Herbal Parc in Chennai verified the legitimacy of the hulled barley grains, which had been obtained at Chengalpattu in Chennai (Voucher No. PARC/2015/3040). After being cleaned and allowed to air dry, the hulled barley grains were ground into a coarse flour and kept at ambient temperature.

Healthy male SD rats weighing 150–200 g were housed in a light/dark cycle with a regulated atmospheric humidity of 44–55% and an average temperature of 22 ± 2°C. The Institutional Animal Ethics Committee (IAEC) granted its approval to this study protocol (XXIII/VELS/PCOL/ 14/2000/CPCSE/IAEC/07.02.2020).

For 14 weeks, males of SD rats were given an HFD so as to develop obesity and hyperlipidemia.

To determine the antihyperlipidemic effect of HBGP and FA against HFD-induced hyperlipidemia, rats were split into seven equal-number groups (Table 1). A normal feed was administered to the rats in groups 1, 2, and 3. From the third week onwards, Group 2 rats were fed HBGP (50:50), whereas Group 3 rats were treated with 200 mg/kg body weight of FA till the end of the experiment. For 14 weeks, rats in groups 4, 5, 6, and 7 were fed an HFD. In addition to 50% of the regular and HFD meals, Group 5 rats started receiving 50% of HBGP added into their feed starting the third week. Rats in Group 6 received 200 mg of FA per kilogram of body weight. Additionally, rats in Group 7 received 10 mg/kg of body weight of rosuvastatin. The rats were given ethoxy ether at the end of the trial period, and they were then put to death via cervical decapitation. For gene expression research, liver and adipose tissue (AT) samples were immediately taken out and suspended in RNA later solution.

The mRNA transcript levels of PPAR-γ, SIRT1, FOXO1, and LXR-α was assessed in the liver and AT samples taken from test animals by RT-PCR analysis. The β-actin gene was selected as the housekeeping gene. Following animal sacrifice, 30 mg of the rat’s liver and AT were removed and immediately suspended in the RNA later solution. By employing the RNeasy Miniprep kit, total RNA has been obtained from the study material.

A post hoc Bonferroni test was performed following the one-way ANOVA method to assess statistical significance. In case the observed P-value was <0.05, statistical significance is then recorded.

PPAR-γ receptors primarily function in AT and control accumulation of fat by regulating the functioning of important genes involved in adipogenesis. Figures 1 and 2, respectively, depicted PPAR-γ activities in liver and AT samples of experimental rats. The concentration of PPAR-γ in both liver and AT samples was lowered (P = 0.001) in HFD-treated rats than in normal rats. In this investigation, PPAR-γ expression in HBGP and FA co-administered rats together showed (P = 0.001) more than rats treated with HFD.

Silence information regulator 1 is a crucial biochemical sensor that directly connects environmental dietary cues to mammalian physiological regulation. It has been connected to insulin production in the pancreas, hepatic gluconeogenesis, and white fat mobilization. Expression of SIRT1 in liver and AT samples of experimental animals was depicted in Figures 3 and 4. SIRT1 expression was found to be downregulated in both the test samples of HFD treated rats. HBGP/FA co-administration significantly (P = 0.001) upregulated SIRT1 gene expression in HFD + HBGP and HFD + FA fed groups, therefore decreasing the adipocyte differentiation in obese conditions.

Forkhead box factors has been linked to glucose and lipid homeostasis in addition to the integration of hormonal and dietary signals for metabolic homeostasis. This gene was recently demonstrated with implications for sirtuin activity via influencing the expression of nicotinamide phosphoribosyl transferase (NAMPT), the enzyme that limits the rate at which NAD is produced and hence modulates sirtuin activity. In the liver, both SIRT1 and SIRT6 have been proven to suppress lipogenesis while promoting fatty acid oxidation. In this study, there was an upregulation in the expression of FOXO1 in the liver and AT samples of HFD-fed rats; on the contrary, FOXO1 activity was found to be restored in the control group as well as in HBGP/FA co-administered rats, as shown in Figures 5 and 6.

LXR-α is a well-known metabolic and cholesterol mobility regulator in the cells of the liver, intestine, and macrophages. It upregulates the gene regulation of HDL-associated apolipoprotein E (ApoE), Cyp7A, the enzyme that limits the rate of bile acid synthesis and the cholesterol transporters ABCA1, ABCG1, and ABCG5/ABCG8. The expression of LXR-α in liver and AT samples of experimental animals is depicted in Figures 7 and 8. LXR-α expression was found to be downregulated in liver as well as AT samples of HFD-given rats. HBGP and FA co-administration upregulated LXR-α gene expression in HFD + HBGP and HFD + FA fed groups, therefore regulating whole-body cholesterol homeostasis.

The main transcription factor linked to adipogenesis among the other PPARs (α, γ, and δ) is PPAR-γ, which has two functions: it stimulates genes unique to AT and expresses the mature adipose phenotype. According to Tontonoz et al. (12), PPAR-γ reduces the incidence of atherosclerosis and fibrotic arteries by suppressing macrophage MMP-9 expression (12). It has been discovered that PPAR-γ is involved in the enhancement of adiponectin that takes place during adipocyte development. In our current study, the elevated levels of PPAR-γ expression in the AT of HBGP- or FA-treated rats (Figures 1 and 2) may be due to PPAR-γ’s major role in the elevation of adiponectin at the time of adipocyte maturation. On the other hand, decreased levels of PPAR-γ expression in HFD-given rats are due to increased calorie intake leading to fat accumulation and oxidative stress. Consequently, PPAR-γ is downregulated with diminished adiponectin concentration, the negative modulator in hyperlipidemic conditions.

High calorie consumption is usually associated with less mitochondrial membrane fluidity as well as more free radical production (13). In adipocytes, fat accumulation and increased oxygen species have been demonstrated to decrease PPAR-γ expression (14). As a result, medicinal drugs that exhibit an increase in the activity of PPAR-γ consequently elevate adiponectin depots, which may be advantageous in the management of hyperlipidemia and overweight. HBGP and its active compound FA are the natural therapy of choice for diet-induced obesity, as per the results of this study. Our findings show that HBGP/FA has hypolipidemic activity, most likely via upregulating PPAR-γ expression to increase the transcription of adiponectin, a negative regulator of obesity and hyperlipidemia.

SIRT1 stimulation can help to improve lipid levels. Nicotinic acid, a precursor of NAD+, elevates cellular NAD+ concentrations along with SIRT1 activation (15), which in turn is utilized to reduce cholesterol levels. Another mechanism to enhance SIRT1 is through CR. In a human investigation, CR was shown to lower TG and cholesterol levels and postpone the onset of many aging-related cardiovascular problems (16).

SIRT1 requires PPAR-γ as a substrate. The deacetylation of lysine residues (268 and 293 K) on PPAR-γ by SIRT1 is required for the co-repressors NCoR and SMRT to regulate its transcriptional activity. SIRT1 can thus limit white adipogenesis by stimulating the binding of PPAR-γ co-repressors NCoR and SMRT, which suppresses PPAR-γ transcriptional activity (17, 18).

Figures 3 and 4 display a decrease in the gene expression of SIRT1 in HFD-treated and untreated rats when compared with control rats. This decrease is an indicator of disturbed lipid and carbohydrate metabolism. Co-administration of HBGP/FA increased SIRT1 expression in the liver and AT of HFD + HBGP and HFD + FA group rats. The lines of evidence that SIRT1 regulates lipid homeostasis are numerous and are explained. CR, prolonged fasting, and natural polyphenolic products like resveratrol (19), fisetin, quercetin, and curcumin lead to the activation of SIRT1, which in turn serves as a checkpoint for the various stages of fat metabolism. In AT SIRT1 represses PPAR-γ via binding docking to the NCoR/SMRT proteins. The final product of NCoR/SMRT/SIRT1 collides with the sequences of DNA known as PPREs and decreases the molecular mechanism of PPAR-γ, thereby initiating fat mobilization (20). Chen and Li (21) reported that decreased SIRT1 levels may be accountable for reduced lipid profiles in chronic kidney failure.

Barley is a versatile grain that competes with other cereal grains in terms of phytochemical content and nutrition. It has high levels of active compounds that work in numerous ways to prevent diseases. The phytochemistry of HBGP reveals the presence of the dominant phenolic compound FA. Therefore, the hypolipidemic activity of HBGP may be due to the presence of FA. Moreover, our findings are supported by Chen et al (22), who demonstrated the potential role of FA in regulating muscle fiber formation through the activation of the SIRT1/AMPK pathway.

Figures 5 and 6 show the gene expression results of FOXO1 in both liver and AT samples of HGBP/FA-treated and untreated rats. FOXO1 expression turns out to be downregulated in HFD-treated rats. HBGP and FA co-administration increased the expression of FOXO1 in HFD + HBGP and HFD + FA fed rats. The mechanism involved in the upregulation of FOXO1 in liver and AT is hypothesized as follows. Firstly, in the liver, SIRT1 removes acetyl groups from gene transcription proteins such as transcription factors such as forkhead box O (FOXO), thus preventing cholesterol production. Moreover, the insulin response element (IRE) sequence in the SREBP-2 promoter region is uniquely recognized by FOXO1, which inhibits SREBP-2 transcription. Li and Wu (23) provided evidence to prove that the upregulation of fatty acids and LDL was detected in FOXO1 knockout mice. Its regulating factor in bile acid metabolism was also discovered in a recent study. In the liver, FOXO1 is necessary for the release of genes associated with bile acid production. On the other hand, inactivation of FOXO1 in hepatic tissues takes place, causing abnormal bile acid synthesis, thus affecting farnesoid x receptor (FXR) capacity to lower TG and cholesterol.

Secondly, in AT SIRT1 deacetylates PGC-1α and activates it. Activated PGC-1α combines with FOXO1, thereby influencing gluconeogenesis and glucose homeostasis in the case of the liver whereas, in the case of AT, FOXO1 upregulates adiponectin gene transcription. Activated FOXO1 binds to the FOXO1-responsive region in the mouse adiponectin promoter, which comprises two neighboring FOXO1 binding sites and also C/EBPα. Therefore, FOXO1 forms a transcription complex with C/EBPα at the mouse adiponectin promoter, improving the expression and secretion of adiponectin in adipocytes, thus increasing serum adiponectin levels in mice (24). Increased adiponectin further stimulates cells to insulin and increases fatty acid oxidation, leading to new energy homeostasis. From the above results, it is highly evident that HBGP/FA co-administration maintained the activity of FOXO1, and this might be by modulating SIRT1 level by both the test drugs, underlining their protection against hyperlipidemia and obesity.

Figures 7 and 8 show the gene expression results of LXR-α in liver and AT samples of HBGP/FA-treated and untreated rats. LXR-α expression displayed a downregulation in HFD-treated rats. HBGP and FA co-administration increased the expression of LXR-α in HFD + HBGP and HFD + FA fed rats. Zeng et al. also found that SIRT1 can prevent atherosclerosis through activation of LXR-ABCA1/ABCG1/CCR7 and by deactivation of NF-κB signaling pathways (25).

In conjunction with fatty acid metabolism, SIRT1 is also shown to regulate LXR through deacetylation reaction. There are two types of LXRs, LXR-α and LXR-β. Human and mouse adipocytes contain both α and β form; however, α form is primarily increased during fat cell proliferation (26). Interestingly, the coregulator PGC-1α is needed for LXR-α activation, which may be beneficial for reducing gastrointestinal cholesterol uptake and increasing centripetal cholesterol flux. Cholesterol 7 α hydroxylase, the enzyme that controls the rate of bile acid synthesis, and ATP binding cassette (ABC) genes associated with hepatic and intestinal genes that LXR-α positively regulates and are necessary for the elimination of cholesterol from the body.

Taken together, we suggest that SIRT1 has a multifunctional role in the body, which is as follows: regulator of whole-body cholesterol and lipid metabolism by activation of PGC-1α, FOXO1, and LXR-α. The overall expression of the amplification plot and fluorescent profile of all target genes during PCR in liver and AT are represented in Figures 9 and 10.