F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.55470.2 Research Article Articles Green Tea and Decaffeinated Light Roasted Green Coffee Extract Combination Improved Cardiac Insulin Resistance through Free Fatty Acids and Adiponectin/FAS Pathways Amelioration in Metabolic Syndrome Rat Model

[version 2; peer review: 1 approved with reservations, 2 not approved]

 Lukitasari Mifetika Conceptualization Data Curation Methodology Writing – Review & Editing 1 Rohman Mohammad Saifur Conceptualization Funding Acquisition Supervision Writing – Review & Editing a 2 Nugroho Dwi Adi Conceptualization Data Curation Methodology Resources https://orcid.org/0000-0002-6195-9771 3 Nur Kholis Mukhamad Data Curation Formal Analysis Investigation Writing – Original Draft Preparation 4 Wahyuni Nila Aisyah Data Curation Investigation Writing – Original Draft Preparation 4 Widodo Nashi Project Administration Supervision Writing – Review & Editing https://orcid.org/0000-0002-1126-498X 5 Department of Nursing, Faculty of Medicine, Brawijaya University, Malang, East java, +62, Indonesia Department of Cardiology and Vascular Medicine, Faculty of Medicine, Brawijaya University, Malang, East java, +62, Indonesia Department of Herbal Medicine, Brawijaya Cardiovascular Research Group, Faculty of Medicine, Brawijaya University, Malang, East java, +62, Indonesia Magister of Biomedical Sciences, Faculty of Medicine, Brawijaya University, Malang, East java, +62, Indonesia Department of Biology Mathematics and Natural Sciences, Brawijaya, Malang, East java, +62, Indonesia ippoenk@ub.ac.id

No competing interests were disclosed.

 14 9 2023 2021 10 990 12 9 2023 Copyright: © 2023 Lukitasari M et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Insulin resistance has been independently associated with cardiac diseases. A free fatty acid is recently known to induce cardiac insulin resistance due to low-grade inflammation. Therefore, the improvement of free fatty acid levels can also improve cardiac insulin resistance. This study investigated the combination of green tea and decaffeinated-light roasted green coffee extract in improvement of free fatty acid-induced cardiac insulin resistance by improving the adiponectin/FAS pathway.

Methods: This study used 25 males Sprague-Dawley rats induced by a high-fat high sucrose diet and injection of low dose streptozotocin to make a metabolic syndrome (MS) rat model and standard chow as healthy control rats. The MS rats were treated with green tea (200 mg/ b. w.), decaffeinated-light roasted green coffee (300 mg/ b. w.), and the combination of both extracts in 9 weeks. Experimental groups in this study were divided into 5 groups: 1) MS (HFHS diet + STZ) group, 2) NC (normal chow) group, 3) GT (green tea extract) group, 4) GC (decaffeinated-light roasted green coffee extract), 5) CM (combination of both extracts) group. Adiponectin and HOMA-IR level was analysed using ELISA, and the gene expression of Adipo-R1, FAS, PI3K, PDK1, Akt, GLUT4 was measured by RT-PCR.

Results: The combination of green tea and decaffeinated-light roasted green coffee showed synergistic effects in improving FFA levels. The adiponectin/FAS pathway was attenuated in the CM group. Moreover, the combination also showed improvement in cardiac insulin resistance markers such as IRS1/2, PI3K, PDK1, Akt, and GLUT4.

Conclusions: The combination of green tea and decaffeinated-light roasted green coffee extract improved cardiac insulin resistance better than green tea and green coffee extract administration alone by reducing free fatty acids levels through adiponectin/FAS pathway modulation.

 metabolic syndrome; green tea; green coffee; cardiac insulin resistance Research and Community Service Departement Faculty of Medicine, Brawijaya University 3125/UN.10.F08/PN/2021 This research was funded by the Research and Community Service Department Faculty of Medicine, Brawijaya University under Grant 3125/UN.10.F08/PN/2021 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

This new version is based on revisions provided via email and comments from reviewers, which include sentence structure, typing errors, and figure legends. Additionally, it incorporates references related to research methods concerning animal modeling and research ethics.

 Editorial note

Editorial Note (4 th August 2023): The F1000 Editorial Team has not yet received a new version of this article, as detailed in the Editorial Note published on 16 th June 2023. The F1000 Editorial Team is actively contacting the authors to request the new version of the article. Peer review activity remains suspended until the authors publish a new version of this article.

Editorial Note (16 th June 2023): Since publication, it has been brought to the attention of the Editorial Team that the article was missing key information regarding animal treatment and ethical approval. The Editorial Team requested further detail and an explanation from the authors in March 2023. The authors provided an adequate response and were requested by the Editorial Team to create a new version of the article to include the additional details. Peer review activity has been suspended until the authors publish a new version of this article.

 Introduction

The incidence of metabolic syndrome (MS) features, such as obesity, hyperglycemia, and insulin resistance has increased and is associated with excessive food consumption. 1 MS prevalence has increased about 50–75% in the last decade. 2 A previous study showed that insulin resistance had been known to be a significant predictor of heart failure (HF) 3 and the major feature in HF patients. 4 Many studies have shown that insulin resistance might develop cardiac remodellings such as hypertrophy, fibrosis, and cardiac dysfunction without coronary artery disease or hypertension. 5 – 9 Lipotoxicity by free fatty acids (FFAs) has been the primary cause of insulin resistance 10 through low-grade inflammation in cardiac tissue. 11 Kim et al. (2009) 12 reported that FFAs might cause inflammation and suppress glucose uptake through inhibition of IRS-1 in cardiac tissue. Meanwhile, reducing FFAs plasma levels could improve cardiac function in the high-fat diet-induced obese rat model. 13 Therefore, improving the lipid profile could prevent the development of cardiac insulin resistance.

Recently, natural compound derivatives revealed beneficial effects in metabolic syndrome improvement. One of the natural compounds that are widely consumed is epigallocatechin-3-gallate (EGCG) which is mainly derived from green tea leaves. 14 Many studies have investigated the beneficial effect of green tea administration and its constituents in alleviating metabolic syndrome. Most of those studies revealed that green tea administration modulated insulin sensitivity, reduced blood glucose level, and improved lipid profile in metabolic syndrome animal models. 15 – 17 Additionally, FFAs-induced insulin resistance and hyperglycemia-induced cardiac fibrosis could be attenuated by EGCG administration in vivo. 18 Moreover, chlorogenic acid (CGA), a phenolic compound in green coffee beans, has been another natural compound derivative that could alleviate metabolic syndrome. 19 , 20 CGA potentiates insulin activity similar to the therapeutic mechanism of metformin 21 but does not induce obesity, unlike thiazolidinedione (TZD) or insulin therapy. 22 A previous study were also showed that CGA improved FFAs metabolism in rats hepatic tissue. 23 Nevertheless, green coffee extract administration has detrimental effects due to the caffeine content, 24 – 26 and the CGA antioxidant activity depends on the roasted level of green coffee beans. 27 , 28 Therefore, this study used decaffeinated light-roasted green coffee beans to avoid the detrimental effects of caffeine content and reach the highest antioxidant activity of CGA. Many studies showed that green tea or green coffee extract administration could treat metabolic syndrome. Still, analysis investigating the beneficial effect of these extract combinations in alleviating metabolic syndrome remains limited, whereas natural compounds have a more significant therapeutic effect when combined. 29 This study investigated the combination of green tea and decaffeinated, light roasted green coffee extract to improve free fatty acid-induced cardiac insulin resistance by improving the adiponectin/FAS pathways.

 Methods Ethics statement

All experimental procedures were approved by the ethical committee of Faculty of Medicine, Brawijaya University with registration number 148/EC/KEPK-S2/06/2021. All efforts were made to ameliorate harm to the animals by using the standard protocol from the Indonesian Ministry of Health ethical research guidelines for animal experimental research. 30

Animals and experimental design

This study is part of a larger study 31 which used 25 males Sprague–Dawley rats (aged 9 weeks, weighed 230 to 340 gram) obtained from the National Agency of Drug and Food Control, Indonesia. The ethical committee approved the experimental protocols of the Faculty of Medicine, Brawijaya University. Rats were maintained and acclimatized in the environmentally controlled standard cage as described in our previous study. 31 Animals were grouped as the previous study as follows: i) normal control group (NC) that were assigned to standard chow (Indofeed™, Indonesia) without STZ injection; metabolic syndrome rats were obtained after HFHS diet and low dose STZ (bioWORLD cat.41910012-4) injection intraperitoneally at the second week of the protocol. NCEP ATP III criteria confirmed metabolic syndrome features. The metabolic syndrome rats were assigned to four groups ii) metabolic syndrome group (MS); iii) green tea group (GT) that were given the HFHS diet, STZ injection, and green tea extract (300 mg/kg b.w.) administration per oral; iv) green coffee group (GC) that were assigned to HFHS diet, STZ injection, and green coffee extract (200 mg/kg b.w.) administration; v) combination group (CM) that were assigned to HFHS diet, STZ injection, and combination of green tea (300 mg/kg b. w.) and green coffee (200 mg/kg b. w.) extract administration, respectively. 32 Green tea and coffee extract were administered in millilitres via oral gavage daily based on weekly measured body weight. 32 After 9 weeks of exact treatment, animals were euthanized by cervical decapitation previously anaesthetized using diethyl ether after a 12-hour fast. As soon as the rats died, the blood samples were obtained from the heart and transferred into a microcentrifuge tube to get serum samples by centrifugation (4,000 × g for 15 minutes at 4°C). 32

Extraction procedures of green tea leaves

The green tea leaves were obtained from Sukawana, Bandung, Indonesia (1550 MAMSL). Protocol of green tea extraction followed the procedure of Saifur Rohman et al. (2021). 32

Extraction procedures of decaffeinated, light roasted green coffee beans

The coffee beans used in this experiment were Coffea canephora/ robusta. The coffee beans were obtained from Dampit, Malang, Indonesia (800 MAMSL). The protocol of green coffee extraction followed the procedure of Saifur Rohman et al. (2021). 32

High-performance liquid chromatography analysis

Bioactive compound levels in green tea extract (EGCG) and green coffee bean extract (caffeine and CGA) were analyzed by high-performance liquid chromatography (HPLC) system using a Shimadzu Brand chromatograph (model SCL10AVP, Japan) that set up with a C-18 reverse-phase column (Shim-pack VP ODS 5μm 150 × 4.6 mm). Details of the method were explained in the previous study. 32

Dose determination

Our previous study determined the exact doses based on the optimal doses for each extract. 32

Physiological measurements

Daily food intake and fluid intake were measured each day, and body weight every week. Foods and fluids intake were measured by subtracting the amount provided by the remaining. More information was written in the previous study. 32

Biochemistry analysis

The fasting blood glucose (BIOLABO, cat no. 80009), triglycerides (TG) (cat no. 80019), and HDL-Cholesterol (BIOLABO, cat no. 86516) were analyzed with commercial kits (Biolabs, France) enzymatically as in the previous study. 32

Enzyme-linked immunosorbent assay (ELISA)

Rat serum was collected at the end of the study and stored at −80 oC. ELISA analysis was conducted for measuring non-esterified fatty acid (NEFA) (Ref E-BC-K014, Elabscience, United States), (Ref E-EL-R2466, Elabscience, United States), and adiponectin level (Ref E-EL-R3012, Elabscience, United States). The measurements were conducted using the manual protocol by the manufacturer and read using ELx808 Absorbance Microplate Reader (BioTek, China) to get the result as ng/mL.

 Homeostatic model assessment for insulin resistance (HOMA-IR)

This study measured HOMA-IR using the Commercial ELISA Kit (Ref E-EL-R2466, Elabscience, United States). The measurement was conducted using the manual protocol by the manufacturer and read using EL×808 Absorbance Microplate Reader (BioTek, China) to get the result as ng/mL. HOMA-IR was used to calculate an index from the product of the fasting concentrations of blood glucose (mg/dL) and insulin (microU/L) levels divided by 14.1. Lower levels of HOMA-IR indicate greater insulin sensitivity, and higher levels indicate insulin resistance. 33

Systolic blood pressure measurements

Blood pressure was measured three times by a tail-cuff sphygmomanometer technique (Ugo Basile 58500) at the baseline and the end of the study. The average of those three measurements was calculated and shown as the final reading for SBP.

 Isolation of total RNA and reverse transcription-polymerase chain reaction

Total RNA was obtained from liver and heart tissues and isolated using the easy-BLUE (Intron Biotechnology, cat no. 17061). Reverse transcription reaction was converted by a ReverTra Ace-α kit (Toyobo, FSK-101). The mRNA expression levels were measured using the touchdown PCR protocol with polymerase chain reaction (PCR) LightCycler 96 system (Takara, cat no. TP600). The PCR process was performed using a GoTaq Green Master PCR Kit (Promega, cat no, M7822) according to the manufacturer's protocols with one of the target gene primers. The primer sequences were as follows: β-actin forward 5′-CGA GTA CAA CCT TCT TGC AG-3′, reverse 5′-CAT TGT AGA AAG TGT GGT GC-3′; FAS forward 5′-TGG AGA AGC CCA GGA ACA ACT CAT-3′, reverse 5′-ACC GAG TAA TGC CGT TCA GTT CCT-3′; Adipo-R1: forward 5′-GAC AGG CCT AGG TGT CCA TCA-3′, reverse 5′-TCG TAT GGG ATG ACC CTC CA-3′; PI3K forward 5′-CCT CTC CTT ATA AAG CTC CTG GAA-3′, reverse 5′-GAT CAC AAT CAA GAA GCT GTC GTA A-3′; IRS1 forward 5′-AAG CAC CTG GTG GCT CTC TA-3′, reverse 5′-TCA GGA TAA CCT GCC AGA CC-3′; IRS2 forward 5′-ATA CCG CCT ATG CCT GTC TG-3′, reverse 5′-AGA AGA AGC TGT CCG AGT GG-3′; PDK1 forward 5′-CGT CCC GCA CGT AGA G-3′, reverse 5′-TCC TCA GCA CTC TTG TCC TTA-3′; AKT forward 5′-TCA CCT CTG AGA CCG ACA CC-3′, reverse 5′-ACT GGC TAG TAG GAG AAC TGG-3′; GLUT4 forward 5′-CTT CCT TCT ATT TGC CGT CCT C-3′, reverse 5′-GCT GCT TTG TCC TTC ATC CTG-3′. Gene expression was defined as the relative expression level after being compared with the housekeeping gene (β-actin).

 Data and statistical analysis

Data were analyzed using SPSS 25 with independent t-test analysis with a confidence level of 95% as significant at p ≤ 0.05.

 Results The concentration of bioactive compounds in green tea and green coffee extract on HPLC analysis

HPLC analysis showed that EGCG concentration in green tea extract was 74.126 μg/g. Meanwhile, CGA, caffeine, and polyphenol concentrations in the green coffee extract was 27.134 μg/g and 43,473 μg/g, respectively. 32

Biochemistry characterization in experimental animals

Our previous study reported that rats with the HFHS diet and low-dose STZ injection had metabolic syndrome. It was proved by the measurement of systolic blood pressure (SBP), fasting blood glucose (FBG) level, triglyceride (TG), and HDL cholesterol (HDL) plasma level in rats induced HFHS diet with STZ injection (MS) group met the metabolic syndrome characteristic in accordance to NCEP-ATP III criteria in 8 weeks of duration. Significant differences in SBP, FBG, TG, and HDL levels were observed between the normal control group (NC) and the metabolic syndrome group (MS) (p < 0.05). 34 The pre- and post-test in all extract-intervention groups showed improvement in SBP, FBG, TG, and HDL levels. Meanwhile, the GC group had no statistically significant difference between pre-and post-test, except the HDL level (data were available in https://doi.org/10.6084/m9.figshare.13249163.v3).

 A combination of green tea and decaffeinated, light roasted green coffee extract lowered plasma non-esterified free fatty acids (NEFA) levels

A significant increase was observed in plasma NEFA levels in the MS group compared tothe NC group (p < 0.05). After extract intervention, the plasma NEFA levels in all extract-treated rats were significantly lower (p < 0.05) compared to that of the MS group. Interestingly, the CM group had the least plasma NEFA levels and a significant difference than GT (p < 0.05) and GC (p < 0.01) was observed in plasma NEFA levels ( Figure 1A). It suggested that combining the green tea extract and decaffeinated light roasted green coffee was more effective in reducing FFAs than green tea or green coffee extract single administration ( Figure 1A).

Free fatty acid levels and insulin resistance status.

A. Serum level of non-esterified free fatty acids; B. HOMA-IR score index was used as representation of insulin resistance status. Data are expressed as mean ± SEM (N = 4-5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with MS. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared with GT. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with GC. MS (metabolic syndrome), NC (normal chow), GT (green tea extract), GC (decaffeinated-light roasted green coffee extract), CM (combination of both extracts).

 A combination of green tea and decaffeinated light roasted green coffee extract improved adiponectin levels and adipo-R1 gene expression

Adiponectin levels were significantly lower in the MS group than in the NC group (p < 0.05). The GT group showed lower levels of adiponectin than the GT group. Still, the combination extract group showed a better effect in improving adiponectin levels significantly compared to that of the GT and GC groups (p < 0.05) ( Figure 2A). Moreover, the relative mRNA expression levels of adiponectin-receptor 1 (Adipo-R1) in this study were also significantly lower in the MS group compared to that of the NC group (p < 0.001) ( Figure 2B). All extract-treated groups, either GT, GC, or CM, revealed a significantly higher relative mRNA expression in Adipo-R1 gene expression compared to that of the MS group (p < 0.001) ( Figure 2B). Moreover, the CM group showed the highest Adipo-R1 mRNA expression levels among other extract-treated groups (p < 0.01) ( Figure 2B).

Adiponectin/FAS Measurement.

A. Adiponectin serum levels; B. Gene expression of Adipo-R1; C. Gene expression of FAS. Data are expressed as mean ± SEM (N = 4-5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with MS. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared with GT. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with GC. MS (metabolic syndrome), NC (normal chow), GT (green tea extract), GC (decaffeinated-light roasted green coffee extract), CM (combination of both extracts).

 A combination of green tea and decaffeinated light roasted green coffee extract improved free fatty acids levels and FAS gene expressions

This study revealed the effect of green tea and green coffee extract administration on the free fatty acid synthesis pathways by analyzing the NEFA levels and adiponectin/Adipo-R1/AMPK/FAS pathways involvement.

There was an increase in fatty acid synthase (FAS) gene expression in the liver. The expression of relative mRNA level of FAS was significantly higher in the MS group compared to that of the NC group (p < 0.001) ( Figure 2C). All extract-treated groups showed a lower relative mRNA level of FAS gene in the GT, GC, or CM groups and significantly different from the MS group (p < 0.05) ( Figure 2C). However, there was no significant difference in FAS gene expression between the GT and GC groups (p > 0.05). Still, the FAS gene expression in the CM group showed a significant difference compared to the GT and GC group (p < 0.01) ( Figure 2C). These results were linear with our previous study; we reported that HFHS diet and low dose STZ injection-induced metabolic syndrome rats had lower AMPK-α2 gene expression (p < 0.05). Meanwhile, the combination of green tea and green coffee extract showed substantially higher AMPK-α2 expression compared to that of single extract either green tea or green coffee extract administration (p < 0.05). 34 Therefore, this study revealed that green tea or green coffee extract administration could ameliorate the FFAs levels by improving the adiponectin/adipo-R1/AMPK/FAS pathways in liver tissue rats. However, the combined extract was more effective.

 A combination of green tea and decaffeinated light roasted green coffee extract improved HOMA-IR

HFHS diet with STZ injection rats showed a higher homeostatic model assessment for insulin resistance (HOMA-IR) index compared to that of the NC group (p < 0.001) ( Figure 1B). All of the extract-treated groups revealed a lower HOMA-IR index compared to the MS group (p < 0.05) ( Figure 1B). Moreover, the CM group showed the lowest level in the HOMA-IR index among other extract-treated groups (p < 0.001). This indicated that the combination revealed a better effect in improving insulin resistance.

 A combination of green tea and decaffeinated light roasted green coffee extract ameliorated cardiac insulin resistance marker expressions

The higher HOMA-IR in HFHS diet with low dose STZ injection-induced rats was accompanied by a decrease of cardiac insulin signalling protein gene expression compared to that of the NC group (p < 0.05) ( Figure 1B). It was illustrated by the lower expression of IRS1/2, PI3K, PDK1, Akt, and GLUT4. A higher cardiac insulin signalling protein gene expression was observed in all extract-treated groups. Nevertheless, the GT group showed higher IRS2, AKT, and GLUT4 gene expressions compared to that of the GC group ( Figure 3B, E, F). However, those markers’ relative mRNA expression levels were higher in the CM group than in the GT or GC group (p < 0.001) ( Figure 3).

Gene expression measurement of cardiac insulin signalling.

A. Relative mRNA expression level of IRS1; B. Relative mRNA expression level of IRS2; C. Relative mRNA expression level of PI3K; D. Relative mRNA expression level of PDK1; E. Relative mRNA expression level of Akt; F. Relative mRNA expression level of GLUT4. Data are expressed as mean ± SEM (N = 4-5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with MS. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared with GT. $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with GC. MS (metabolic syndrome), NC (normal chow), GT (green tea extract), GC (decaffeinated-light roasted green coffee extract), CM (combination of both extracts).

 Discussion

Metabolic syndrome is a metabolic disturbance characterized by insulin resistance and a decrease of adiponectin as a biomarker. Adiponectin has essential roles in metabolic profile, such as improved glucose and lipid metabolism and insulin resistance. 35 Adiponectin, an inducer of AMP-activated protein kinase (AMPK) has a regulatory effect on glucose and lipid metabolism that had an essential role in free fatty acid production through inhibition of fatty acid synthetase (FAS). 36 The present study showed lower adiponectin and higher FFA levels in MS rats than the normal rats and intervention rat groups. The administration of green coffee and green tea in MS rats improved adiponectin and FFA serum levels. The improvement of adiponectin and FFA levels were related to the modulation of adipo-R1 and AMPK gene expression in hepatic tissue. 37 – 39 This study showed improvement in adipoR1 in the extract-treated groups. Additionally, FAS gene expression in hepatic tissue was lower in either the green tea or green coffee group. Furthermore, our previous study showed that hepatic AMPK expression was improved in all intervention groups, and the highest level showed in the combination extract group. 34

Lower adiponectin levels and higher FFA levels are related to insulin resistance in hepatic, skeletal, and cardiac tissue. 35 , 40 This study showed that higher HOMA-IR levels reflected insulin resistance conditions. The highest HOMA-IR was shown in MS group rats compared to that of intervention group rats. Moreover, green tea and green coffee extract administration had significantly lower HOMA-IR levels compared to that of MS group rats. Lower HOMA-IR in the extract-treated group was accompanied by improvement of insulin signalling in cardiac tissue. Our study showed cardiac tissue gene expression improvement related to insulin signalling, such as through IRS, Akt, PI3K, PDK1, and GLUT4 in MS rat with green tea and green coffee administration. The results showed that insulin signaling gene expressions were significantly higher in the intervention group compared to MS group rats. Moreover, the combination of green tea and green coffee had the highest expressions.

The present study aimed to investigate the effect of combining green tea and green coffee on cardiac insulin resistance caused by the elevation of FFA serum and lower adiponectin levels. This study showed that the administration of green tea and green coffee improved cardiac insulin resistance by reducing serum free fatty acids levels and elevating serum adiponectin levels by modulation of adipo-R1, AMPK, and FAS gene expressions. These results were similar to previous studies that improving serum FFA and adiponectin signalling pathways could attenuate insulin resistance. 41 – 44 Although animal and human studies have shown that lowering serum FFAs directly improved insulin resistance in some tissues such as muscles, adipose, hepatic, and endothelial tissues, few studies showed the effect on cardiac tissues. Moreover, green coffee administration could improve blood lipids and increase metabolic rates, fatty acid oxidation, and hepatic triglyceride in obese animal rat or mice models using diet or genetic modification. 12 , 45 – 47 In addition, a chlorogenic acid compound in the green coffee extract was associated with the improvement of adiponectin levels and had an important role in insulin sensitivity and inflammation. 48 Besides, green tea administration was known to have similar effects. 49 Li et al. (2018) study showed that the EGCG compound in green tea extract inhibited gene expression involved in synthesizing de novo fatty acids, such as FAS, ACC, and SC. 50 Other studies showed that EGCG had a beneficial effect on insulin resistance by improving the AMPK pathways. 18

Administration of a combination of green coffee and green tea extract in this study showed a higher adiponectin level compared to that of green coffee or green tea single extract administration. Additionally, the Adipo-R1 gene expression was higher in the CM group. Previous studies showed that green tea extract had a beneficial effect on the adiponectin signalling pathways through improving adiponectin levels and modulating adipo-R1 expressions. 37 , 38 On the other hand, few studies have revealed the effect of green coffee on the improvement of FFA levels. 51 , 52 Our study were also showed that green tea and green coffee administration were adequate to reduce serum FFA levels. This study was in accordance with previous studies that revealed the improvement of adiponectin and adipo-R1 is involved in lower serum FFA levels. 18 , 41 , 45 – 47 Interestingly, the combination of both extracts exhibited lower serum FFA levels than green tea or decaffeinated-light roasted green coffee alone.

Elevation of serum FFA levels is caused by excess nutrition intake, adipose lipolysis, and de novo fatty acid synthesis (glucose utilization), mainly in the hepatic tissues. 39 The present study showed that hepatic de novo fatty acid synthesis was increased in metabolic syndrome rats. It was illustrated by increasing FAS mRNA expressions in the MS group (P < 0.01 vs. NC group). This finding was linear with our previous study that AMPK mRNA expressions were reduced in MS rats. 34 AMPK has an inhibitory effect on FAS 53 ; thus, the reduction of AMPK showed a decrease in inhibitory regulation effect. Nevertheless, green tea or green coffee intervention in the present study showed a decrease in FAS expressions, and these were similar to other previous studies 45 , 46 , 51 , 54 , 55 ; however, our findings showed no significant difference between green tea and green coffee extract rat groups. Meanwhile, the combination of light roasted decaffeinated green coffee and green tea extract significantly reduced FAS mRNA expression compared to that of single extract groups. These findings showed that green tea and green coffee had more practical effects on improving hepatic de novo fatty acid synthesis, thus reducing serum FFAs levels.

It was known that FFA induced insulin resistance. 44 , 56 This study revealed HOMA-IR accompanied the increase of serum FFA levels in MS rats. Compared with previous studies, 57 – 59 we had similar findings that administration of green tea decaffeinated-light roasted green coffee had beneficial effects on improving insulin resistance. It was illustrated by a lowering of HOMA-IR in all intervention groups. Meanwhile, the administration of the combination of both extracts showed a lower level of HOMA-IR than green tea or green coffee administration alone. A previous study showed that the administration of EGCG 54 , 60 attenuated insulin sensitivity in skeletal and adipose tissue.. Moreover, CGA and other polyphenols in green coffee improved insulin sensitivity in HFD Mice. 51 HOMA-IR reflects insulin resistance conditions from multi organs such as adipose, heart, and muscle, including skeletal and cardiac muscle. 61 , 62

Many reviews have revealed that FFA could induce alteration of cardiac structure and function without coronary disease or hypertension, 7 which is called diabetic cardiomyopathy, and insulin resistance is the early step in the development of the disease. 63 Many studies suggest that an increase of serum FFA levels can induce insulin resistance by inhibiting IRS1/PI3K/Akt in cardiac muscle, 64 – 66 and associated with cardiac remodelling and dysfunction. 67 , 68 It was recently known that FFAs could interact with toll-like receptors (TLR) and induce insulin resistance through the disturbance of insulin signalling pathways in cardiac tissues. 11 , 69 Our findings observed that improvement of serum FFA levels was accompanied by improved cardiac insulin signalling pathways. All treatment groups showed a high of IRS1/2, PI3K, PDK1, Akt, and GLUT4 mRNA expression, but the GT group showed higher IRS2, Akt, and GLUT4 than the GC group. Rebollo-Hernanz et al. (2019) recently showed that phenolic compounds in green coffee, especially CGA, modulate adipogenesis and insulin resistance via PI3K/Akt signalling pathways in adipocytes. 70 Another study showed that chlorogenic acid stimulates glucose transport in skeletal muscle via AMPK activation in db/db mice. 58 Besides, the green tea administration showed the ameliorating mechanism of hyperglycemia by promoting GLUT4 translocation in skeletal muscle of diabetic rodents. 60 Moreover, another study revealed that EGCG administration induced GLUT4 translocation in skeletal muscle through PI3K- and AMPK-dependent pathways. 16 Nevertheless, the combination of green tea and decaffeinated-light roasted green coffee extract had the highest expressions. It showed that green tea and green coffee extract had synergistic interaction that consequently improved insulin sensitivity.

This study had several limitations. Firstly, we did not investigate the biomarker in adipose tissues, which might give a comprehensive understanding of green tea and green coffee administration’s effect on lipid metabolism. Secondly, we could not exhibit the molecular mechanisms for the effects of green tea and green coffee combination administration.

 Conclusion

Our study revealed clear evidence that a combination of green tea and decaffeinated light roasted green coffee extracts improved cardiac insulin resistance better than single extract administration by ameliorating free fatty acids through adiponectin/FAS pathways modulation.

 Data availability

Figshare: Underlying data for ‘Green Tea and decaffeinated light roasted green coffee extract combination improved cardiac insulin resistance through free fatty acids and adiponectin/FAS pathways amelioration in metabolic syndrome rat model’ https://doi.org/10.6084/m9.figshare.13249163.v3.

This project contains the following underlying data:

Data of Metabolic Syndrome Rat Model.xlsx (This file contains the analyzed data)

RAW data metabolic syndrome.xlsx (This file contains the actual observed values of the variables)

Chlorogenic acid Coffee HPLC.pdf (This file contains the value of coffee chlorogenic acid levels as measured by HPLC)

Caffeine coffee HPLC.pdf (This file contains the value of coffee caffeine levels as measured by HPLC)

Green Tea Catechin HPLC.pdf (This file contains the value of the catechin levels in the tea)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Author contributions

M.L: Conceptualization, Data Curation, Methodology, Writing – Review & Editing

M.S.R: Conceptualization, Funding acquisition, Supervision, Writing – Review & Editing

D.A.N: Conceptualization, Data Curation, Methodology, Resources

M.N.R: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation

N.A.W: Data Curation, Investigation, Writing – Original Draft Preparation

N.W: Project Administration, Supervision, Writing – Review & Editing

 Acknowledgements

Thanks to the Medical Faculty of Brawijaya University, the Molecular Biology Laboratory of the Department of Biology Mathematics and Natural Sciences of Brawijaya University, and the Ministry of Education, Culture, Research, and Technology.

 References Hotamisligil GS Erbay E : Nutrient sensing and inflammation in metabolic diseases. Nat Rev Immunol. 2008 Dec;8(12):923–934. 19029988 10.1038/nri2449 PMC2814543 Ranasinghe P Mathangasinghe Y Jayawardena R : Prevalence and trends of metabolic syndrome among adults in the Asia-pacific region: a systematic review. BMC Public Health. 2017 Dec;17(1):101. 28109251 10.1186/s12889-017-4041-1 PMC5251315 Ingelsson E Sundström J Ärnlöv J : Insulin Resistance and Risk of Congestive Heart Failure. JAMA. 2005 Jul 20;294(3):334. 16030278 10.1001/jama.294.3.334 Paolillo S Rengo G Pellegrino T : Insulin resistance is associated with impaired cardiac sympathetic innervation in patients with heart failure. Eur Heart J Cardiovasc Imaging. 2015 Oct;16(10):1148–1153. 25845954 10.1093/ehjci/jev061 Rather MJ Thibault HB Buys ES : A short duration of high-fat diet induces insulin resistance and predisposes to adverse left ventricular remodeling after pressure overload. Am J Physiol Heart Circ Physiol. 2008 Dec;295(6):H2495–H2502. 18978196 10.1152/ajpheart.00139.2008 PMC2614531 Deng J-Y Huang J-P Lu L-S : Impairment of cardiac insulin signaling and myocardial contractile performance in high-cholesterol/fructose-fed rats. Am J Physiol Heart Circ Physiol. 2007 Aug;293(2):H978–H987. 17400720 10.1152/ajpheart.01002.2006 Jia G Hill MA Sowers JR : Diabetic Cardiomyopathy: An Update of Mechanisms Contributing to This Clinical Entity. Circ Res. 2018 Feb 16;122(4):624–638. 29449364 10.1161/CIRCRESAHA.117.311586 PMC5819359 Schüttler D Clauss S Weckbach LT : Molecular Mechanisms of Cardiac Remodeling and Regeneration in Physical Exercise. Cells. 2019 Sep 23;8(10):1128. 31547508 10.3390/cells8101128 PMC6829258 Zheng L Li B Lin S : Role and mechanism of cardiac insulin resistance in occurrence of heart failure caused by myocardial hypertrophy. Aging. 2019 Aug 28;11(16):6584–6590. 31461405 10.18632/aging.102212 PMC6738426 Lambert JE Ramos–Roman MA Browning JD : Increased De Novo Lipogenesis Is a Distinct Characteristic of Individuals With Nonalcoholic Fatty Liver Disease. Gastroenterology. 2014 Mar;146(3):726–735. 24316260 10.1053/j.gastro.2013.11.049 PMC6276362 Fuentes-Antrás J Ioan AM Tuñón J : Activation of Toll-Like Receptors and Inflammasome Complexes in the Diabetic Cardiomyopathy-Associated Inflammation. Int J Endocrinol. 2014;2014:1–10. 24744784 10.1155/2014/847827 PMC3972909 Ko HJ Zhang Z Jung DY : Nutrient Stress Activates Inflammation and Reduces Glucose Metabolism by Suppressing AMP-Activated Protein Kinase in the Heart. Diabetes. 2009 Nov 1;58(11):2536–2546. 19690060 10.2337/db08-1361 PMC2768176 Sun X Pan H Tan H : High free fatty acids level related with cardiac dysfunction in obese rats. Diabetes Res Clin Pract. 2012 Feb;95(2):251–259. 22088789 10.1016/j.diabres.2011.10.028 Yuan H Ma Q Ye L : The Traditional Medicine and Modern Medicine from Natural Products. Molecules. 2016 Apr 29;21(5):559. 27136524 10.3390/molecules21050559 PMC6273146 Yang CS Zhang J Zhang L : Mechanisms of body weight reduction and metabolic syndrome alleviation by tea. Mol Nutr Food Res. 2016 Jan;60(1):160–174. 26577614 10.1002/mnfr.201500428 PMC4991829 Ueda-Wakagi M Hayashibara K Nagano T : Epigallocatechin gallate induces GLUT4 translocation in skeletal muscle through both PI3K- and AMPK-dependent pathways. Food Funct. 2018;9(8):4223–4233. 29998274 10.1039/c8fo00807h Bose M Lambert JD Ju J : The Major Green Tea Polyphenol, (-)-Epigallocatechin-3-Gallate, Inhibits Obesity, Metabolic Syndrome, and Fatty Liver Disease in High-Fat–Fed Mice. J Nutr. 2008 Sep 1;138(9):1677–1683. 18716169 10.1093/jn/138.9.1677 PMC2586893 Li Y Zhao S Zhang W : Epigallocatechin-3-O-gallate (EGCG) attenuates FFAs-induced peripheral insulin resistance through AMPK pathway and insulin signaling pathway in vivo. Diabetes Res Clin Pract. 2011 Aug;93(2):205–214. 21514684 10.1016/j.diabres.2011.03.036 Qin L Zang M Xu Y : Chlorogenic Acid Alleviates Hyperglycemia-Induced Cardiac Fibrosis through Activation of the NO/cGMP/PKG Pathways in Cardiac Fibroblasts. Mol Nutr Food Res. 2021 Jan;65(2):2000810. 33200558 10.1002/mnfr.202000810 Bagchi D Moriyama H Swaroop A : Green Coffee Bean Extract in Human Health. 0ed. CRC Press;2016 [cited 2021 Jul 4]. Reference Source McCarty MF : A chlorogenic acid-induced increase in GLP-1 production may mediate the impact of heavy coffee consumption on diabetes risk. Med Hypotheses. 2005 Jan;64(4):848–853. 15694706 10.1016/j.mehy.2004.03.037 Alonso-Castro AJ Miranda-Torres AC González-Chávez MM : Cecropia obtusifolia Bertol and its active compound, chlorogenic acid, stimulate 2-NBDglucose uptake in both insulin-sensitive and insulin-resistant 3T3 adipocytes. J Ethnopharmacol. 2008 Dec;120(3):458–464. 18948178 10.1016/j.jep.2008.09.019 S HV V K Patel D : Biomechanism of chlorogenic acid complex mediated plasma free fatty acid metabolism in rat liver. BMC Complement Altern Med. 2016 Dec;16(1):274. 27495925 10.1186/s12906-016-1258-y PMC4974694 Verhoef P Pasman WJ van Vliet T : Contribution of caffeine to the homocysteine-raising effect of coffee: a randomized controlled trial in humans. Am J Clin Nutr. 2002 Dec 1;76(6):1244–8. 12450889 10.1093/ajcn/76.6.1244 Turnbull D Rodricks JV Mariano GF : Caffeine and cardiovascular health. Regul Toxicol Pharmacol. 2017 Oct;89:165–185. 28756014 10.1016/j.yrtph.2017.07.025 Lim D Chang J Ahn J : Conflicting Effects of Coffee Consumption on Cardiovascular Diseases: Does Coffee Consumption Aggravate Pre-existing Risk Factors? Processes. 2020 Apr 8;8(4):438. 10.3390/pr8040438 Choi S Jung S Ko K : Effects of Coffee Extracts with Different Roasting Degrees on Antioxidant and Anti-Inflammatory Systems in Mice. Nutrients. 2018 Mar 16;10(3):363. 29547558 10.3390/nu10030363 PMC5872781 del Castillo MD Ames JM Gordon MH : Effect of Roasting on the Antioxidant Activity of Coffee Brews. J Agric Food Chem. 2002 Jun 1;50(13):3698–703. 12059145 10.1021/jf011702q Tulp M Bruhn JG Bohlin L : Food for thought. Drug Discovery Today. 2006 Dec;11(23–24):1115–1121. Kemenkes RI : Pedoman dan standar etik penelitian dan pengembangan kesehatan nasional. Kementerian Kesehatan RI. 2017:1–58. Saifur Rohman M Lukitasari M Adi Nugroho D : Ida Panca Nugraheini N, Wahyu Sardjono eguh. Development of an Experimental Model of Metabolic Syndrome in Sprague Dawley Rat. RJLS. 2017 Apr 1;4(1):76–86. Saifur Rohman M Lukitasari M Nugroho DA : Decaffeinated light-roasted green coffee and green tea extract combination improved metabolic parameters and modulated inflammatory genes in metabolic syndrome rats. F1000Res. 2021 Jun 14;10:467. 10.12688/f1000research.27921.1 Van Dijk TH Laskewitz AJ Grefhorst A : A novel approach to monitor glucose metabolism using stable isotopically labelled glucose in longitudinal studies in mice. Lab Anim. 2013 Apr;47(2):79–88. 23492513 10.1177/0023677212473714 Lukitasari M Nugroho D Rohman M : Beneficial effects of green coffee and green tea extract combination on metabolic syndrome improvement by affecting AMPK And PPAR-α gene expression. J Adv Pharm Technol Res. 2020;11(2):81. 32587821 10.4103/japtr.JAPTR_116_19 PMC7305783 Achari A Jain S : Adiponectin, a Therapeutic Target for Obesity, Diabetes, and Endothelial Dysfunction. IJMS. 2017 Jun 21;18(6):1321. 28635626 10.3390/ijms18061321 PMC5486142 Horman S Beauloye C Vanoverschelde J-L : AMP-activated Protein Kinase in the Control of Cardiac Metabolism and Remodeling. Curr Heart Fail Rep. 2012 Sep;9(3):164–173. 22767403 10.1007/s11897-012-0102-z Santamarina AB Oliveira JL Silva FP : Green Tea Extract Rich in Epigallocatechin-3-Gallate Prevents Fatty Liver by AMPK Activation via LKB1 in Mice Fed a High-Fat Diet. Aspichueta P, editor. PLoS ONE. 2015 Nov 4;10(11):e0141227. 26536464 10.1371/journal.pone.0141227 PMC4633218 Ferreira LT de Sousa Filho CPB Marinovic MP : Green tea polyphenols positively impact hepatic metabolism of adiponectin-knockout lean mice. J Functional Foods. 2020 Jan;64:103679. 10.1016/j.jff.2019.103679 Sobczak AIS Blindauer CA Stewart AJ : Changes in Plasma Free Fatty Acids Associated with Type-2 Diabetes. Nutrients. 2019 Aug 28;11(9):2022. 31466350 10.3390/nu11092022 PMC6770316 Balsan GA da C Vieira JL de Oliveira AM : Relationship between adiponectin, obesity and insulin resistance. Rev Assoc Med Bras. 2015 Feb;61(1):72–80. 25909213 10.1590/1806-9282.61.01.072 Hamdaoui MH Snoussi C Dhaouadi K : Tea decoctions prevent body weight gain in rats fed high-fat diet; black tea being more efficient than green tea. J Nutr Intermediary Metabolism. 2016 Dec;6:33–40. 10.1016/j.jnim.2016.07.002 Storz P Doppler H Wernig A : Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells. Palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake. Eur J Biochem. 1999 Nov 15;266(1):17–25. 10542046 10.1046/j.1432-1327.1999.00809.x Dresner A Laurent D Marcucci M : Effects of free fatty acids on glucose transport and IRS-1–associated phosphatidylinositol 3-kinase activity. J Clin Invest. 1999 Jan 15;103(2):253–259. 9916137 10.1172/JCI5001 PMC407880 Boden G : Obesity, insulin resistance and free fatty acids: Current Opinion in Endocrinology. Diabetes Obes. 2011 Apr;18(2):139–143. 21297467 10.1097/MED.0b013e3283444b09 PMC3169796 Murase T Misawa K Minegishi Y : Coffee polyphenols suppress diet-induced body fat accumulation by downregulating SREBP-1c and related molecules in C57BL/6J mice. Am J Physiol Endocrinol Metab. 2011 Jan;300(1):E122–E133. 20943752 10.1152/ajpendo.00441.2010 Cho A-S Jeon S-M Kim M-J : Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice. Food Chem Toxicol. 2010 Mar;48(3):937–943. 20064576 10.1016/j.fct.2010.01.003 Ilmiawati C Fitri F Rofinda ZD : Green coffee extract modifies body weight, serum lipids and TNF-α in high-fat diet-induced obese rats. BMC Res Notes. 2020 Dec;13(1):208. 32276657 10.1186/s13104-020-05052-y PMC7149906 Zhang Z Wang S Zhou S : Sulforaphane prevents the development of cardiomyopathy in type 2 diabetic mice probably by reversing oxidative stress-induced inhibition of LKB1/AMPK pathway. J Mol Cell Cardiol. 2014 Dec;77:42–52. 25268649 10.1016/j.yjmcc.2014.09.022 Yuan H Li Y Ling F : The phytochemical epigallocatechin gallate prolongs the lifespan by improving lipid metabolism, reducing inflammation and oxidative stress in high-fat diet-fed obese rats. Aging Cell. 2020 Sep [cited 2021 Jul 17];19(9). Reference Source Li F Gao C Yan P : EGCG Reduces Obesity and White Adipose Tissue Gain Partly Through AMPK Activation in Mice. Front Pharmacol. 2018 Nov 22;9:1366. 30524290 10.3389/fphar.2018.01366 PMC6262053 Song SJ Choi S Park T : Decaffeinated Green Coffee Bean Extract Attenuates Diet-Induced Obesity and Insulin Resistance in Mice. Evid Based Complement Alternat Med. 2014;2014:1–14. 24817902 10.1155/2014/718379 PMC4003760 Li Y Shen D Tang X : Chlorogenic acid prevents isoproterenol-induced hypertrophy in neonatal rat myocytes. Toxicol Lett. 2014 May;226(3):257–263. 24583048 10.1016/j.toxlet.2014.02.016 Day EA Ford RJ Steinberg GR : AMPK as a Therapeutic Target for Treating Metabolic Diseases. Trends Endocrinol Metab. 2017 Aug;28(8):545–560. 28647324 10.1016/j.tem.2017.05.004 Li F Gao C Yan P : EGCG Reduces Obesity and White Adipose Tissue Gain Partly Through AMPK Activation in Mice. Front Pharmacol. 2018 Nov 22;9:1366. 30524290 10.3389/fphar.2018.01366 PMC6262053 Zhang R Xiao W Wang X : Novel inhibitors of fatty-acid synthase from green tea (Camellia sinensis Xihu Longjing) with high activity and a new reacting site. Biotechnol Appl Biochem. 2006 Jan 1;43(1):1. 15943584 10.1042/BA20050064 Capurso C Capurso A : From excess adiposity to insulin resistance: The role of free fatty acids. Vascul Pharmacol. 2012 Sep;57(2–4):91–97. 22609131 10.1016/j.vph.2012.05.003 Lukitasari M Nugroho DA Rohman MS : Light-Roasted Green Coffee Extract Improved Adiponectin, Insulin Resistance, And Metabolic Profile Of Metabolic Syndrome Rat Model. Asian J Pharm Clin Res. 2017 Sep 1;10(9):279. 10.22159/ajpcr.2017.v10i9.19329 Ong KW Hsu A Tan BKH : Chlorogenic Acid Stimulates Glucose Transport in Skeletal Muscle via AMPK Activation: A Contributor to the Beneficial Effects of Coffee on Diabetes. Calbet JAL , editor. PLoS ONE. 2012 Mar 7;7(3):e32718. 22412912 10.1371/journal.pone.0032718 PMC3296733 Yu J Song P Perry R : The Effectiveness of Green Tea or Green Tea Extract on Insulin Resistance and Glycemic Control in Type 2 Diabetes Mellitus: A Meta-Analysis. Diabetes Metab J. 2017;41(4):251. 28868822 10.4093/dmj.2017.41.4.251 PMC5583402 Ueda-Wakagi M Nagayasu H Yamashita Y : Green Tea Ameliorates Hyperglycemia by Promoting the Translocation of Glucose Transporter 4 in the Skeletal Muscle of Diabetic Rodents. IJMS. 2019 May 16;20(10):2436. 31100973 10.3390/ijms20102436 PMC6566303 Zabielski P Błachnio-Zabielska AU Wójcik B : Effect of plasma free fatty acid supply on the rate of ceramide synthesis in different muscle types in the rat. Chan C , editor. PLoS ONE. 2017 Nov 2;12(11):e0187136. 29095868 10.1371/journal.pone.0187136 PMC5667851 Xin Y Wang Y Chi J : Elevated free fatty acid level is associated with insulin-resistant state in nondiabetic Chinese people. DMSO. 2019 Jan;12:139–147. 30705599 10.2147/DMSO.S186505 PMC6342222 Palomer X Pizarro-Delgado J Vázquez-Carrera M : Emerging Actors in Diabetic Cardiomyopathy: Heartbreaker Biomarkers or Therapeutic Targets? Trends Pharmacol Sci. 2018 May;39(5):452–467. 29605388 10.1016/j.tips.2018.02.010 Witteles RM Fowler MB : Insulin-Resistant Cardiomyopathy. J Am College Cardiol. 2008 Jan;51(2):93–102. Cook SA Varela-Carver A Mongillo M : Abnormal myocardial insulin signalling in type 2 diabetes and left-ventricular dysfunction. Eur Heart J. 2010 Jan;31(1):100–111. 19797329 10.1093/eurheartj/ehp396 PMC2800920 Muniyappa R Montagnani M Koh KK : Cardiovascular Actions of Insulin. Endocr Rev. 2007 Aug 1;28(5):463–491. 17525361 10.1210/er.2007-0006 Ouwens DM Diamant M Fodor M : Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification. Diabetologia. 2007 Sep;50(9):1938–1948. 17639306 10.1007/s00125-007-0735-8 PMC2039861 Waddingham MT : Contractile apparatus dysfunction early in the pathophysiology of diabetic cardiomyopathy. WJD. 2015;6(7):943. 26185602 10.4239/wjd.v6.i7.943 PMC4499528 de Luca C Olefsky JM : Inflammation and Insulin Resistance. 2009;17. Rebollo-Hernanz M Zhang Q Aguilera Y : Phenolic compounds from coffee by-products modulate adipogenesis-related inflammation, mitochondrial dysfunction, and insulin resistance in adipocytes, via insulin/PI3K/AKT signaling pathways. Food Chem Toxicol. 2019 Oct;132:110672. 31306686 10.1016/j.fct.2019.110672 10.5256/f1000research.155894.r444953 Reviewer response for version 2 Reda Doha 1 Referee https://orcid.org/0000-0002-3654-9340 Clinical pathology, Mansoura University Faculty of Veterinary Medicine (Ringgold ID: 158400), Mansoura, Dakahlia Governorate, Egypt

Competing interests: No competing interests were disclosed.

 7 1 2026 Copyright: © 2026 Reda D 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

General Impression

The authors investigated whether a combination of green tea and decaffeinated light-roasted green coffee extract has a superior effect on improving cardiac insulin resistance compared with single treatments. It presents an interesting research concept with a specific focus on cardiac tissue, clear figures, and a well-structured manuscript. However, it also has several weaknesses, which are outlined below:

Abstract

The control group should be mentioned first, followed by the metabolic syndrome group, to maintain consistency throughout the manuscript.

This study uses a relatively small sample size.

The abbreviations HFHS and STZ should be defined.

The statistical significance is not included.

 Introduction

      There are some grammatical errors, such as: “A pervious study were” in the 20 th line should be “A pervious study was”; “by improving the adiponectin/FAS pathways” should be “improving the adiponectin/FAS pathway.”

Method

The number of animals per group should be specified.

The composition of both the normal and high-fat, high-sucrose diets should be detailed.

The dose of streptozotocin and the frequency of administration per week should be clearly stated.

In line 9, “administration per oral” should be corrected to “administered orally.”

The description of the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) is unclear. The authors mention that it was used to calculate an index, but they do not explain what this index represents.

For clarity, the primer sequences in the PCR protocol should be presented in a table.

One-way ANOVA, rather than independent t-tests, should be applied to evaluate statistical differences among all tested groups, since t-tests can only compare two groups at a time.

 Results

Data on food intake and body weight are missing.

The manuscript lacks reference to the NCEP-ATP III criteria, which should be cited and justified for defining metabolic syndrome.

The authors should clarify whether free fatty acids were measured in the plasma or in the serum.

The figure numbering does not align with the text; specifically, the legend of Figure 2 should be replaced with Figure 3, and vice versa.

 Discussion

This sentence should be added: “Future studies are needed to translate these findings to humans.”

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

No

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

No

Reviewer Expertise:

Metabolic diseases, Nutrition, Clinical chemistry

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.59048.r138844 Reviewer response for version 1 Riedel Sylvia 1 Referee Biomedical Research and Innovation Platform, South African Medical Research Council (MRC), Tygerberg, South Africa

Competing interests: No competing interests were disclosed.

 18 7 2022 Copyright: © 2022 Riedel S 2022 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

General impression: The authors determined whether green tea, decaffeinated green coffee, and a combination thereof can reduce features of metabolic syndrome in rats, with a specific focus on insulin resistance. The manuscript in its current form lacks important information as well as clarity. Grammar and sentence construction need to be reviewed critically throughout the manuscript. The work has merit, but there are too many errors and omissions in this version, the authors should rethink and revise the manuscript thoroughly. The referencing requires attention. Specific comments are highlighted below:

Abstract

Second sentence - grammar. What does “improvement of free fatty acid” mean?

Abbreviations need to be explained when first used (e.g. FAS).

It is unclear how many rats were used per group, is it 25 for all groups or per group?

From the abstract, it is not clear whether groups 3, 4, and 5 were given the high-fat high-sucrose (HFHS) diet and streptozotocin (STZ).

What is the rationale for the combination group of green tea and green coffee?

Was circulating adiponectin measured and in which tissues were the mentioned genes assessed?

The results should be stated clearly and vague statements such as “showed synergistic effects” should be avoided.

The p values should be included. A clear description of which parameters increased and which decreased in which groups should be included.

Introduction

There is a specific set of diagnostic criteria for metabolic syndrome which should be detailed and referenced in the introduction. Reference 10 describes non-alcoholic fatty liver disease (NAFLD) and not insulin resistance (IR) as such, it seems inappropriate to cite here. There are a few sentences that do not make sense. The authors should draw a distinction between cardiac dysfunction and cardiac insulin resistance and define what cardiac insulin resistance entails.

Methods

The study outline is not clear enough from the description, an illustration would be helpful. Critical details regarding the animal work are missing, such as the number of animals per group and dosage of STZ applied. What are the “NCEP ATP III” criteria? These need to be explained and referenced and how the rats used for this study meet these criteria needs to be presented with data (can be supplementary data).

What is the composition of the HFHS diet?

It seems that this study and the study by Rohman et al. 2021 (reference 31) are using the same animals. If this is the case, then this should be stated clearly. Both manuscripts could benefit from combining the results to provide a more substantial and comprehensive overview of the study in one single paper.

It would be preferable if critical references such as 31 were already published/accepted articles, rather than cited while awaiting peer review.   

If the present study and reference 31 are different experiments, then critical data on the animal’s metabolic status (fasting blood glucose, body weights, and data showing that metabolic syndrome was induced, etc.) needs to be provided in the manuscript (the data is shared, but clear visuals would be preferable) and all paragraphs in the methods including extract preparation and the animal study need to clearly reflect this.

There is also mention that the study is part of a study published previously (reference 30) – it needs to be clarified in which way. It seems that some of the confusion derives from an unclear formulation of sentences.

The preparation of the extracts can be referenced if the same method was followed as in reference 31, however, it is not clear what was used to gavage the animals and how the solutions were prepared. Were the animals in the normal control group and the metabolic syndrome control also gavaged and if so, with what? For future studies, alternative methods such as administering the extracts in the diet should be considered to replace gavaging. Extracts can be freeze-dried and mixed into the diet.

It is not clear how the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was analyzed using a kit. How was fasting blood glucose determined? This section requires clarification.

Statistical analysis using a t-test is inappropriate. To assess group differences at least a one-way analysis of variance (ANOVA) or better yet a two-way ANOVA should be used for the MS-induced treatment groups. The MS treatment groups fit a 2x2 factorial design, and a two-way ANOVA will give more appropriate insight into the data, especially the combination of treatments. The negative control and positive control can be compared using a t-test. It is unclear whether the authors report standard deviations (SD) as stated in the statistics paragraph or standard error of the mean (SEM) as stated in the graph legends. The distinction is important and requires clarification.

 Results

Legends to the graphs require more details, such as an explanation of abbreviations and a clear description in which tissues gene expression was analyzed. In general, graph legends should provide enough information to understand the data presented without reading the text.

It was stated that decaffeinated green coffee was used. An explanation is required as to why the extract seems to contain caffeine. There is a chromatogram for caffeine in the data section. The second sentence in the HPLC results section is not clear, it mentions CGA, caffeine, and polyphenols but only two values are given. These values should also be presented with standard deviations of replicate measurements. This needs clarification.

Graph numbers do not align with the references in the text. For example, on page 6 the first paragraph refers to Fig 2, but the data is displayed in Fig 3. In the last paragraph on page 10, the authors also refer to Fig 2 which should be Fig 3.

There was no protein expression included or reported. This is an important aspect to add as gene expression does not necessarily correlate with protein levels and function.

 Discussion

Once the manuscript structure has been clarified and revised, the discussion should be refocused and written concisely to clearly work out what new knowledge this study brings to the body of literature. Grammar should be checked.

The last sentence in the limitations section “…we could not exhibit the molecular mechanisms for the effects of green tea and green coffee combination administration…”, is unclear but seems to contradict the authors' conclusion.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

No

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Metabolic diseases, inflammation, gastrointestinal physiology, food chemistry

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.59048.r98201 Reviewer response for version 1 Arumugam Vijaya Anand 1 Referee https://orcid.org/0000-0001-7485-1586 Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India

Competing interests: No competing interests were disclosed.

 10 11 2021 Copyright: © 2021 Arumugam VA 2021 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The authors intended to evaluate the effects of Green Tea and Decaffeinated Light Roasted Green Coffee Extract metabolic syndrome. It is an interesting and useful study. Firstly, this manuscript is well-organised and well-structured. Secondly, this study observed the differentially expressed genes and provided potential target candidates for the treatment of the metabolic syndrome.

1. There are some grammatical and syntax errors in the manuscript. For example, the words “A free fatty acid is” may be as “Free fatty acids are”; “improvement” as “the improvement” (in the 7th line of the abstract); “IRS-1” as “of IRS-1”; “Combination green” as “A combination of green”; “study also” as “study were also”; “higher of” as “higher”; “lower of” as “lower”; “different with” as “different from”. The grammar mistakes which are not mentioned here also to be checked and corrected properly.

2. There are some typing mistakes as well, and authors are advised to carefully proof-read the text. For example, the word “prodcedure” may be as “procedure”; “compound level” as “compound levels”; “tothe” as “to the”; “extract treated” as “extract-treated”; “theMSgroup” as “the MS group”; “theNCgroup” as “the NC group”; “GTgroup” as “GT group”; “theGTandGCgroups” as “the GT and GC groups”; “theCMgroup” as “the CM group”; “injection induced” as “injection-induced”; “pathway” as “pathways”; “tissue..” as “tissue.”; “decaffeinatedlight” as “decaffeinated light”. The typos not mentioned here also to be checked and corrected properly.

3. The keywords should not be from the title and also the remaining part of the manuscript. The keywords should assist computer searches to find your specific article.

4. Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the text and then use only the abbreviation (For example, high-fat high sucrose (HFHS), streptozotocin (STZ), metabolic syndrome (MS), chlorogenic acid (CGA), high-fat diet (HFD), etc.). And it should be in both abstract as well as in the remaining part of the manuscript. Make a term abbreviated in the article when it is repeated at least three times in the text, not all of them need to be.

6. The weight of the animals should be included in the materials and methods under the heading “Animals and experimental design”. The composition of the standard diet may be included in the materials and methods and also where it has been obtained.

7. In the statistical analysis the authors should include the software and version of the software used in the present findings under the heading “Data and statistical analysis”.

8. The referencing in this paper is inconsistent. The authors have not given the references as per the journal format. In many places, the authors used numbers and authors with year and in few places used only numbers and in some places the authors' used names and years only. For example, “Van Dijk et al., 2013” and it should be corrected properly.

9. The figures should be improved and a proper footnote should be given. All legends should have enough description for a reader to understand the figures without having to refer back of the main text of the manuscript. The authors have used the abbreviations in the axis and expansions that are not given with the graph. The quality of the image is not to be changed since it seems good. The only suggestion here is the authors may include the expansion of the terms used like 1) MS (metabolic syndrome), 2) NC (normal chow) 3) GT (green tea extract) 4) GC (decaffeinated-light roasted green coffee extract), 5) CM (combination of both extracts) as legends.

10. The author should justify how the dose fixation is done (is it based on toxicity test or previous reference, if previous should be included in the reference section) in materials and methods or discussion section of the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Phytothreapeutics, Clinical Biochemistry, Medical Genetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Nugroho Dwi Adi Brawijaya University, Indonesia

Competing interests: No competing interests were disclosed.

 10 9 2023

Dear A. Vijaya Anand, 

Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India

I would like to express my sincere gratitude for taking the time to review our manuscript titled "Green Tea and Decaffeinated Light Roasted Green Coffee Extract Combination Improved Cardiac Insulin Resistance through Free Fatty Acids and Adiponectin/FAS Pathway Amelioration in Metabolic Syndrome Rat Model." Your thoughtful feedback and constructive comments have been invaluable in improving the quality of our work.

I have made the necessary revisions to the manuscript as per your recommendations. You will find these changes clearly marked in the revised manuscript. Additionally, I have included a detailed response to each comment in the attached document.

Once again, thank you for your time and expertise in reviewing our work. We believe that your feedback has significantly enhanced the quality and rigor of our research. We look forward to your further insights and guidance during the next round of review.

Sincerely,

M.N. Kholis