented goji berry in diet by 2% (w/w); level primarily by enhancing anti-inflammatory and anti - HF/GB: HF with non‑fermented goji berry in diet by 2% (w/w); IL: Interleukin; oxidant responses. LF: Low fat; PPARγ: Peroxisome proliferator‑activated receptor gamma; ROS: There are some considerations to this study that Reactive oxygen species; SOD1: Superoxide dismutase; SREBP1c: Sterol regula‑ tor element‑binding protein 1c; TG: Triglyceride; TNFα: Tumor necrosis factor deserve more attention. First, we employed a HF regime alpha; MCP‑1: Macrophage chemoattractant protein. for six weeks to induce diet-induced obesity and related metabolic disturbances. This protocol was sufficient to Supplementary Information induce obese phenotypes (increases in body weight and The online version contains supplementary material available at https:// doi. visceral fat mass), which was further supported by HF- org/ 10. 1186/ s13765‑ 021‑ 00642‑1. induced dysregulation of hepatic metabolism (IL-1β, PPARγ, and SREBP1c). However, we failed to find any Additional file 1: Table S1. Composition of experimental diets. Table S2. remarkable differences between the LF and HF groups Primer sequences used for RT‑PCR. for the other metabolic parameters at the molecular level including those for inflammation, antioxidant actions, Acknowledgements and hepatic functions. Thus, a study with longer duration This research was supported by the Ministry of Trade, Industry and Energy in 2019 [Grant Number P0010311]. than six weeks would better enable the establishment of other measures for the development of HF-induced Authors’ contributions obesity. In the meanwhile, despite little difference the Conceptualization: HS (Hee‑jae Suh), HP, SL. Animal experiment: SL, SJ, YP, CY, UH. Sample preparation: SJ, YP, HS (Hyunji Seo), CY, UH. Sample analysis: SL, SJ. LF and HF groups in those metabolic parameters, both Data analysis: SL. Writing, review, and editing: HS (Hee‑jae Suh), HP, SL. Funding GB and FGB supplementation demonstrated metabolic acquisition: HS (Hee‑jae Suh). All authors have read and approved the final benefits compared to the HF group, even compared manuscript. to the LF group for the regulation of the antioxidant defense system. Notably, these beneficial effects were Declarations more outstanding in the HF/GB group. 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Supplementation of non-fermented and fermented goji berry (Lycium barbarum) improves hepatic function and corresponding lipid metabolism via their anti-inflammatory and antioxidant properties in high fat-fed rats

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Abstract

Development of obesity is associated with excessive fat accumulation and oxidative stress along with chronic inflam‑ mation. Goji berries (Lycium barbarum) are high in polyphenolic compounds and have anti‑inflammatory, anti‑ oxidant, and hypolipidemic properties that may alleviate the pathogenesis of obesity and related metabolic complications. Thus, the aim of this study was to investigate potential metabolic benefits of GB supplementation against high fat (HF) diet‑induced obesity and its comorbidities in HF diet ‑fed rats (male Sprague–Dawley, n = 8/group, 6 weeks old). We also sought to examine the potential metabolic benefits of fermented GB (FGB) with L. plantarum CB3 and possi‑ ble distinctions in the degree and/or mechanism of action compared to GB. GB and FGB supplementation suppressed the gene expression of inflammation indices at the local (adipose tissues) and systemic (liver) levels. In addition, GB and FGB supplementation upregulated the gene expression of antioxidant enzymes compared to the HF and/or even low fat (LF) group with more remarkable antioxidant effects by GB supplementation. Also, GB and FGB supplementa‑ tion protected from HF‑induced damages of the liver and dyslipidemia. In conclusion, we demonstrated that GB and FGB supplementation protected from HF‑induced metabolic complications primarily by improving hepatic function and corresponding lipid metabolism via their anti‑inflammatory and antioxidant properties. To our knowledge, this is the first in vivo study confirming metabolic benefits of GB in a fermented form. Thus, these findings support the potential application of both GB and FGB to ameliorate obesity‑associated metabolic abnormalities. Keywords: Dyslipidemia, Fermentation, Goji berry (Lycium barbarum), Hepatic function, Inflammation, Obesity, Oxidative stress Introduction Obesity is primarily characterized by abnormal or excessive fat accumulation and a low-level systemic inflammation [1]. An increase in visceral adipos- ity serves as a strong predictor of local and systemic inflammation with its association with macrophage *Correspondence: suhhj@sunmoon.ac.kr infiltration and subsequent secretion of pro-inflamma- Department of Food Science, Research Center for Food and Bio tory cytokines [2]. The pivotal role of pro-inflamma- Convergence, Sun Moon University, Asan, Chungchengnam‑do 31460, Republic of Korea tory cytokines such as interleukin-1beta (IL-1β) and © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lee et al. Appl Biol Chem (2021) 64:70 Page 2 of 11 tumor necrosis factor alpha (TNFα) has been identi- Materials and methods fied in the development of obesity and related meta- Materials bolic dysregulation [3]. Further, the inflammatory Commercial kits for animal tests were purchased cascade can facilitate the formation of toxic reac- from Elabscience (Houston, Texas, USA). Goji berries tive oxygen species and the subsequent generation (Lycium barbarum) were purchased from local mar- of oxidative stress, which inhibits the expression of kets in Cheongyang Korea. Pectinex Ultra SP-L was antioxidant enzymes and consequently impairs the obtained from Vision Biochem (Seongnam, Korea). antioxidant defense system [3]. Excessive oxidative Lactobacillus plantarum CB3, isolated from kimchi in stress also can impair liver functions, in the regulation our laboratory, was used as a starter culture for fermen- of hepatic lipid metabolism in particular [4]. Previous tation of goji berries. studies have reported that HF-induced hepatic dys- function demonstrated by the elevated level of alkaline Preparation of GB and FGB powder phosphatase (ALP) and aspartate transaminase (AST) For the preparation of GB extract, 10  g of dried GB enzyme activities in circulation was associated with was added to 100  mL of 90  °C water and extracted for the upregulation of adipogenesis and lipogenesis tran- 6  h. Twenty mL of GB extract was taken and used as scription factors, resulting in hepatic fat accumulation a substrate for the production of FGB. Pectinex Ultra and dyslipidemia [5, 6]. SP-L (0.5%, w/v) was added into the GB extract, and Extensive attention has been paid to the role of ber- the mixture was reacted at 55 °C for 6 h with shaking at ries in the prevention of obesity and related meta- 160  rpm and sterilized at 100  °C for 10  min. The steri - bolic disturbances. Goji berries (Lycium barbarum) lized GB suspension was inoculated with 5% (v/v) of L. are high in polyphenolic compounds and have anti- plantarum CB3 culture [1 × 10 colony-forming units inflammatory, anti-oxidant, and hypolipidemic prop- (CFU)/mL] to give a final cell concentration of about erties that may affect disease pathogenesis [7]. Dietary 1 × 10   CFU/mL, and fermented at 37  °C for 72  h supplementation with goji berry (GB) reduced bio- with shaking at 160 rpm. The GB and FGB suspensions markers of oxidative stress and lipid peroxidation as were lyophilized to 88% solids using an EYELA freeze well as inflammatory gene expression in both experi- dryer (FDU-1200, Tokyo Rikakikai Co.) and used for mental and clinical studies [8–10]. In addition, Yang experiments. et  al. reported that GB supplementation ameliorated HF-induced insulin resistance via activation of phos- Analysis of betaine phatidylinositol-3 kinase/protein kinase B/nuclear fac- Quantitative analysis of betaine was performed using tor-E2-related factor 2 (PI3K/AKT/Nrf2) pathway [11]. the method proposed by Lee et al. with some modifica - Despite a growing evidence on metabolic benefits tions [15]. Betaine from GB and FGB samples (lyophi- of GB, there is limited knowledge on the potential lized to 88% solids) was detected using a 1260 Infinity benefits of GB in obesity and related metabolic dis- system (Agilent Technologies, Santa Clara, CA, USA) orders. Thus, the aim of this study was to investigate attached with an evaporative light scattering detector metabolic benefits of GB supplementation against HF (ELSD; Agilent Technologies, Santa Clara, CA, USA) diet-induced obesity and its comorbidities in rats. We and a Discovery C18 column (4.6 × 250  mm, 5  μm, hypothesized that GB supplementation would improve SUPELCO, PA, USA). The mobile phase contained HF-induced obese phenotypes and dysregulation of water and acetonitrile and was used in isocratic elution lipid metabolism in association with anti-inflamma- as at a 95:5 ratio (v/v %), respectively. The flow rate of tory and anti-oxidant mechanisms. Further, consid- the elution was 0.5 mL/min and the evaporator and the ering that fermentation of bioactive prebiotics with nebulizer temperature for the ELSD were 50 and 70 °C, probiotics such as Lactobacillus or Bifidobacterium respectively. has been confirmed to enhance the pharmacological efficacy and metabolic functions of the original non- Measurement of total phenolics and antioxidant activity fermented prebiotics in previous studies [12–14], we The total phenolic content of GB and FGB samples also sought to examine the potential metabolic ben- (lyophilized to 88% solids) was determined by modify- efits of fermented GB (FGB) with L. plantarum CB3 as ing the method of previous studies [16]. To determine probiotics and possible distinctions in the degree and/ total phenolic compound and antioxidant activity, or mechanism of action compared to GB. lyophilized GB and FGB samples were diluted with 80% methanol to a concentration of 5%, respectively. For the calibration curve of phenolic compounds, L ee et al. Appl Biol Chem (2021) 64:70 Page 3 of 11 gallic acid stock solution (5  mg/mL) was prepared Animals and experimental design and diluted with water. Five-hundred microliters Animals were maintained and handled in accordance of each sample extract was mixed with 300  μl of 1  N with protocols approved by the Institutional Ani- Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) mal Care and Use Committee (Sun Moon University; in a tube. After reacting for 3  min at room tempera- SM-2020-02-01). Male Sprague–Dawley rats (n = 8/ ture, and 3  mL of 2% sodium carbonate was added in group; 6  weeks old; Samtako Co., Osan, Korea) were the same tube. After reacting at room temperature for individually housed in a controlled environment at 30  min, the absorbance of the mixture was measured 23 ± 1  °C at 50 ± 5% relative humidity under a 12  h at 720 nm in glass cuvettes using a spectrophotometer light/dark cycle. After acclimation for a week on low- (Libra S22, Biochrom Ltd., Cambridge, UK). The same fat (LF) diet, animals were split into four weight- procedure was performed for diluted gallic acid (as a matched groups and fed either a low-fat (LF; 10% kcal standard). A calibration curve for gallic acid was used as fat), high-fat (HF; 45% kcal as fat), or HF diet sup- to determine the total phenolic contents in the sam- plemented with non-fermented (HF/GB) or fermented ples, and expressed as gallic acid equivalent (GAE). goji berry at 2% (w/w) in diet for 6  weeks (Additional The antioxidant activities of GB and FGB samples file  1: Table S1). Selection of the 2% goji berry concen- were determined using diphenylpicrylhydrazyl (DPPH) tration was based on previously published studies [21– radical scavenging activity and ferric reducing anti- 23]. Body weight and food intake were measured on a oxidant power assay (FRAP). DPPH radical scavenging weekly and daily basis, respectively. Food efficiency activity was performed with a 100  μM DPPH solution ratio (FER) was determined as weight gain (g)/energy proposed by Liang et  al. and Park et  al. [17, 18]. One intake (kcal). After 6 weeks on respective diets, animals and half mL of each methanol extract of GB and FGB were fasted overnight and euthanized by carbon diox- samples was dispensed into a tube and added to 1.5 mL ide inhalation. Blood was placed into a sterile Vacu- of 100  μM DPPH solution. After vortexing and react- tainer plastic tube (BD Vacutainer, Plymouth, UK) and ing for 30  min in the dark, absorbance was measured centrifuged at 1000×g for 10 min at 4 °C for serum col- at 517  nm using a spectrophotometer (Libra S22, bio- lection. The liver and visceral fat pads (retroperitoneal chrom Co.). 80% of methanol (1.5 mL) and DPPH solu- and epididymal) were collected and weighed; an adi- tion (1.5  mL) were collected as a control sample. All posity index was determined. Serum and all the tissues samples were measured in triplicate. DPPH scavenging were snap-frozen and stored − 80 °C until analysis. activity was calculated using the following equation: Antioxidant activity (%) = 1 − [(As − Ab)/Ac] × 100 RNA extraction and quantitative RT‑PCR Total RNA from liver and epididymal fat tissues was where antioxidant activity-DPPH radical scaveng- extracted using the RNeasy Mini Kit (Qiagen, Hilden, ing activity; As, Ab and Ac represent the absorbance of Germany) per the manufacturer’s instructions. cDNAs DPPH with specific samples, blank, and DPPH solutions, were synthesized from 2  μg of purified RNA samples respectively. using TOPscript RT DryMIX (dT18 plus; Enzynomix, The FRAP analysis is based on reduction of ferric- 3+ Daejeon, Korea) following the manufacturer’s protocol. tripyridyltriazine (Fe -TPTZ) complex into ferrous 2+ Real-time PCR was performed with the CFX96 Touch tripyridyltriazine (Fe -TPTZ) by interacting with Real-Time PCR Detection System (Bio-Rad, Hercules, antioxidants in the sample [19]. FRAP assay was per- CA, USA) using ToprealTM qPCR 2 × PreMIX SYBR formed using the modified Benzie and Strain method Green (Enzynomix) for detection. GAPDH was used as [20]. The FRAP reagent was prepared by mixing a housekeeping gene. Genes of interest were analyzed 300 mM sodium acetate buffer (pH3.6), 10 mM TPTZ −ΔΔCT according to the 2 method [24] and compared with solution, and 20  mM F eCl ∙6H O solution in a ratio 3 2 control samples. Primer sequences are provided in Addi- of 10:1:1 (v/v). 50  μL of each of the methanol extract tional file 1: Table S2. of GB and FGB was mixed with 1.5  mL of the FRAP reagent and reacted for 5  min at room temperature in the dark. Then, the absorbance of the mixtures was Biochemical analysis measured at 450  nm. The calibration curve was pre- The levels of serum alkaline phosphatase (ALP) and pared with ferric sulfate and FRAP results for samples aspartate aminotransferase (AST) were measured via col- are expressed in μg/mL. All samples were measured in orimetric assay kits (ALP: E-BC-K091-S and AST: E-BC- triplicate. K236-M, Elabscience, Houston, TX, USA) according to the manufacturer’s guideline. Lee et al. Appl Biol Chem (2021) 64:70 Page 4 of 11 Statistical analysis On the other hand, little difference was observed in Unless stated otherwise (microbiome analysis), statistical cumulative food intake and food efficiency among analysis was performed by using Prism software (Prism groups (Fig. 1B, C). Visceral adiposity was evaluated from 8.4.3; GraphPad Software, La Jolla, CA, USA). Two-factor epididymal and retroperitoneal fat depots (Fig.  1D). HF repeated-measures analysis of variance (ANOVA) was feeding significantly increased fat mass compared to the used to analyze body weight and one-factor ANOVA was LF group (LF vs. HF, p < 0.0001) and little difference was performed to analyze the rest of the parameters. Differ - found among HF-fed. ences between groups were analyzed by using Fisher’s least-significant-difference test. Differences were consid - Supplementation of GB and FGB attenuates HF‑induced ered significant if p < 0.05. Data are presented as means inflammation ± standard error of the mean (SEMs). In adipose tissues, both GB and FGB supplementa- tion significantly down-regulated the gene expression Results of macrophage chemoattactrant protein 1 (MCP-1), as a Betaine, total polyphenol contents and antioxidant marker of macrophage infiltration, and pro-inflammatory activities cytokines, IL-6 and TNFα, compared to the HF group The concentrations of betaine, total polyphenols and (MCP-1: HF vs. GB or FGB, p < 0.01; IL-1β: HF vs. GB antioxidant activity were measured in GB and FGB sam- or FGB, p < 0.05; TNFα: HF vs. GB or FGB, p < 0.05; ples (lyophilized to 88% solids) (Table  1). After lyophili- Fig. 2A–C). zation to 88% solids, the betaine contents of the GB and At the systemic level, HF feeding significantly upregu - FGB samples were measured to be 129.29  mg/mL and lated the gene expression of IL-1β in the liver compared 165.51  mg/mL, respectively, indicating that the betaine to the LF group, which was suppressed by GB (signifi - concentration was increased by about 1.3-fold by fer- cantly) and FGB (partially) supplementation (LF vs. HF, mentation (p < 0.05). p < 0.01; HF vs. GB, p < 0.05; HF vs. FGB, p = 0.075; Total phenolic contents, DPPH radical scavenging abil- Fig. 2D). Further, the gene expression of IL-10 as an anti- ity, and FRAP assay were significantly increased by fer - inflammatory cytokine was upregulated by GB and FGB mentation (p < 0.05). The total phenolic contents of the supplementation by 98% and 97% compared to the HF GB and FGB were 230.76  mg/mL and 280.53  mg/mL, group although this did not reach statistical significance respectively, indicating that the total phenolic contents of (HF vs. GB, p = 0.075; HF vs. FGB, p = 0.076; Fig. 2E). the GB sample were significantly increased by about 1.2- fold that of initial during fermentation. The FRAP assay in the GB and FGB were 273.03  μg/mL and 479.56  μg/ Supplementation of GB and FGB exerts antioxidant effects mL, respectively. This means that the FRAP assay of GB To investigate the antioxidant properties of GB and FGB was increased dramatically to about 1.8-fold after fer- supplementation against the HF challenge, the gene mentation. On the other hand, the DPPH radical scav- expression of antioxidant enzymes was examined. While enging ability of the GB was slightly increased by about HF feeding showed little effect on the gene expression of 3.6% after fermentation. antioxidant enzymes, both GB and FGB supplementation significantly upregulated their gene expression compared Supplementation of GB and FGB improves hyperphagic to the HF and/or even LF group with more remarkable phenotypes antioxidant effects by GB supplementation (CAT: LF or After six weeks on respective diets, all HF fed groups HF vs. HF/GB, p < 0.001; HF/GB vs. HF/FGB, p < 0.05; showed significantly (HF, HF/FGB) or partially (HF/GB) GPX: LF or HF vs. HF/GB, p < 0.0001; HF vs. HF/FGB, higher body weight than the control LF group (LF vs. HF p < 0.01; HF/GB vs. HF/FGB, p < 0.01; GSR: LF or or HF/FGB, p < 0.05; LF vs. HF/GB, p = 0.057; Fig. 1A). HF vs. HF/GB, p < 0.01; SOD1: LF or HF vs. HF/GB, Table 1 Betaine, total phenol contents and antioxidant activities of GB and FGB Sample Betaine (mg/mL) Total polyphenol (mg/mL) DPPH (%) FRAP (μg/mL) b b b b GB 129.29 ± 0.49 230.76 ± 0.59 64.93 ± 0.57 273.03 ± 6.40 a a a a FGB 165.51 ± 1.37 280.52 ± 1.01 68.51 ± 0.68 479.56 ± 6.61 Data are expressed as mean ± SD Different superscript letters in the same column mean significantly different at p < 0.05 by Duncan’s multiple range test GB Freeze-dried goji berry sample (88% solids); FGB Freeze-dried goji berry samples (88% solids) pretreated with Pectinex Ultra SP-L and fermented with L. plantarum CB3 L ee et al. Appl Biol Chem (2021) 64:70 Page 5 of 11 Fig. 1 Supplementation of non‑fermented and fermented goji berry has little effects on hyperphagic phenotypes. Body weight (A), cumulative energy intake (B), feed efficiency [weight gain (g)/energy intake (kcal); (C)], and visceral adiposity (D) in rats fed an LF or HF with or without 1% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. For Fig. 1A, significant # /#/ /## differences are denoted as an asterisk (*) for LF vs. HF, sharp ( ) for LF vs. HF/GB, and dagger (†) for HF vs. HF/FGB at * †p < 0.05 and ** p < 0.01. For Fig. 1B–D, significant differences are denoted as an asterisk (*) at *p < 0.05 and **p < 0.01. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat p < 0.0001; LF vs. HF/FGB, p < 0.001; HF vs. HF/FGB, p (PPARγ) was significantly upregulated by HF feeding < 0.01; Fig. 3). compared to the LF group, which was normalized to the LF level by both GB and FGB supplementation (LF vs. Supplementation of GB and FGB improves hepatic HF, p < 0.0001; HF vs. GB or FGB, p < 0.0001; Fig.  5). functions Similarly, the gene expression of hepatic sterol regula- Hepatic functions were examined by the level of ALP and tor element-binding protein 1c (SREBP1c), as a marker AST in circulation as markers of liver damage (Fig. 4). HF of adipogenesis, was partially upregulated by HF feed- feeding had little effect on the level of serum ALP but the ing compared to the LF group and this was significantly level was significantly decreased by 2% GB supplemen - suppressed by both GB and FGB supplementation (LF tation compared to the HF group (Fig.  4A). The serum vs. HF, p = 0.059; HF vs. GB, p < 0.01; HF vs. FGB, p < ALP level in the HF/GB group was even lower than the 0.001; Fig. 5B). The similar effect of GB and FGB supple - LF group by 27%. Similarly, the serum AST level was sig- mentation on lipid profile was also confirmed at the sys - nificantly reduced by both GB and FGB supplementation temic level; the serum TG level was significantly reduced compared to the HF group (HF vs. GB, p < 0.001; HF vs. by both GB and RGB supplementation compared to the FGB, p < 0.05; Fig. 4B). HF group (HF vs. GB or FGB, p < 0.01; Fig. 5C). Supplementation of GB and FGB improves lipid profiles Discussion In addition to visceral adiposity, lipid profile was fur - In this present study, we investigated the beneficial effects ther examined in the liver and in circulation (Fig.  5). As of GB supplementation on obesity and related metabolic a marker of lipogenesis, the gene expression of hepatic disorders in HF diet-fed rats. We also sought to examine peroxisome proliferator-activated receptor gamma the beneficial effects of fermented GB with L. Plantarum Lee et al. Appl Biol Chem (2021) 64:70 Page 6 of 11 Fig. 2 Supplementation of non‑fermented and fermented goji berry attenuates HF‑induced inflammation. Gene expression of IL ‑6 and TNFα in epididymal fat (A–C) and liver (D, E) tissues of rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05 and **p < 0.01. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); IL interleukin; LF low fat; MCP-1 macrophage chemoattractant protein 1; TNFα tumor necrosis factor alpha CB3, elucidating the possible metabolic benefits result - it functions as a methyl donor and has the effects of ing from the fermentation process. Our hypothesis was improving liver function, cell replication, detoxification, GB and FGB supplementation as a part of HF diet would and kidney protection [25, 26]. improve the HF-induced disturbances in lipid, inflam - In this study, we observed that betaine concentra- matory, and oxidative profiles along with hepatic func - tion of goji berry was significantly increased by enzyme tions, possibly preventing the development of obesity and (Pectinex Ultra SP-L) treatment and L. plantarum CB3 related metabolic abnormalities, and that these effects fermentation, confirming the previous finding that the would be enhanced by fermentation. betaine concentration increases after fermenting goji ber- As major bioactive components of GB, concentrations ries with various lactic acid bacteria [27]. Total phenolic of betaine and total polyphenolic and their correspond- contents, DPPH radical scavenging ability, and FRAP ing antioxidant activities were measured in GB and FGB assay were significantly increased after fermentation with samples. Betaine has similar functions to choline, folic L. plantarum CB3 inoculation, which is corresponds to acid, vitamin B , and the amino acid methionine known the finding of the previous studies [28]. This seems to be as SAMe (S-adenosylmethionine). In the human body, because the enzyme and carboxylic acid produced by L. L ee et al. Appl Biol Chem (2021) 64:70 Page 7 of 11 Fig. 3 Supplementation of non‑fermented and fermented goji berry exerts antioxidant effects. Gene expression of antioxidant enzymes, CAT (A), GPX (B), GSR (C), and SOD1 (D), in the liver of rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. CAT catalase; GPX glutathione peroxidases; GSR glutathione reductase; HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat; SOD1 superoxide dismutase 1 Fig. 4 Supplementation of non‑fermented and fermented goji berry protects hepatic functions; levels of serum ALP (A) and AST (B) in rats fed an LF or HF with or without 2% non‑fermented or fermented goji berry supplementation diet for 6 weeks. Values are means ± SEMs; n = 8/group. Significant differences are denoted as an asterisk (*) at *p < 0.05 and ***p < 0.001. HF high fat; HF HF without goji berry; HF/FGB HF with fermented goji berry in diet by 2% (w/w); HF/GB HF with non‑fermented goji berry in diet by 2% (w/w). ALP alkaline phosphatase; AST aspartate transaminase; HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low fat plantarum destroy the cellular structure of GB and cause systemic (IL-1β in the liver) levels. In addition, GB and the release of phenolic substances in the fermentation FGB supplementation upregulated the gene expression process [29]. of antioxidant enzymes, catalase (CAT), glutathione per- In vivo, GB and FGB supplementation suppressed oxidases (GPX), glutathione reductase (GSR), superoxide the gene expression of inflammation indices at the dismutase (SOD1) compared to the HF and/or even LF local (MCP-1, IL-1β, TNFα in the adipose tissues) and group with more remarkable antioxidant effects by GB Lee et al. Appl Biol Chem (2021) 64:70 Page 8 of 11 GB and FGB supplementation improved HF-induced metabolic disturbances and these improvements were associated with their anti-inflammatory and anti-oxidant properties. It is widely demonstrated that consumption of a HF diet can lead to increases in body weight, energy intake, and visceral fat mass [30]. In this present study, HF feed- ing for six weeks significantly increased the body weight. While little difference was observed in overall energy intake and feed efficiency between LF and HF fed groups, an increase in adiposity was still observed in HF-fed rats compared to the LF group, confirming the previous finding that total and visceral body fat weights increases proportionally to the level of fat in the diet even when fed isocalorically [31]. It was unexpected that GB and FGB supplementation did not prevent HF-diet induced increases in body weight gain and adiposity. Thus, to bet - ter understand the limited effects of supplementation on these obesity phenotypes, we further investigated the related parameters at the molecular level. The pathogenesis of obesity involves infiltration of macrophages into expanding adipose tissue and the acti- vated macrophages can contribute to local (adipose tis- sue) and systemic (liver) inflammation, which is primarily promoted by the release of pro-inflammatory cytokines [2]. In particular, studies have demonstrated that HF diet- induced obese animals developed chronic inflammation in adipose and hepatic tissues [32–34]. In this present study, the HF group showed the highest expression of MCP-1 gene, which was remarkably down-regulated by GB and FGB supplementation. Correspondingly, GB and FGB supplementation also exerted anti-inflammatory effects against other pro-inflammatory cytokines, IL-1β and TNFα and in favor of IL-10 as an anti-inflammatory cytokine at the local and systemic levels, which corre- sponds to the finding of the previous study [35]. The inflammatory cascade facilitates the formation of toxic reactive oxygen species and the subsequent produc- Fig. 5 Supplementation of non‑fermented and fermented goji berry tion of oxidative stress, which inhibits the expression of improves lipid profiles. Gene expression of PPARγ (A) and SREBP1c antioxidant enzymes and consequently impairs the anti- (B) and serum TG level (C) in rats fed an LF or HF with or without oxidant defense system [3]. Here, we observed that GB 2% non‑fermented or fermented goji berry supplementation diet and/or FGB supplementation remarkably upregulated for 6 weeks. Values are means ± SEMs; n = 8/group. Significant the gene expression of antioxidant enzymes in the liver differences are denoted as an asterisk (*) at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HF high fat; HF HF without goji including CAT, GPX, GSR, and SOD1 compared both the berry; HF/FGB HF with fermented goji berry in diet by 1% (w/w); LF and HF groups, which is consistent to the previous HF/GB HF with non‑fermented goji berry in diet by 2% (w/w); LF low findings of antioxidant effects of GB and FGB in vitro and fat; PPARγ peroxisome proliferator‑activated receptor gamma; SREBP1c in vivo [10, 36, 37] sterol regulator element‑binding protein 1c; TG triglyceride Excessive oxidative stress also can cause tissue damage to the liver [4]. It is known that ALP and AST enzymes are reliable markers of hepatic function and increased supplementation. Also, GB and FGB supplementation levels of circulating ALP and AST indicate hepatic dam- protected from HF-induced damages of the liver and ages [38, 39]. Here, the highest levels of serum ALP and dyslipidemia. Taken together, these data show that both AST were observed in the HF group and their activities L ee et al. Appl Biol Chem (2021) 64:70 Page 9 of 11 were significantly inhibited by GB and/or FGB sup - metabolic effects between non-fermented and fermented plementation. It is important to note that the elevation prebiotics resulting from fermentation with probiotics in these hepatic enzymes have been associated with [45–47]. In these studies, metabolic benefits were more abnormal hepatic lipid metabolism [5]. Further, studies enhanced or different mechanism of action was resulted have previously shown that HF feeding caused ectopic by fermentation compared to the original prebiotics. In fat deposition, which was closely associated with the this regard, it is possible that different pathways might upregulated expression of hepatic PPARγ as a transcrip- have been involved in metabolic benefits observed in the tion factor for adipogenesis [32]. Considered as the mas- HF/FGB group. Thus, further investigation into multiple ter regulator of adipogenesis [40], its overexpression has possible metabolic pathways would identify the potential been known to be sufficient for inducement of hepatic fat mechanism of action by FGB for its metabolic benefits. deposition [41] in concert with SREBP1c as a transcrip- Further, since the gut microbiota has been identified the tion factor of lipogenesis and also a mediator of PPARγ primary target by prebiotics and probiotics for their met- expression [42]. In this study, both GB and FGB signifi - abolic benefits, metagenomic and metabolomic analyses cantly suppressed HF-induced upregulation of PPARγ would provide a better understanding of distinct mecha- and SREBP1c gene expression in the liver. This was also nism of action on metabolic profiles between GB and confirmed at the systemic level. As a well-known indica - FGB. tor of obesity, dyslipidemia is primarily characterized by In conclusion, we demonstrated that GB and FGB sup- the elevation of triglycerides in circulation [6]. In this plementation protected from HF-induced metabolic present study, the serum TG level was increased in the complications primarily by improving hepatic function HF group by 19% compared to the LF group and this ele- and corresponding lipid metabolism via their anti-inflam - vation was significantly suppressed by both GB and FGB matory and antioxidant properties. To our knowledge, supplementation. The beneficial effects of GB supple - this is the first in  vivo study confirming metabolic ben - mentation also has been reported in previous studies via efits of GB in a fermented form. u Th s, these findings inhibiting the expression of hepatic lipogenesis factors support the potential application of both GB and FGB to and/or decreasing the level of triglyceride in circulation, ameliorate obesity-associated metabolic abnormalities. which confirms the findings of this study [43, 44]. Taken together, it can be confirmed that GB and FGB Abbreviations supplementation improved HF-induced metabolic dys- ALP: Alkaline phosphatase; AST: Aspartate transaminase; ANOVA: Analysis of regulation in adipose and liver tissues at the molecular variance; CAT : Catalase; GPX: Glutathione peroxidases; GSR: Glutathione reduc‑ tase; HF: High fat; HF/FGB: HF with fermented goji berry in diet by 2% (w/w); level primarily by enhancing anti-inflammatory and anti - HF/GB: HF with non‑fermented goji berry in diet by 2% (w/w); IL: Interleukin; oxidant responses. LF: Low fat; PPARγ: Peroxisome proliferator‑activated receptor gamma; ROS: There are some considerations to this study that Reactive oxygen species; SOD1: Superoxide dismutase; SREBP1c: Sterol regula‑ tor element‑binding protein 1c; TG: Triglyceride; TNFα: Tumor necrosis factor deserve more attention. First, we employed a HF regime alpha; MCP‑1: Macrophage chemoattractant protein. for six weeks to induce diet-induced obesity and related metabolic disturbances. This protocol was sufficient to Supplementary Information induce obese phenotypes (increases in body weight and The online version contains supplementary material available at https:// doi. visceral fat mass), which was further supported by HF- org/ 10. 1186/ s13765‑ 021‑ 00642‑1. induced dysregulation of hepatic metabolism (IL-1β, PPARγ, and SREBP1c). However, we failed to find any Additional file 1: Table S1. Composition of experimental diets. Table S2. remarkable differences between the LF and HF groups Primer sequences used for RT‑PCR. for the other metabolic parameters at the molecular level including those for inflammation, antioxidant actions, Acknowledgements and hepatic functions. Thus, a study with longer duration This research was supported by the Ministry of Trade, Industry and Energy in 2019 [Grant Number P0010311]. than six weeks would better enable the establishment of other measures for the development of HF-induced Authors’ contributions obesity. In the meanwhile, despite little difference the Conceptualization: HS (Hee‑jae Suh), HP, SL. Animal experiment: SL, SJ, YP, CY, UH. Sample preparation: SJ, YP, HS (Hyunji Seo), CY, UH. Sample analysis: SL, SJ. LF and HF groups in those metabolic parameters, both Data analysis: SL. Writing, review, and editing: HS (Hee‑jae Suh), HP, SL. Funding GB and FGB supplementation demonstrated metabolic acquisition: HS (Hee‑jae Suh). All authors have read and approved the final benefits compared to the HF group, even compared manuscript. to the LF group for the regulation of the antioxidant defense system. Notably, these beneficial effects were Declarations more outstanding in the HF/GB group. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Dyslipidemia; Fermentation; Goji berry (Lycium barbarum); Hepatic function; Inflammation; Obesity; Oxidative stress

References