Beet (Beta vulgaris L.) Stalk and Leaf Supplementation Improves Glucose Homeostasis and Insulin Resistance Markers in Liver of Mice Exposed to a High-Fat Diet

doi:10.21203/rs.3.rs-40368/v1

PDF

Research

Beet (Beta vulgaris L.) Stalk and Leaf Supplementation Improves Glucose Homeostasis and Insulin Resistance Markers in Liver of Mice Exposed to a High-Fat Diet

https://doi.org/10.21203/rs.3.rs-40368/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 03 Aug, 2020

You are reading this latest preprint version

Background Although beet stalks and leaves are not consumed and are usually discarded, they are an important source of bioactive flavonoids possessing antioxidant and anti-inflammatory activity, which could be explored to prevent metabolic disorders associated with an unhealthy diet. The aim of this study was to assess the effect of supplementation with beet (Beta vulgaris L.) stalks and leaves on metabolic parameters and glucose homeostasis in mice exposed to a high-fat diet.

Methods Six-week-old male Swiss mice were randomly divided into five experimental groups submitted to either standard diet (CT) or high-fat diet (HF), and HF-fed mice were subdivided into three treatment groups supplemented with oven-dehydrated beet stalks and leaves (SL), lyophilized beet stalks and leaves (Ly) or beet stalk and leaf extract (EX).

Results Supplementation with SL was able to ameliorate glucose homeostasis (P < 0.05) with no alteration in hepatic triglyceride content. It remains to be clarified if the enhancement in the glucose homeostasis observed in HFSL could be a consequence of improvement in pancreatic insulin secretion and/or glucose uptake from skeletal muscle and white adipose tissues.

Conclusions The current results suggest that beet stalks and leaves could be used as adjuvants to improve parameters related to glucose metabolism in the liver.

Nutrition & Dietetics

Endocrinology & Metabolism

Obesity

Phenolic compounds

Glucose homeostasis

Liver

Food waste

Beetroot

Obesity is associated with a high incidence of many metabolic disorders, including type 2 diabetes mellitus (DM2) and cardiovascular diseases. DM2 may be due to evolution of the glucose intolerance observed in metabolic syndrome, which is also associated with excessive weight gain and a sedentary lifestyle [1]. Hepatic disorders, such as non-alcoholic fatty liver disease (NAFLD), could be associated with obesity and have been considered by many authors the hepatic manifestation of metabolic syndrome [2–4]. One alternative to minimize the obesity associated diseases is to change the lifestyle with improvements in the usual diet quality [5–7].

It is known that diet characteristics are one of the main causes associated with obesity and that consumption of fruits and vegetables provides health benefits. One example is beetroot (Beta vulgaris L.), which presents a high concentration of phenolic compounds and other substances, such as betalains, in its composition which have a positive effect on health [8–12]. Additionally to the antioxidative properties and hepatoprotective activity [12], some authors have observed that consumption of beetroot or its derivative products is able to improve sports performance, glycaemia control and blood pressure [13–19]. However, despite the positive effects of beetroot or its derivatives on human health, there are few studies on non-conventional parts of beetroot such as stalks and leaves.

In some countries, some parts of the beetroot such as stalks and leaves are considered food waste or a by-product commonly discarded. Some fruits and vegetables and some fruits yield between 25% and 30% of nonedible products [20–22]. However, different types of flavonoids (myricetin, quercetin, rutin and kaempferol) considered bioactive phytochemicals are found in beet leaves [23]. Recent studies have shown different positive effects on metabolic parameters from treatment or supplementation with these phenolic compounds. Ferulic acid treatment to DM2 in rats, for example, improves insulin sensitivity and hepatic glycogenesis [24]. Kulabas et al. [25] observed that Lavandula stoechas L. extract was effective in the treatment/prevention of insulin resistance and inflammation, attributed to the presence of caffeic acid, apigenin, luteolin, rosmarinic acid and its methyl ester, 4-hydroxybenzoic acid, vanillic acid, ferulic acid and salicylic acid. Other flavonoids, such as vitexin-2-O-rhamnoside and other vitexin derivatives, are also found in beet stalks and leaves. These compounds are able to attenuate the deleterious effects of a high-fat diet on oxidative damage in the liver in mice [26]. According to Bumke-Vogt et al. [27], administration of the flavones apigenin and luteolin to cells from human liver carcinomas has shown antidiabetic potential and reduces hepatic steatosis.

Thus, supplementation with beet stalks and leaves, or even extracts rich in the bioactive compounds, may improve the metabolic parameters involved in gluconeogenesis and liver function. So, our aim was to evaluate the effect of supplementation with beet (Beta vulgaris L.) stalks and leaves on glucose homeostasis and insulin resistance markers in liver of mice exposed to a high-fat diet.

Organic beetroot (Beta vulgaris L.) stalks and leaves, grown in May and April 2016, were obtained from a producer in the city of Limeira, São Paulo state, Brazil. All reagents used for analysis were of analytical grade. High-performance liquid chromatography (HPLC)-grade acetonitrile was obtained from J.T. Baker (Phillipsburg, NJ, USA), and phosphoric acid was obtained from Labsynth (Diadema, SP, Brazil). Vitexin-2-rhamnoside standard was purchased from Sigma-Aldrich (São Paulo, Brazil).

Preparation of the raw material used in the experimental diets

An overview of the preparation of the beet stalks and leaves and extract to be used for supplementation of the diets is presented in Fig. 1. Beet stalks and leaves were sanitized, and excess moisture was removed at room temperature. Stalks/leaves were divided into two groups; one group was placed on aluminum trays and taken to an oven to perform the dehydration process (Clarice Brand, Pinhalzinho, SC, Brazil) at 180 °C for 45 min (SL). The samples were homogenized and stored in hermetically sealed containers at − 80 °C. The other group was placed in aluminum trays, frozen and submitted to the lyophilization process (Ly) (Laboratório de Apoio Central – LAC, Faculty of Food Engineering, University of Campinas, SP, Brazil) (Fig. 1). The samples were stored in hermetically sealed containers at − 80 °C. The beet stalk and leaf extract that was used to supplement the mice diet (EX) was prepared only using dehydrated beet stalks and leaves (SL). These two extracts (lyophilized and dehydrated at 180 °C/45 min) were used to determine the total and individual phenolic compound concentration in the samples. The extract was prepared using 1.0 ± 0.1 g of oven-dried beet stalks and leaves. The raw material was placed in a conical centrifuge tube with 10 mL of ultrapure water and stirred on a tube shaker (Phoenix Luferco AP56) for 5 min, followed by centrifugation (5810 R Centrifuge, Germany) at 4,000 rpm for 15 min at room temperature (25 ± 1 °C). After centrifugation, the supernatant was filtered through Whatman no. 3 filter paper, 10 mL of 80% ethanol was added to the solid residue in the conical tube, and the process was repeated under the same conditions. The extracts obtained by the two sequential extractions of the same raw material were mixed, and the volume was completed to 20 mL with 80% ethanol in a volumetric flask. The extract was stored at − 80 °C in amber glass until used for HPLC analysis, and the extract (EX) obtained from dehydrated beet stalks and leaves (SL) was stored at − 80 °C until used to supplement the diet (EX group).

Sample preparation for the analysis of beet stalks and leaves by HPLC

Freeze-dried and oven-dried beet stalk and leaf samples were prepared using an extraction protocol similar to that used for preparation of the extract applied for supplementation of the experimental diets. The samples (1.0 g) were extracted by two sequential extractions. The first extraction was carried out using 10 mL of water and by stirring for 5 min. After centrifugation, the supernatant was collected, and the solid residue re-extracted with 10 mL of 80% ethanol by stirring for 5 min. Both extracts were combined, and the volume brought up to 20 mL in a volumetric flask. The extracts were filtered through syringe filters (nylon, 25 mm, 0.22 µm; Analytica, Barueri, São Paulo, Brazil) before chromatographic analysis.

Identification of the compounds present in the extracts by ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC–MS/MS)

The compounds present in the extract were identified using a UHPLC–MS/MS 8040 instrument (Shimadzu, Kyoto, Japan) consisting of a liquid chromatography system coupled to a triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source. Chromatographic separation was performed on a Kinetex C18 column (2.6 µm, 3.0 mm i.d., 100 mm; Phenomenex, California, USA) using a binary mobile phase. Solvent A was water, and solvent B was acidified acetonitrile (0.1% formic acid). The elution gradient used at 40 °C was as follows: 0 min, 98% A; 5 min, 98% A; 15 min, 85% A; 20 min, 80% A; 25 min, 65% A; 30 min, 20% A; 34 min, 20% A; 35 min, 98% A, at a flow rate of 0.3 mL/min. The autosampler temperature was maintained at 10 °C, and the injection volume was 10 µL. The ESI source parameters were as follows: capillary voltage, − 3.5 kV; heat block temperature, 500 °C; desolvation line temperature, 250 °C; drying gas flow (N₂), 10 L/min; nebulizing gas flow (N₂), 1.5 L/min; collision-induced dissociation gas pressure (Ar), 224 kPa. For each compound, ESI(−)–MS/MS data were first collected for the identification of deprotonated molecules [M − H], and two of the most selective product ions were chosen for the MRM transitions using a dwell time of 20 ms. Data were acquired and processed with Labsolution software (version 5.53 SP2, Shimadzu). The recorded masses were processed throughout the chromatogram during the time interval of the peaks present at different wavelengths of detection (260, 290, 335, 360 and 484 nm). The extract was then reinjected, the detected masses were examined for fragmentation using different capillary voltages, and the detected fragments were recorded. The main compound present in the extracts was identified as vitexin-2-rhamnoside based on its molecular weight (MW 578) and the presence of the fragments (m/z) 457, 274, 413 and 293. Most compounds present in the extracts were related to vitexin, an apigenin flavone glucoside, presenting the characteristic 293 ion in the MS/MS spectra.

HPLC analysis

The HPLC analysis of the samples was carried out in an EXTRACT-US analysis system (FAPESP 2013/04304-4, patent pending), consisting of an HPLC pump (PU2080, Jasco, Kyoto, Japan), an HPLC ternary gradient unit (LG 2080-2, Jasco, Japan), a three-line degasser (DG 2080-55, Jasco, Kyoto, Japan), a UV-Vis detector (UV-7075, Jasco) and five automatic two-position 10-port valves (Waters Corporation, Milford, MA, USA). The compounds were separated by an adaptation of the method developed by Rostagno et al. using a fused core-type column (Kinetex C18, 2.6 μm, 100 A, 100 × 4.6 mm; Phenomenex, Torrance, CA, USA) [28]. The column was maintained at room temperature. The mobile phase consisted of water with 1% v/v phosphoric acid (solvent A), and acetonitrile with 1% v/v phosphoric acid (solvent B). The gradient profile was: 2 min, 88% A; 4 min, 80% A; 6 min, 70% A; 8 min, 40% A; 10 min, 20% A; 13 min, 20% A; and 14 min, 95% A. The equilibration time between runs was 3 min. Flow rate was 1.2 mL/min, and injection volume was 5 μL. Peaks were recorded and integrated at 320 nm. The software for the control of the system was developed by Kalatec (Campinas, SP, Brazil). ChromNav software from Jasco was used for data acquisition and processing. The vitexin-2-O-rhamnoside compound was identified by comparing the retention times of the peak obtained in analysis of the extracts to the peak obtained in analysis of the authentic standard. The vitexin-2-O-rhamnoside standard solution (100 mg/L) was diluted in a mixture of methanol and water (90 : 10 v/v) to prepare the calibration curve (six points: 100, 50, 25, 2.5, 1 and 0.5 ppm). The calibration curve for the compound was prepared by plotting concentration versus area. All compounds present in the samples were expressed as vitexin-2-rhamnoside equivalents (VRE). The analysis was performed in duplicate.

Experimental protocol

The experiments involving animal procedures followed the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health, and the guidelines of the Brazilian College for Animal Experimentation. Experiments were approved by the Ethics Committee on Animal Use – CEUA, UNICAMP, under protocol 4239-1. Forty Swiss male mice, 21 days old, were obtained from the UNICAMP Animal Centre. The animals were allowed to acclimate for 3 weeks before the beginning of the experiment and were then divided randomly into five groups with a similar mean weight and standard deviation: (CT) standard group (n = 8); (HF) high-fat diet group (n = 8); (HFEX) group fed high-fat diet supplemented with extract of dried beet stalks and leaves (n = 8); (HFSL) group fed high-fat diet supplemented with dried beet stalks and leaves (n = 8); and (HFLy) group fed high-fat diet supplemented with lyophilized beet stalks and leaves. Diet and water were given ad libitum throughout the experiment. The animals were kept in a temperature-controlled environment (25 ± 1 °C) with a 12-h light cycle. An analytical balance (Mark 500, Bel Engineering, Italy) was used to weigh the animals and to analyze the food intake and was checked once a week during the 8-week experiment. The experimental protocol of supplementation with beet stalks and leaves is presented in Figure 2; 0.5% of dehydrated or lyophilized beet stalks and leaves was mixed directly into the diet (HFSL and HFLy) (Figure 1). The beet stalk and leaf extract were added directly into the diet (HFEX), and the total volume (10 mL/100 g) was adjusted to enhance the total phenolic compounds expressed as VRE/100 g diet compared to the phenolic concentration in the HFSL diet (Figure 1). Therefore, the group supplemented with dehydrated (powder) beet stalks and leaves (HFSL) received 2.04 mg VRE/100 g of diet, the animals supplemented with beet stalk and leaf extract (HFEX) received 2.04 mg VRE/100 g of diet, and the group supplemented with lyophilized beet stalks and leaves received 4.10 mg VRE/100 g of diet. All groups were treated for 8 weeks. The composition and nutritional value of the control diet and the high-fat diet are shown in Table 1.

Table 1.
Composition and nutritional value of the diets.
Ingredients (g/100g)	Standard (g)	High-fat diet 60%
	Standard (g)	High-fat diet (g)	Extract of stalks and leaves (g)	Dried stalks and leaves (g)	Lyophilized stalks and leaves (g)
	(CT)	(HF)	(HFEX)	(HFSL)	(HFLy)
Corn starch	41.07	2.00	2.00	2.00	2.00
Casein	11.00	16.10	16.10	16.10	16.10
Wheat flour	20.00	24.00	24.00	24.00	24.00
Extract of beet stalks and leaves (mL)	--	--	10.00	--	--
Dehydrated beet stalks and leaves	--	--	--	0.50	0.50
Dextrinized corn starch	11.50	3.91	3.91	3.91	3.91
Sucrose	2.50	10.00	10.00	10.00	10.00
Soybean Oil	4.00	4.00	4.00	4.00	4.00
Lard	-	30.00	30.00	30.00	30.00
Cellulose microfiber (fiber)	5.00	5.00	5.00	4.50	4.50
Mineral mix	3.50	3.50	3.50	3.50	3.50
Vitaminmix	1.00	1.00	1.00	1.00	1.00
L-cistine	0.18	0.24	0.24	0.24	0.24
Choline bitartrate	0.25	0.25	0.25	0.25	0.25
Total	100	100	100	100	100
Energy value (kcal/g)	3.90	5.35	5.35	5.35	5.35
Composition of standard diet (CT), high-fat diet 60% (HF), high-fat diet 60% supplemented with extract of stalks and leaves (HFEX), high-fat diet 60% supplemented with dried stalks and leaves (HFSL) and high-fat diet 60% supplemented with lyophilized stalks and leaves (HFLy). *CT diet contains 10% of dietary energy as lipids, and high-fat diets (HF, HFEX, HFSL and HFLy) contain 60% of dietary energy as lipids.

Intraperitoneal glucose tolerance test (ipGTT)

Basal blood glucose levels were measured after 4 h of fasting with an Accu-Check Performa glucometer (Roche). The test was done after an intraperitoneal injection of glucose solution (1.0 g glucose/kg body weight). Blood glucose concentration was measured in blood from tail-tip bleedings and were used to determine glucose levels at 0, 15, 30, 60 and 120 min. Area under the curve values were determined by the trapezoidal method.

Western blotting

At the end of the experiment, the animals were anesthetized with a solution of sodium ketamine (0.1 g/kg), diazepam (5 mg/kg) and xylazine (3 mg/kg), and decapitation was used to cull mice that had been starved for 12 h. The negative control liver samples were weighed, frozen in liquid nitrogen and stored at −80 °C for further analysis. To evaluate insulin signalling, a bolus injection of regular insulin (5 UI) was administered (Humulin, Eli Lilly and Company, USA) through the abdominal cava vein; subsequently, hepatic samples were extracted after 45 s, this tissue being the positive control for the stimulus. The samples were homogenized in freshly prepared ice-cold buffer [1% (v/v) Triton X-100, 0.1 mol/L Tris, pH 7.4, 0.1 mol/L sodium pyrophosphate, 0.1 mol/L sodium fluoride, 0.01 mol/L EDTA, 0.01 mol/L sodium vanadate, 0.002 mol/L PMSF and 0.01 mg/mL aprotinin] using a tissue homogenizer (Bead Ruptor 12 Homogenizer, Omni International, Kennesaw, GA, USA). Insoluble material was removed by centrifugation (10,000 g) for 30 min at 4 °C. The protein concentration of the supernatant was determined using the Biuret dye-binding method. The supernatant was suspended in Laemmli sample buffer (1 mmol sodium phosphate/L, pH 7.8, 0.1% bromophenol blue, 50% glycerol, 10% sodium dodecyl sulphate (SDS), 2% mercaptoethanol). Immunoblotting was performed by using protein extract (50 μg) from each mouse sample and polyacrylamide gels (SDS-PAGE) using a miniature gel apparatus (BioRad, Richmond, CA, USA). Electrotransfer of proteins from the gel to a nitrocellulose membrane was performed for 120 min at 120 V (constant) in a transfer buffer that contained methanol and SDS. Membranes were then blocked with a solution containing 5% fat-free milk in Tris-buffered saline (TBS)–Tween 20 (TTBS; 10 mmol Tris/L, 150 mmol NaCl/L, 0.5% Tween 20) for 2 h at room temperature. Nitrocellulose membranes were probed overnight at 4 °C with specific antibodies [pAKT (#4060), AKT (#4691) and β-actin (ab8227)]. The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1 : 5000 in TTBS containing 3% dry fat-free milk) at room temperature, which was followed with a 2-h incubation. Bands were detected by chemiluminescence (Thermo Scientific #34078) and quantified by densitometry (UN-Scan-it Gel 6.1, Silk Scientific Inc, Orem, UT, USA).

Haematoxylin and eosin staining

The hepatic tissues of the mice (n = 3 per group) were perfused with 4% paraformaldehyde solution during sacrifice. Then, liver sections of approximately 3 cm were fixed in 6% formaldehyde for approximately 6 h. The liver sections were then washed in running water for 40 min and processed in the following solutions: 70% alcohol solution; 80% alcohol solution; 90% alcohol solution; 99% alcohol solution; xylol I; xylol II; paraffin I; paraffin II, at 65 °C. After being embedded in paraffin, the slices were incorporated into a mould, cut into sections of 5 μm thickness using a microtome and finally stained with haematoxylin and eosin (H&E) for visualization of hepatocyte morphology. NAFLD activity score [29] was applied for semiquantitative analysis of the three defined criteria of NASH: steatosis (0–3), ballooning (0–3) and lobular inflammation (0–2) using a Leica DMI 4000 B (Switzerland) microscope.

Fatty acid profile

Hepatic total lipids were extracted from 200 mg of tissue following the method of Folch [30]. The samples were homogenized in 1 mL Folch solution (2 :1 v/v chloroform/methanol) and centrifuged at 1,000 rpm for 10 min. The supernatants were collected, and the lipid-containing fraction was dried. Lipids were transmethylated, and the resulting fatty acid methyl esters were analyzed by gas chromatography as previously described [31].

Data presentation and statistical analysis

The results are expressed as the mean ± SE of the indicated number of experiments. The Levene test for the homogeneity of variances was initially used to check the fit of data to the assumptions for parametric ANOVA. Data were analyzed by one-way ANOVA followed by LSD post hoc tests if there were significant interactions among CT, HF and HFEX; CT, HF and HFSL; or CT, HF and HFLy groups. The level of significance was set at P < 0.05.

The concentration of phenolic compounds was different in the samples obtained from beet stalks and leaves

Analysis of the samples showed a complex matrix made of several different compounds at different concentrations (Table 2). The main compound present in the samples was the flavonoid vitexin-2-rhamnoside (V2R), an apigenin derivative. The UHPLC–MS/MS indicated that most compounds were associated with vitexin due to the presence of the molecular ion m/z 293, characteristic of this compound, and therefore were expressed as VRE.

Table 2

Concentration of the main compounds present in the extracts.
Peak*	EX	SL	Ly
Peak*	(mg/10 mL)^a	(mg/g)^b	(mg/g)^c
1	0.01	0.02	0.03
2	0.03	0.03	0.03
3	0.03	0.03	0.03
4 (Vitexin- 2- rhamnoside)	0.82	0.82	1.47
5	0.04	0.04	0.09
6	0.09	0.10	0.26
7	0.37	0.37	1.76
8	0.10	0.10	0.22
9	0.04	0.04	0.04
10	0.31	0.31	0.13
11	0.03	0.03	0.03
12	0.03	0.03	0.02
13	0.15	0.15	0.04
Total	2.04	2.04	4.10
* Indicates the results expressed in vitexin-2-hamnoside equivalents. ^a Concentration (mg/10 mL) in the extract obtained from dehydrated beet stalks and leaves (EX); ^b Concentration (mg/g) in the dehydrated beet stalks and leaves (SL); ^c Concentration (mg/g) in the freeze-dried beet stalks and leaves (Ly).

Analysis of the samples revealed that freeze-dried and oven-dried beetroot stalks and leaves have a different chemical profile (Fig. 3). Basically, the same compounds were detected but differences were observed in the amount of individual compounds, possibly caused by degradation of compounds present due to exposure to high temperatures.

V2R (peak #4) was significantly affected by the high temperature used in the oven for drying the raw material, and a much lower concentration of this compound was found in the SL sample (1.63 mg/g) as compared to the freeze-dried sample (2.93 mg/g). There was also a drastic reduction in the concentration of another vitexin derivative (peak #7), from 3.51 (Ly) to 0.74 mg VRE/g when comparing the freeze-dried sample with the oven-dried sample (SL). In contrast, the concentrations of several V2R-related compounds (peaks #5, #6 and #8) were higher in Ly when compared to SL, which can be explained by the thermal degradation of the compounds present, promoting interconversion between chemical forms.

Additionally, a lower total amount of the compounds detected by HPLC was observed in the SL sample (4.08 mg VRE/g) when compared to the HFSL sample (8.21 mg VRE/g).

The chemical profile of the extract was the same as that of the SL sample since the same raw material was used in both cases. Considering the amount of extract used to supplement the diet (10 mL), the EX group received an estimated total of 2.04 mg VRE/100 g of feed, of which 0.82 mg was V2R.

High-fat diet led to body mass gain after 8 weeks

Eight weeks of high-fat (HF) diet led to increased body mass gain (P < 0.05) after the third week of the experiment compared to the normocaloric group (CT) (Fig. 4A and Table 3). There was no difference in body mass (P > 0.05) among groups with beet stalks and beet leaves (HFEX, HFSL and HFLy). There was no difference in food intake (P > 0.05), with a mean consumption of: HF = 3.57 g/day (18.19 calories/day); CT = 5.25 g/day (18.92 calories/day); HFEX = 4.87 g/day (24.82 calories/day); HFSL = 3.41 g/day (17.38 calories/day) and HFLy = 3.43 g/day (17.50 calories/day), and the animals did not presented adverse symptoms as diarrhea. These results suggest that the supplementation with beet stalks and leaves could not suppress the body mass gain of diet induced obesity (DIO) mice.

Table 3

Body mass gain, fasting glucose and area under glucose curve (AUC) during the ipGTT test.
	CT	HF	HFEX	HFSL	HFLy
Total weight gain (g)	3.68 ± 0.43^a	18.75 ± 2.24^b	18.70 ± 2.12^b	13.66 ± 1.31^b	17.60 ± 1.99^b
Fasting glucose (mmol/L)	9.8 ± 0.6^a	11.7 ± 0.4^b	11.6 ± 0.4^b	9.9 ± 0.6^a	11.6 ± 0.7^b
Area under the curve (mg/dL × 120 min × 10³)	28.75 ± 0.88^a	46.68 ± 2.00^b	45.37 ± 3.75^b	35.10 ± 2.24^a	41.61 ± 1.65^b
*Body mass and fasting glucose (n = 8 animals per group), area under glucose curve (AUC) (n = 5–8 animals per group). Control (CT); high-fat diet (HF); high-fat diet with extract of stalks and leaves (HFEX); high-fat diet with dried stalks and leaves (HFSL); and high-fat diet with lyophilized stalks and leaves (HFLy). Data are shown as mean ± SEM. One-way ANOVA, P < 0.05.

Supplementation with beet stalks and leaves (HFEX, HFSL and HFLy) did not ameliorate hepatic fatty liver

Eight weeks of HF diet feeding dramatically induced hepatic steatosis, which was evidenced by H&E staining of liver sections and NAFLD activity score (NAS) (Table 4), when compared to the control group (Fig. 5A and B). The group treated with EX or Ly (Fig. 5C and 4E) showed qualitative evidence of lower fat accumulation and ballooning compared to the HF diet group (Table 4). There was no difference between SL supplemented group compared to the HF in the liver score of steatosis (Fig. 5D). Our results show that the HF diet was able to promote hepatic steatosis indistinctly and the score NAS evidenced only mildly ameliorated in mice treated with EX or Ly but the SL could not rescue the damage caused by lipid overload.

Table 4

NAFLD activity score (NAS) in mice.
Histological features	CT	HF	HFEX	HFSL	HFLy
Steatosis	0.67 ± 0.76^a	1.75 ± 0.65^a	1.00 ± 0.00^a	2.17 ± 0.29^a	1.50 ± 0.70^a
Hepatocellular ballooning	0.33 ± 0.28^b	1.75 ± 0.65^ab	1.00 ± 0.00^ab	2.17 ± 0.29^a	1.25 ± 1.06^ab
Lobular inflammation	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
n = 3 animals per group. Control (CT); high-fat diet (HF); high-fat diet with extract of stalks and leaves (HFEX); high-fat diet with dried stalks and leaves (HFSL); and high-fat diet with lyophilized stalks and leaves (HFLy). Data are shown as mean ± SEM. One-way ANOVA, P < 0.05. Data are presented in mean ± SD. Different letters at the same graphic mean statistical differences among experimental groups. Kruskal-Wallis, Dunn post-hoc test, P < 0.05. n = 3 per group.

Dehydrated powder of beet stalks and leaves ameliorated glucose homeostasis

As expected, there was an increase in fasting glucose in HF animals when compared to that of the CT group (P < 0.05). Interestingly, even without differences in body mass and steatosis among HF diet groups, supplementation with SL (HFSL group) caused a decrease in fasting glucose levels (P < 0.05) (Table 3). In order to analyze the glucose tolerance, a GTT was performed after 7 weeks of treatment (Fig. 4B). The HF group presented a greater area under the curve during the test when compared to the CT group (P < 0.05). As observed for fasting glucose, the HFSL group presented a decreased area under the curve (P < 0.05) (Table 3). These results confirm that consumption of a high-fat diet increased fasting glucose and impaired glucose homeostasis in HF, HFEX and HFly groups, but evidenced that SL treatment improved glucose homeostasis.

Supplementation with beet stalks and leaves did not alter the liver fatty acid profile after 8 weeks

In order to understand the link between our results above with the treatments, we performed analysis of the lipid profile in the liver after the experimental period (8 weeks). There was no difference in total fatty acid profile in the supplemented groups compared to the HF group. The polyunsaturated fatty acid (PUFA – DHA C22:6 and ARA C20:4) content increased in the liver of the HFSL group (Table 5). Overall, with this result we could not relate the mild alterations in the lipid profile shown here with the metabolic parameter results.

Table 5

Fatty acid composition obtained from hepatic lipids.
Fatty acid	CT (%)	HF (%)	HFEX (%)	HFSL (%)	HFLy (%)
C 12:0 (Lauric)	0.10	0.23	0.27	0.10	0.15
C 14:0 (Miristic)	0.50	0.50	0.52	0.28	0.39
C 15:0 (Pentadecanoic)	0.08	0.10	0.10	0.09	0.10
C 16:0 (Palmitic)	23.95	22.53	21.16	21.99	22.19
C 16:1 (Palmitoleate)	3.85	1.77	1.61	1.27	1.49
C 17:0 (Margaric)	0.12	0.29	0.25	0.26	0.27
C 17:1 (Heptadecenoic acid)	0.12	0.18	0.14	0.11	0.16
C 18:0 (Stearic)	6.00	5.76	6.02	6.00	5.98
C 18:1 (Oleic)	33.32	32.81	32.40	28.88	29.10
C 18:2 (Linoleic)	18.02	25.56	25.50	27.67	26.94
C 18:3 (Linolenic)	0.53	0.60	0.55	0.60	0.60
C 20:0 (Arachidic)	0.27	0.51	0.56	0.62	0.48
C 20:1 (Gadoleic)	0.92	1.03	1.09	0.92	0.84
C 20:4 (ARA)	6.97	4.54	6.10	6.52	6.11
C 22:0 (Behenic)	0.07	0.20	0.31	0.37	0.20
C 20:5 (EPA)	0.17	0.15	0.20	0.17	0.15
C 22:5 (DPA)	0.21	0.34	0.35	0.31	0.48
C 22:6 (DHA)	4.67	2.86	2.80	3.75	4.23
C 24:0 (Lignocérico)	0.13	0.04	0.07	0.09	0.14
SFA	31.22	30.16	29.26	29.80	29.90
MUFA	38.21	35.79	35.24	31.18	31.59
PUFA	30.57	34.05	35.50	39.02	38.51
Total (%)	100.00	100.00	100.00	100.00	100.00
Standard group (CT), high-fat diet group (HF), high-fat diet supplemented with extract of stalks and leaves (HFEX), high-fat diet supplemented with dried stalks and leaves (HFSL) and high-fat diet supplemented with lyophilized stalks and leaves (HFLy).

Supplementation with beet stalks and leaves could modulate a key gluconeogenic enzyme and an important insulin signaling pathway protein

Dietary fat intake is one of the most important environmental factors leading to the development of insulin resistance and consequent metabolic dysfunction. After assessing metabolic parameters, we analyzed the hepatic tissue in order to determine if supplementation with beet stalks and leaves could modulate the insulin pathway. The improvement in glucose homeostasis by HFSL supplementation could be a result of its effects on the insulin signaling. Our data suggest that HFEX, HFSL and HFLy exhibited higher activation of the insulin pathway in the liver, as evaluated by AKT phosphorylation at Thr308, after insulin administration compared to animals maintained on an HF diet with supplementation (Fig. 6).

Dietary manipulation is the basal and first-line intervention in combating obesity and obesity-associated metabolic disorders, because of access and lower cost. Beetroot (Beta vulgaris L.) and its non-conventional parts such as stalks and leaves are rich in phenolic compounds and may have functional properties. Our results suggest that supplementation with dehydrated beet stalks and leaves may prevent some of the alterations from dietary-induced obesity in mice, decreasing fasting glucose and cholesterol levels and attenuating the oxidative stress generated by HF diet (HFD) [26].

In the current study, we evaluated if supplementation with beet stalks and leaves could revert metabolic parameters and ameliorate hepatic steatosis of experimental animals submitted to an HFD. We compared the effect of three types of supplementation on HFD, namely an ethanolic extract of dehydrated beet stalks and leaves (EX), dehydrated powder of beet stalks and leaves (SL) and lyophilized powder of beet stalks and leaves (Ly). In our study, we observed that animals fed an HFD for 8 weeks presented increased body mass gain and fasting glucose, greater area under the curve during the GTT and evidence of hepatic insulin resistance compared to the normocaloric group (CT). Our findings are in agreement with the literature reporting that long-term exposure to lipid and carbohydrate overload places a heavy burden on adaptive responses that leads to continuous chronic inflammation and oxidative stress, resulting ultimately in a metabolic imbalance [32, 33].

Regarding supplemented groups, after 8 weeks, we observed no differences in body mass and food intake (data not shown) among any groups (EX, SL and Ly groups). However, supplementation with SL attenuated the deleterious effect of HF and restored fasting glucose levels to those of the CT group.

In addition, all supplemented group seemed to be more sensitive to insulin stimulus in the liver compared to HF-fed mice, but only SL group presented improvement on glucose homeostasis. Taken together, our results indicate improvement of insulin resistance in liver and extrahepatic tissues after supplementation with SL.

Moreover, AKT phosphorylation was restored in the liver of animals from supplemented groups, suggesting an improvement in insulin signaling. The reasons for the disconnection between liver fat /AKT signaling and the benefit for glucose homeostasis are unclear. The improvement in the glucose homeostasis found in HFSL could be a consequence of pancreatic insulin secretion and/or glucose uptake from skeletal muscle and white adipose tissues [34]. One possibility is that the HFSL group has both mechanisms occurring, which either may not have occurred to HFEX and HFLy groups or occurred in a lower intensity, leading to a minor change in blood glucose homeostasis.

Our results could be associated with the chemical profile of the beet stalks and leaves. Vitexin and its derivatives were the major bioactive compounds presented in the beet stalks and leaves. Vitexin (apigenin-8-C-glucoside), a flavonoid glycoside derived from apigenin, is an active component of many medicinal plants and is receiving increased attention due to its range of pharmacological effects.

Vitexin-2-O-rhamnoside-related compounds, as demonstrated in previous studies [25–27, 35–39], have been shown to reduce inflammation and promote beneficial effects against many disorders caused by HFD, including the improvement of glucose homeostasis, HOMA-IR, plasma insulin and hepatic lipid metabolism, and a significant decrease in hepatic PEPCK and G6Pase activity in HFD-fed mice [35, 40–43]. It is important to mention that the dose of bioactive compounds, like vitexin derivatives, used in our study may be insufficient for suppressing body weight gain and body fat accumulation (data not shown). Further studies are needed to evaluate the dose response and duration of treatment with these bioactive compounds.

Surprisingly, our results suggest that dehydrated leaves (SL) were more biologically active than the ethanolic extract (EX) or lyophilized material (Ly). The differences in the biological effects observed between treatments (SL vs EX or Ly) may be related to interactions of the phenolic compounds with other components of the sample, especially proteins, present in the food matrix. According to Borgwardt et al. [44], the interaction between bioactive compounds, such as genistein, and acidic peptides potentialized the inhibitory effect on platelet aggregation. The formation of genistein conjugates may improve their biological activity [45] and, consequently, their bioavailability during the digestion process [44]. In this context, the dehydration process at high temperature can promote cell disruption in the beet leaf matrix and release bioactive compounds, improving the biological activity of beet stalks and leaves. Therefore, considering our results obtained so far, it is important to investigate whether bioactive components present in the beet stalks and leaves used in our study could be involved in the regulation of hepatic gluconeogenesis and/or the insulin signaling pathway in the liver of mice. However, we could not rule out the possibility of the metabolic improvement due to the indirect effect of changes in body mass, rather than to more direct pharmacological effects in glucose homeostasis.

In summary, our data indicate the addition of phenolic compounds in the form of dehydrated stalks and beet leaves in the animals fed an HFD was positive, considering some of the parameters evaluated. We observed that the form of administration, the dose used, and the time of intervention may influence the effects associated with fasting glucose and glucose tolerance. Our results suggest that there is a positive effect of the food matrix on the bioavailability of the phenolics present in dehydrated beet leaves and stalks. It is known that phenolic compounds with antioxidant potential may be in a free form and/or in a bound form in foods of plant origin. Thus, by supplementing the diet with the extract, only the free and soluble phenolic compounds are capable of being absorbed and made available to the body. Furthermore, it is necessary to carry out future studies that evaluate the dose response as a function of time. In addition, the isolation of some compounds presents in beet stalks and leaves and their administration by different means and in different experimental protocols could result in interesting data that would help to understand the possible functional properties of beet stalks and leaves. Although, some limitations of our study should be noted. Despite the treatment with EX, SL or Ly, we observed that fatty liver occurs in all four HF-fed groups. Here, we presented only qualitative evidence of hepatic triglyceride content because we focused the samples on the hepatic fatty acid composition analysis. Thus, we were unable to assess whether improvement of glucose homeostasis in the SL-treated group is mediated by alterations on hepatic triglyceride content. However, semiquantitative analysis of NASH (Fig. 4A) suggests it could not be explained by decreases in liver steatosis, since the SL group presented the same score as the HF group. Future studies would be necessary in order to clarify if the enhancement in the glucose homeostasis found in HFSL could be a consequence of improvement pancreatic insulin secretion and/or glucose uptake from skeletal muscle and white adipose tissues.

Our results suggest that stalks and leaves could be used as adjuvants in obesity to mitigate the damage generated by oxidative stress and to improve parameters related to glucose homeostasis. The dose–response relationship between the consumption of beet stalks and leaves and the health-related benefits remains to be elucidated.

CT: Standard diet

HF: High-fat diet

SL: Dehydrated beet stalks and leaves

Ly: Lyophilized beet stalks and leaves

EX: Beet stalk and leaf extract

DM2: Type 2 Diabetes Mellitus

NAFLD: Non-alcoholic Fatty Liver Disease

HPLC: High-performance liquid chromatography

UHPLC-MS/MS: Ultra-high performance liquid chromatography coupled with mass spectrometry

ESI: Electrospray ionization

VRE: Vitexin-2-rhamnoside equivalents

HFSL: High-fat diet supplemented with dehydrated beet stalks and leaves

HFLy: High-fat diet supplemented with lyophilized beet stalks and leaves

HFEX: High-fat diet supplemented with beet stalk and leaf extract

ipGTT: Intraperitoneal glucose tolerance test

H&E: Haematoxylin and eosin

V2R: Vitexin-2-rhamnoside

DIO: Diet induced obesity

NAS: NAFLD activity score

PUFA: Polyunsaturated fatty acid

HFD: HF diet

Ethics approval and consent to participate

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing Interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Funding

The present study was supported by São Paulo Research Foundation (FAPESP) processes 2011/51205-6, 2013/04304-4, 2013/18498-5, 2015/15035-0 and 2016/24768-3 National Council for Scientific and Technological Development (CNPq) processes 445552/2014-0 and 303568/2016-0, and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Author Contributions

I.M.L. and J.E.M.: contributed to design research, conducted research, reviewed the literature, conducted the statistical analysis and wrote the manuscript. A.L.F.V. and B.R.S.: conducted research and experimental planning. R.M.N.B., M.A.T., A.S.T., M.A.R. and M.M.: provided essential reagents, materials and financial support besides providing advice regarding interpretation of the results and editing the manuscript. M.M. and C.D.C.: were responsible for design research, provided essential reagents, materials and other financial support besides providing advice regarding interpretation of the results, conduct of the research and data collection; they also provided advice regarding the manuscript, and wrote and edited it.

Acknowledgments

This work was supported by grants from the São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES).

World Health Organization (WHO). Obesity and overweight, 2020. Available online: https://www.who.int/en/news-room/fact-sheets/detail/obesity-and-overweight (acessed on 01^st April 2020).
Bugianesi E, McCullough AJ, Marchesini G. Insulin resistance: A metabolic pathway to chronic liver disease. Hepatology 2005;42:987–1000. https://doi.org/10.1002/hep.20920.
Perry RJ, Samuel VT, Petersen KF, Shulman GI. The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes. Nature 2014;510:84–91. https://doi.org/10.1038/nature13478.
Lottenberg AM, Afonso M da S, Lavrador MSF, Machado RM, Nakandakare ER. The role of dietary fatty acids in the pathology of metabolic syndrome. J Nutr Biochem 2012;23:1027–40. https://doi.org/10.1016/j.jnutbio.2012.03.004.
Pan MH, Lai CS, Tsai ML, Ho CT. Chemoprevention of nonalcoholic fatty liver disease by dietary natural compounds. Mol Nutr Food Res 2014;58:147–71. https://doi.org/10.1002/mnfr.201300522.
Bagherniya M, Nobili V, Blesso CN, Sahebkar A. Medicinal plants and bioactive natural compounds in the treatment of non-alcoholic fatty liver disease: A clinical review. Pharmacol Res 2018;130:213–40. https://doi.org/10.1016/j.phrs.2017.12.020.
Salomone F, Ivancovsky-Wajcman D, Fliss-Isakov N, Webb M, Grosso G, Godos J, et al. Higher phenolic acid intake independently associates with lower prevalence of insulin resistance and non-alcoholic fatty liver disease. JHEP Reports 2020;2:100069. https://doi.org/10.1016/j.jhepr.2020.100069.
Vidal PJ, López-Nicolás JM, Gandía-Herrero F, García-Carmona F. Inactivation of lipoxygenase and cyclooxygenase by natural betalains and semi-synthetic analogues. Food Chem 2014;154:246–54. https://doi.org/10.1016/j.foodchem.2014.01.014.
Ninfali P, Angelino D. Nutritional and functional potential of Beta vulgaris cicla and rubra. Fitoterapia 2013;89:188–99. https://doi.org/10.1016/j.fitote.2013.06.004.
Vulić J, Čanadanović-Brunet J, Ćetković G, Tumbas V, Djilas S, Četojević-Simin D, et al. Antioxidant and cell growth activities of beet root pomace extracts. J Funct Foods 2012;4:670–8. https://doi.org/10.1016/j.jff.2012.04.008.
Kujala TS, Loponen JM, Klika KD, Pihlaja K. Phenolics and betacyanins in red beetroot (Beta vulgaris) root: Distribution and effect of cold storage on the content of total phenolics and three individual compounds. J Agric Food Chem 2000;48:5338–42. https://doi.org/10.1021/jf000523q.
Vulić JJ, ć;ebović TN, ćanadanović-Brunet JM, ćEtković GS, Čanadanović VM, Djilas SM, et al. In vivo and in vitro antioxidant effects of beetroot pomace extracts. J Funct Foods 2014;6:168–75. https://doi.org/10.1016/j.jff.2013.10.003.
Gilchrist M, Winyard PG, Fulford J, Anning C, Shore AC, Benjamin N. Dietary nitrate supplementation improves reaction time in type 2 diabetes: Development and application of a novel nitrate-depleted beetroot juice placebo. Nitric Oxide - Biol Chem 2014;40:67–74. https://doi.org/10.1016/j.niox.2014.05.003.
Daab W, Bouzid MA, Lajri M, Bouchiba M, Saafi MA, Rebai H. Chronic Beetroot Juice Supplementation Accelerates Recovery Kinetics following Simulated Match Play in Soccer Players. J Am Coll Nutr 2020;0:1–9. https://doi.org/10.1080/07315724.2020.1735571.
Mirmiran P, Houshialsadat Z, Gaeini Z, Bahadoran Z, Azizi F. Functional properties of beetroot (Beta vulgaris) in management of cardio-metabolic diseases. Nutr Metab 2020;17:1–15. https://doi.org/10.1186/s12986-019-0421-0.
Jajja A, Sutyarjoko A, Lara J, Rennie K, Brandt K, Qadir O, et al. Beetroot supplementation lowers daily systolic blood pressure in older, overweight subjects. Nutr Res 2014;34:868–75. https://doi.org/10.1016/j.nutres.2014.09.007.
Clifford T, Howatson G, West DJ, Stevenson EJ. The potential benefits of red beetroot supplementation in health and disease. Nutrients 2015;7:2801–22. https://doi.org/10.3390/nu7042801.
Coles LT, Clifton PM. Effect of beetroot juice on lowering blood pressure in free-living, disease-free adults: A randomized, placebo-controlled trial. Nutr J 2012;11:1–6. https://doi.org/10.1186/1475-2891-11-106.
Baião D dos S, d’El-Rei J, Alves G, Fritsch Neves M, Perrone D, Del Aguila EM, et al. Chronic effects of nitrate supplementation with a newly designed beetroot formulation on biochemical and hemodynamic parameters of individuals presenting risk factors for cardiovascular diseases: A pilot study. J Funct Foods 2019;58:85–94. https://doi.org/10.1016/j.jff.2019.04.041.
Ajila CM, Aalami M, Leelavathi K, Rao UJSP. Mango peel powder: A potential source of antioxidant and dietary fiber in macaroni preparations. Innov Food Sci Emerg Technol 2010;11:219–24. https://doi.org/10.1016/j.ifset.2009.10.004.
Amaya-Cruz DM, Rodríguez-González S, Pérez-Ramírez IF, Loarca-Piña G, Amaya-Llano S, Gallegos-Corona MA, et al. Juice by-products as a source of dietary fibre and antioxidants and their effect on hepatic steatosis. J Funct Foods 2015;17:93–102. https://doi.org/10.1016/j.jff.2015.04.051.
Ajila CM, Bhat SG, Prasada Rao UJS. Valuable components of raw and ripe peels from two Indian mango varieties. Food Chem 2007;102:1006–11. https://doi.org/10.1016/j.foodchem.2006.06.036.
Koubaier HBH, Snoussi A, Essaidi I, Chaabouni MM, Thonart P, Bouzouita N. Betalain and phenolic compositions, antioxidant activity of Tunisian red beet (Beta vulgaris L. conditiva) roots and stems extracts. Int J Food Prop 2014;17:1934–45. https://doi.org/10.1080/10942912.2013.772196.
Narasimhan A, Chinnaiyan M, Karundevi B. Ferulic acid exerts its antidiabetic effect by modulating insulin-signalling molecules in the liver of high-fat diet and fructose-induced type-2 diabetic adult male rat. Appl Physiol Nutr Metab 2015;40:769–81. https://doi.org/10.1139/apnm-2015-0002.
Kulabas SS, Ipek H, Tufekci AR, Arslan S, Demirtas I, Ekren R, et al. Ameliorative potential of Lavandula stoechas in metabolic syndrome via multitarget interactions. J Ethnopharmacol 2018;223:88–98. https://doi.org/10.1016/j.jep.2018.04.043.
Lorizola IM, Furlan CPB, Portovedo M, Milanski M, Botelho PB, Bezerra RN, et al. Beet Stalks and Leaves (Beta vulgaris L.) Protect Against High-Fat Diet-Induced Oxidative Damage in the Liver in Mice. Nutrients 2018;10:872. https://doi.org/10.3390/nu10070872.
Bumke-Vogt C, Osterhoff MA, Borchert A, Guzman-Perez V, Sarem Z, Birkenfeld AL, et al. The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells. PLoS One 2014;9. https://doi.org/10.1371/journal.pone.0104321.
Rostagno MA, Manchón N, D’Arrigo M, Guillamón E, Villares A, García-Lafuente A, et al. Fast and simultaneous determination of phenolic compounds and caffeine in teas, mate, instant coffee, soft drink and energetic drink by high-performance liquid chromatography using a fused-core column. Anal Chim Acta 2011;685:204–11. https://doi.org/10.1016/j.aca.2010.11.031.
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313–21. https://doi.org/10.1002/hep.20701.
Folch J, Lees M, Stanley SGH. A simple method for the isolation and purification of total lipides from animal tissue. J Biol Chem 1957;224:497–509.
Miyamoto JÉ, Ferraz ACG, Portovedo M, Reginato A, Stahl MA, Ignacio-Souza LM, et al. Interesterified soybean oil promotes weight gain, impaired glucose tolerance and increased liver cellular stress markers. J Nutr Biochem 2018;59:153–9. https://doi.org/10.1016/j.jnutbio.2018.05.014.
Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114:1752–61. https://doi.org/10.1172/JCI200421625.1752.
Gregor MF, Hotamisligil GS. Inflammatory Mechanisms in Obesity. Annu Rev Immunol 2011;29:415–45. https://doi.org/10.1146/annurev-immunol-031210-101322.
2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes - 2018. Diabetes Care 2018;41:S13–27. https://doi.org/10.2337/dc18-S002.
Jung UJ, Cho YY, Choi MS. Apigenin ameliorates dyslipidemia, hepatic steatosis and insulin resistance by modulating metabolic and transcriptional profiles in the liver of high-fat diet-induced obese mice. Nutrients 2016;8. https://doi.org/10.3390/nu8050305.
Gengatharan A, Dykes GA, Choo WS. Betalains: Natural plant pigments with potential application in functional foods. Lwt 2015;64:645–9. https://doi.org/10.1016/j.lwt.2015.06.052.
Choo CY, Sulong NY, Man F, Wong TW. Vitexin and isovitexin from the Leaves of Ficus deltoidea with in-vivo α-glucosidase inhibition. J Ethnopharmacol 2012;142:776–81. https://doi.org/10.1016/j.jep.2012.05.062.
He M, Min JW, Kong WL, He XH, Li JX, Peng BW. A review on the pharmacological effects of vitexin and isovitexin. Fitoterapia 2016;115:74–85. https://doi.org/10.1016/j.fitote.2016.09.011.
Nurdiana S, Goh YM, Ahmad H, Dom SM, Syimal’ain Azmi N, Noor Mohamad Zin NS, et al. Changes in pancreatic histology, insulin secretion and oxidative status in diabetic rats following treatment with Ficus deltoidea and vitexin. BMC Complement Altern Med 2017;17:1–17. https://doi.org/10.1186/s12906-017-1762-8.
Escande C, Nin V, Price NL, Capellini V, Gomes AP, Barbosa MT, et al. Flavonoid apigenin is an inhibitor of the NAD+ase CD38: Implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome. Diabetes 2013;62:1084–93. https://doi.org/10.2337/db12-1139.
Kim MA, Kang K, Lee HJ, Kim M, Kim CY, Nho CW. Apigenin isolated from Daphne genkwa Siebold et Zucc. inhibits 3T3-L1 preadipocyte differentiation through a modulation of mitotic clonal expansion. Life Sci 2014;101:64–72. https://doi.org/10.1016/j.lfs.2014.02.012.
Shukla S, Gupta S. Apigenin: a promising molecule for cancer prevention. Pharm Res 2010;27:962–78. https://doi.org/10.1007/s11095-010-0089-7.Apigenin.
Ono M, Fujimori K. Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells. J Agric Food Chem 2011;59:13346–52. https://doi.org/10.1021/jf203490a.
Borgwardt K, Bonifatius S, Gardemann A. Acidic peptides enhanced genistein-dependent inhibition of human platelet aggregation: potential protective effect of digestible peptides plus genistein against atherosclerosis. Nutr Res 2008;28:523–31. https://doi.org/10.1016/j.nutres.2008.03.017.
Rimbach G, Weinberg PD, De Pascual-Teresa S, Alonso MG, Ewins BA, Turner R, et al. Sulfation of genistein alters its antioxidant properties and its effect on platelet aggregation and monocyte and endothelial function. Biochim Biophys Acta - Gen Subj 2004;1670:229–37. https://doi.org/10.1016/j.bbagen.2003.12.008.

PDF

Version 1

posted 03 Aug, 2020

You are reading this latest preprint version

Beet (Beta vulgaris L.) Stalk and Leaf Supplementation Improves Glucose Homeostasis and Insulin Resistance Markers in Liver of Mice Exposed to a High-Fat Diet

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1