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. 2020 Nov 25;19(2):1583–1592. doi: 10.1007/s40200-020-00694-y

Four weeks exercise training enhanced the hepatic insulin sensitivity in high fat- and high carbohydrate-diet fed hyperinsulinemic rats

Anu Joseph 1,✉, S Parvathy 1, Koyikkal Karthikeya Varma 1, Aiswarya Nandakumar 1
PMCID: PMC7843715  PMID: 33520854

Abstract

Aim

Hyperinsulinemia is considered the primary defect underlying the development of type 2 diabetes. The liver is essential for the regular glucose homeostasis. In this study, we examined the effect of physical training on the insulin signaling, oxidative stress enzymes and Glucose-6-phosphatase(G6Pase) activity in the liver of Wistar rats.

Methods

Adult male Wistar rats were divided into Control diet group(C), High carbohydrate diet(HCD), High fat diet(HFD), HCD and HFD with training(HCD Ex & HFD Ex). HFD Ex and HCD Ex were trained on a small animal treadmill running at 20 m/min for 30 min, 5 days/wk. The present work investigated the effect of training on hepatic insulin receptor(InsR) signaling events, oxidative stress marker expressions and G6Pase activity in hyperinsulinemic rats.

Results

High carbohydrate and fat feeding led to hyperinsulinemic status with increased hepatic G6Pase activity and impaired phosphorylation of insulin receptor substrate 1(IRS1) and reduced expression of antioxidant enzymes.Training significantly reduced hepatic G6Pase activity, upregulated phosphoinositide 3 kinase(PI3K) docking site phosphorylation and downregulated the negative IRS1 phosphorylations thereby increasing the glucose transporter(GLUT) expressions (aa(P < 0.001) when compared to HFD, b(P < 0.01),bb (P < 0.001 when compared to HCD). Anti oxidant enzymes like CAT, SOD, eNOS expression were increased with reduction in the expression of inflammatory enzymes like TNF-α and COX-2 (*(P < 0.05),**(P < 0.01),***(P < 0.001) when compared to control, †(P < 0.05),††(P < 0.01),†††(P < 0.001) when compared to HFD and HCD).

Conclusion

Thus, our study shows that four weeks training enhanced the hepatic insulin sensitivity in high fat and high carbohydrate-diet fed hyperinsulinemic rats.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40200-020-00694-y.

Keywords: Exercise, Hepatic Insulin resistance, Hyperinsulinemia, G6Pase, Insulin signaling pathway, Oxidative stress

Introduction

Physical exercise has shown significant improvement in insulin sensitivity as well as prevention of type 2 diabetes mellitus [1–3]. The main mechanism underlying these phenomena appears to be an improvement in insulin sensitivity of peripheral tissues. The liver, muscle and adipose tissue are the three major tissues largely responsible for clearing glucose from the blood in healthy individuals. The liver is vital for the preservation of normal glucose homeostasis, it produces glucose during fasting and stores glucose postprandially [4, 5]. Although several studies have described the effects of exercise on hepatic metabolism, only fewer studies [6, 7] have analysed the direct effect of exercise on hepatic insulin sensitivity and glucose metabolism. Hepatic insulin action requires a synchronized transmit of intracellular signals. The insulin receptor substrate (IRS) proteins are key mediators of insulin signaling. Of the six IRS proteins identified, IRS1 and IRS2 integrate essential signals from the Insulin Receptor (InsR) that regulate a variety of processes including metabolism, cellular growth, development, and survival. Activation of the InsR results in the tyrosine phosphorylation of IRS1 and IRS2. Then, phosphorylated IRSs bind to Src homology 2 (SH2) domains, such as the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase and play an important role in the metabolic actions elicited by insulin [8, 9]. Oxidative stress has been recently recognized as a key mechanism in insulin resistance. Reactive species, especially reactive oxygen species (ROS) like superoxide, hydrogen peroxide, and hydroxyl radical ions are the agents of oxidative stress and are produced at low physiological levels mostly in the mitochondria and peroxisomes. ROS damage has direct roles in the development and progression of many chronic hepatic diseases [10]. Glucose metabolism in liver is tightly controlled and insulin exerts its action by down regulating hepatic gluconeogenesis. Key gluconeogenic enzymes are phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase (G6Pase). G6Pase is a critical enzyme in the last step of the glycogenolytic pathway [11]. Hepatic insulin resistance is expressed as defective insulin signal transduction and as an increase in hepatic glucose output because of inability of insulin to inhibit the activation of gluconeogenic enzymes [12–15]. Diet and lifestyle modification has been proven to be an effective approach for the improvement in insulin sensitivity. Studies have shown that exercise training periods of ~ 12–16 weeks can enhance hepatic insulin sensitivity. Exercise training is known to reduce the adverse effects of dietary fat on insulin levels and improves insulin sensitivity in high-fat fed animals. Regular physical exercise training has become an important strategy for the nonpharmacological treatment of insulin resistance But it remains unclear whether these training-induced improvements in hepatic insulin sensitivity extend to enhanced hepatic glucose metabolism and reduced oxidative stress levels [16, 17]. The aim of the present study was to characterise the influence of physical training on expression of insulin signaling molecules, biochemical markers of oxidative stress and G6Pase activity in liver of male Wistar rats.

Materials and methods

All animal care and procedures were in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Govt. Of India and was approved by MIMS Research Foundation Ethics Committee.

Animals

The type of rats used, diet and methods of estimating body weight, blood glucose and total insulin levels were as described by Joseph et al.. [18].

Adult male Wistar rats were purchased from Sree Chitra Thirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala and used for all experiments. 180–200 g body weight was maintained at their housing conditions with a controlled humidity (55%) and a 21 ± 1 °C temperature, under 12 hours light and 12 hours dark periods and were maintained on standard food pellets and water ad libitum. Control, high carbohydrate and high fat feed were purchased from the Animal Nutrition Department, Kerala Veterinary and Animal Sciences University, Mannuthy, Kerala composing of 18.6% proteins, 44.2% carbohydrates and 6.2% fat for control group. High-fat diets contained as percentage of calories 18% proteins, 24% carbohydrates, 58% fat and high-carbohydrate diets: 21.8% proteins, 75.6% carbohydrate, and 2.5% fat.

After a week of acclimatization, the rats were randomly grouped into five groups with 10–12 animals in each group.

  • Control Group (control diet)- C.

  • High fat group (58% high Fat diet)- HFD.

  • High carbohydrate group (75.6% high Carbohydrate)-HCD.

  • HFD with exercise group (HFD + 30 minutes exercise training for two weeks)- HFD Ex.

  • HCD with exercise group (HCD + 30 minutes exercise training for two weeks)- HCD Ex.

The control group was fed a standard diet and the other four groups received a high fat or carbohydrate diet. After 4 weeks, the exercise groups HFD Ex and HCD Ex rats, were trained on a small animal treadmill. All rats were firstly accustomed to exercise for 2 days, by running on a treadmill at a speed of 10 m/min for 15 minutes/day. Subsequently, all the animals were regularly trained according to the running protocol (20 m/min for 30 min, 5 days/wk).

Blood glucose, total circulating insulin and body weight were monitored and recorded periodically. At the end of 6 weeks, fasting blood was collected from vena caudalis for biochemical analysis. The rats were then weighed and sacrificed by Intra-peritoneal Barbiturate Overdose (200 mg/kg) after an overnight fast and 48 h after the last training session in the case of the trained rats. Under sterile conditions, tissues were dissected and stored at − 80 °C for further analysis.Glucose and cholesterol levels were determined by commercial colorimetric kits (Span Diagnostics) and total circulating serum insulin was estimated using Insulin ELISA kit (Mercodia developing Diagnostics) according to the manufacturer’s instructions.

Western blotting

The liver tissues were lysed on ice in RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitor, and 1 mM PMSF. After homogenisation the preparation was centrifuged at 20,000 g for 20 minutes at 4 °C. The supernatant was then stored at -80 °C for later analysis. The protein content in each sample was measured by Bradford method. Equal amounts of lysate proteins (40 µg) were separated in 12% SDS PAGE gels before being transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk or BSA (according to primary antibody used) and then immunoblotted with antibodies 1:1000 Anti InsR, 1:1000 Anti IRS1 (phospho Y612), 1:1000 Anti GLUT2 (Abcam), 1:500 pIRS1 (Ser 636), 1:500 pIRS1 (Ser 639) (Santa Cruz), 1:1000 Anti eNOS (Abcam), 0.2 µg/ml Anti Superoxide dismutase 1(Abcam), 1 µg/ml Anti MMP-2(Abcam), 1:2000 Anti Glutathione Peroxidase 1 (Abcam), 1:2000 Anti Catalase (Abcam), 1:1000 Anti TNF-α (Abcam), 1 µg/ml Anti pan Akt (Abcam) and 1:1000 Anti Cyclo oxygenase 2(COX-2) (Abcam), overnight at 4 °C. After washes with 0.1% Tween 20 in TBS, the membranes were incubated with horseradish peroxidase linked secondary antibody. The protein bands were visualized after developing with BCIP/NBT reagent. Protein content was normalized by Monoclonal Anti β Actin (1:2000, Sigma-Aldrich).

Glucose 6 phosphatase analysis

The liver (~ 800 mg) was minced in homogenization buffer (250 mM Sucrose, 5 mM Hepes, pH 7.0). The homogenate was filtered through gauze and centrifuged at 2500 g during 10 min at 4 °C. The supernatant was collected and centrifuged at 17,000 g for 20 min. Supernatant transferred to fresh tube. Protein estimated by Bradford assay. Approximately 300 µg of protein was used for the further kinetic assay. 10 mM glucose 6 phosphate (G6P) and 100 mM cacodylate buffer solutions were mixed and equilibrated at 37 °C. The assay was initiated with the addition of 300 µg protein. The assay was prepared to determine Pi production at 0, 1.0, 2.5, 5.0, 7.5, 10, 15, 30 and 45 min. The reactions were terminated by the addition of colorimetric reagent (six volumes of acid molybdate, two volumes of 5% SDS and one volume of 10% ascorbic acid). The absorbance of the phosphate-molybdate complex was determined against a reagent blank at 820 nm [19]. The kinetic activity is reported based on the amount of Pi liberated. The concentration of Pi can be obtained from the linear standard curve. Pi concentrations plotted on Y axis against time on X axis. Linear Standard curves were prepared with 0-150 nmol Pi (using 1 mM KH2P04) containing l00mM cacodylate, pH 6.5. Reaction stopped by addition of colorimetric reagent. Absorbance read at 820 nm. A standard curve was plotted with OD on the Y axis and Pi concentration (nmoles) on the X axis.

Statistical analysis

Data are presented as Mean ± SE. Statistical analysis was conducted using Graph Pad Prism 5.0 software (Graph Pad Software Inc., USA). Statistical evaluation was done by 2-way ANOVA. Differences between groups were considered statistically significant if P < 0.05.

Results

Standardization of hyperinsulinemic rat model

Our studies showed that body weight, total cholesterol and total circulating insulin of high fat and high carbohydrate fed rats increased compared to control (Suppl Table 1,3,4) (P < 0.001 when compared to control) whereas the blood glucose levels were within the borderline range (Suppl Table 2). With exercise the body weight, total cholesterol, total circulating insulin and blood glucose levels were reduced to near to control (P < 0.001 HFD Ex when compared to HFD, P < 0.01 HCD Ex when compared to HCD). Thus, rat model with pre-diabetic high circulating insulin was standardised. Exercise training showed significant role in controlling insulin levels.

Hepatic insulin signaling response

Insulin receptor expression

The total InsR expression was studied in liver tissue. Our results revealed no significant changes in the InsR expression in liver tissues of control, HFD, HCD and exercise group rats (Fig. 1). This shows that the expression of overall number of InsRs is not affected by high fat and high carbohydrate diet with or without exercise. These results lead us to further analyse the phosphorylation sites on the IRS1.

Fig. 1.

Fig. 1

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Protein expression of InsR in Liver. Lane 1: C (Control), lane 2: HFD (High Fat Diet), lane 3: HFD Ex (High Fat Diet with Exercise), lane 4: HCD (High Carbohydrate Diet), lane 5: HCD Ex (High Carbohydrate Diet with Exercise)

Tyrosine p-y612 Expression

Tyrosine p-y612 is the PI3 docking site. Phosphorylation of this site activates the downstream process in insulin signaling pathway. In liver tissues tyrosine p-y612 phosphorylation was significantly decreased in HFD rats and HFD Ex group showed increased expression near to the control (P < 0.001). The phosphorylation in HCD and HCD Ex groups showed almost same level of expression (Fig. 2).

Fig. 2.

Fig. 2

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Protein expression of IRS1 Tyrosine p-y612 in Liver. Tyrosine p-y612- is the PI3 docking site; Phosphorylation activates the downstream process in insulin signaling pathways. Lane 1: C (Control), lane 2: HFD (High Fat Diet), lane 3: HFD Ex (High Fat Diet with Exercise), lane 4: HCD (High Carbohydrate Diet), lane 5: HCD Ex (High Carbohydrate Diet with Exercise). aa (P < 0.001) when compared to HFD

Serine p636/639 Expression

Phosphorylation at Serine 636 and 639 is inhibitory to insulin signal transduction. In liver tissues, HCD showed an increased phosphorylation which was reduced with exercise (P < 0.01). But in HFD groups the phosphorylation was significantly decreased indicating an alternative pathway for inhibition (Fig. 3). HFD showed an increased phosphorylation which was reduced with exercise (P < 0.001). But in HCD groups the phosphorylation was significantly decreased indicating that the inhibitory mechanism for HCD and HFD groups in liver tissues is by two different pathways. The inhibitory mechanism in HFD groups is through ser p639 whereas in HCD it is through ser p636 (Fig. 3).

Fig. 3.

Fig. 3

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Protein expression of IRS1 Serine 636/639 in Liver. On phosphorylation of Serine p-636/639 it inhibits the insulin signal transduction. Lane 1: C (Control), lane 2: HFD (High Fat Diet), lane 3: HFD Ex (High Fat Diet with Exercise), lane 4: HCD (High Carbohydrate Diet), lane 5: HCD Ex (High Carbohydrate Diet with Exercise). aa (P < 0.001) when compared to HFD, b (P < 0.01), bb (P < 0.001) when compared to HCD

GLUT2 Expression

GLUT2 is the major transporter in liver. Expression of GLUT2 was decreased in the liver of HFD and HCD fed rats. Exercise produced an increased expression of GLUT2 thereby increasing the glucose utilization (Fig. 4: P < 0.001 HFD Ex when compared to HFD, P < 0.01 HCD Ex when compared to HCD).

Fig. 4.

Fig. 4

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Protein expression of GLUT2 in Liver. GLUT2 is the main glucose transporter in liver. Lane 1: C (Control), lane 2: HFD (High Fat Diet), lane 3: HFD Ex (High Fat Diet with Exercise), lane 4: HCD (High Carbohydrate Diet), lane 5: HCD Ex (High Carbohydrate Diet with Exercise) aa (P < 0.001) when compared to HFD, b (P < 0.01) when compared to HCD

Measurements of biochemical markers of oxidative stress

High doses of ROS determine oxidative stress responsible for serious metabolic dysfunctions and damage to biological macromolecules. To minimise the damage caused by free radicals the organism utilizes enzymatic (Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidise (GPx) etc.) and non enzymatic (Vit A, Vit C, Vit E) antioxidant systems. Oxidative stress can increase ROS reducing the formation of antioxidant defences. The antioxidant activities of the liver tissue associated with enzymes SOD, CAT, eNOS (endothelial nitric oxide synthase), MMP-2 (Matrix Metallopeptidase 2) were significantly reduced during hyperinsulinemic state (Fig. 5a-d). The Akt protein expression was also examined as an important key molecule in the PI3-K/Akt pathway and in the activation of eNOS. The Akt levels were also reduced significantly in hyperinsulinemic state (Fig. 5e).

Fig. 5.

Fig. 5

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Measurements of biochemical markers of oxidative stress in Liver. Western blot analysis for the protein expression of (a) Catalase (b) Superoxide dismutase (c) MMP-2 (D) eNOS (e) Akt (f) COX-2 (g) TNF-α (h) Glutathione peroxidise normalised with β- actin. Values are mean ± SEM of 4–6 separate experiments. *(P < 0.05), **(P < 0.01), *** (P < 0.001) when compared to control, † (P < 0.05), †† (P < 0.01), †††(P < 0.001) when compared to HFD and HCD. Lane 1: C (Control), lane 2: HFD (High Fat Diet), lane 3: HFD Ex (High Fat Diet with Exercise), lane 4: HCD (High Carbohydrate Diet), lane 5: HCD Ex (High Carbohydrate Diet with Exercise)

Increased TNF-α (Tumor Necrosis Factor – α) production is a consequence of metabolic disturbances. Since expression of COX-2 has been detected in several liver pathologies, COX-2 expression was also examined. Both TNF-α and COX-2 levels were significantly increased in hyperinsulinemic state (Fig. 5f, g). GPx is an important anti oxidant mechanism against ROS. Increased GPx levels reflect an appropriate activity of antioxidant barrier enzyme as a response to increased oxidative stress. But in our study, GPx expression remained unchanged across the group (Fig. 5h).* (P < 0.05), ** (P < 0.01), *** (P < 0.001).

Hepatic G6Pase activity

Liver G6Pase contributes to blood glucose homeostasis by catalysing the dephosphorylation of G6P to glucose, the terminal reaction of gluconeogenesis and glycogenolysis. In mammals, the hepatic expression of G6Pase is regulated by hormonal and nutritional status. We studied the role of the time course in the G6Pase activity in the liver of Control, HFD, HCD and exercise groups at 10 mM G6P concentration. After 30 min of incubation, HFD and HCD showed a significant increase in quantity of phosphate released (P < 0.05) when compared to the control and exercise ((P < 0.05) when compared to HFD) and (P < 0.01) when compared to HCD) group (Fig. 6) showing that during prediabetic hyperinsulinemia, the activity of G6Pase is increased several times.

Fig. 6.

Fig. 6

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Kinetic Activity of G6Pase enzyme. Amount of phosphate (nmol) liberated at the end of 30 minutes among the groups Comparative analysis of the time course of G6Pase activity in the liver with 10 mM G6P. Values are mean ± SEM of 4–6 separate experiments. *(P < 0.05) when compared to control, † (P < 0.05) †† (P < 0.01) when compared to HFD and HCD respectively. Control, High Fat diet (HFD), High fat diet exercise (HFD Ex), High Carbohydrate diet (HCD), High Carbohydrate diet Exercise (HCD Ex)

Discussion

The present study tries to understand the changes occurring in the insulin signaling pathway and the metabolic and oxidative stress mechanisms occurring during the development of hyperinsulinemia and the effect of exercise on these signaling and metabolic pathways. An intervention done during an early hyperinsulinemic stage can have high beneficial effects than after the development of diabetes. Various clinical studies from our hospital show that high insulin resistance and high fasting plasma insulin levels can be found at an early stage. High insulin resistance and plasma insulin is observed in patients even before hyperglycemia is evident [20]. Physical exercise is an accepted treatment and preventive method for insulin resistance and type 2 diabetes mellitus [21]. However, while the effects of exercise on muscle and fat have been widely studied, less direct information exists on hepatic response to exercise and its association with the prevention of type 2 diabetic mellitus. Also, while most of the studies focus on the diabetes stage, the prevalence of hyperinsulinemia and associated multiple abnormalities are less studied. Here, we established pre-diabetic hyperinsulinemia in rats by feeding a high fat and carbohydrate diet, resulting in higher body weight, serum insulin level and total cholesterol levels whereas the glucose levels were within normal compared to control rats (suppl table 1–4).

As for the insulin signaling response InsR expression were similar in all experimental groups indicating that the receptor numbers do not play significant role for a substantial reduction in insulin action during insulin resistant states. We also provide proof for down regulated tyrosine phosphorylation of IRS1 [22] and significantly up regulated phosphorylation of IRS1 on serine 636 and 639 during hyperinsulinemic prediabetic states. The phosphorylation patterns however differed among the HFD and HCD fed rats. HCD showed an increased phosphorylation of serine p636 whereas HFD showed an increased phosphorylation of serine p639 indicating that the inhibitory mechanism for HCD and HFD groups in liver tissues is by two different pathways.

Oxidative disturbances in the liver cells may play a significant role in the genesis of the diabetes induced liver damages like chronic liver disease, including the non-alcoholic fatty liver disease and its occasional progression to steatohepatitis and cirrhosis [23, 24]. In our study, expression levels of COX-2 and TNF-α were significantly increased whereas CAT, SOD, MMP-2 and eNOS were reduced in hyperinsulinemic state. It is widely known that oxidative stress plays an important role in the mechanisms of regulation of cellular adherence, proliferation, migration and signaling of the extracellular matrix, thus, being able to change the structure and permeability of the cell membranes and intracellular organelles [25]. Evidences from the literature suggests that chronic oxidative stress of the liver not only can initiate molecular alterations that lead to fat accumulation in the organ, but also of promoting alterations resulting in cirrhosis [23, 24, 26]. Enzymes like CAT, SOD helps in protecting the cell from oxidative damage by reactive oxygen species (ROS). Hence reduced expression of these enzymes during hyperinsulinema makes the hepatocytes more vulnerable to accumulation of ROS. GPx expression is usually increased as a protective mechanism during stress. Our results showing a similar GPx expression across the group may indicate a mechanism to balance the oxidative stress. Furthermore, these ROS stimulates the secretion of MMPs, facilitating the degradation of extracellular matrix (ECM) components and simultaneously distorts the hepatic tissue architecture. Akt is an important key molecule that phosphorylates and activates eNOS, leading to the production of nitric oxide (NO) and also activating the PI3-K/Akt pathway. NO generated by eNOS is clearly beneficial for liver function regulating blood flow and blood cell interactions [27]. Hence reduced eNOS activity and NO production can activate Kupffer cells, which initiate inflammatory processes and potentiate hepatic insulin resistance [28]. COX-2 is dramatically up regulated by ROS production and contributes to producing prostaglandins (PGs), which mediate a number of the characteristic features of inflammation and reactions leading to the tissue damage [29]. Our study on G6Pase showed an increased activity in HCD and HFD compared to the control. The persistence of a signaling pathway dysregulations and chronic stress in liver cells seems to be one of the most important predictive factors for hepatic complications like cirrhosis development.

While most of the current literature addresses effects of exercise training on skeletal muscle comparatively fewer interesting studies on liver adaptations and responses to exercise training have been performed. Liver is remarkably important during exercise training outcomes, including modulation of ROS and inflammatory mediators [30]. In our study, many of the signaling defects seen during pre-diabetic hyperinsulinemic states were reversed with regular vigorous exercise training shown by increased expression of GLUT2 in liver. Some studies have demonstrated that exercise training induced a rapid and regulated cellular insulin signal transduction leading to improved hepatic insulin sensitivity [31–36]. It was interesting to find that during exercise training even with lower level of expression of y612, the GLUT2 expression was increased in liver of HCD and HCD EX rats. We assume there might be another parallel pathway activated through enhanced IRS2 signaling mediated PI3K activity during exercise training which finally led to increase in GLUT2 expression without y612 expression. In liver IRS 1 functions primarily after refeeding, whereas IRS2 signaling is mostly dominating in the period directly after food intake and during fasting [37]. Study by Titchenell et al. showed that in the absence of Foxo1, insulin signals via an intermediary extra hepatic tissue to regulate liver glucose production i.e., a hepatic mechanism distinct from the IRS-Akt-Foxo1 axis exist to regulate glucose production [38]. Exercise groups showed a G6Pase value near to control or significantly less compared to control. Reductions in the G6Pase activity with exercise training in liver cells indicate that sensitivity to insulin was improved. G6Pase activity is known to increase in type 2 diabetes mellitus [39]. This is a critical enzyme during the last step in the production of glucose by both the glycogenolytic and gluconeogenetic pathways [40]. Several studies have shown that insulin failed to inhibit G6Pase activity during the pre-diabetic state [41, 42]. Our study also shows that regular exercising induces hepatic antioxidant and anti-inflammatory improvements. CAT, SOD, Akt and eNOS levels were increased with exercise training whereas TNF-α and COX-2 expressions were reduced thereby increasing the hepatic insulin sensitivity. Several lines of studies reported that exercise training can decrease ROS production and pro-inflammatory cytokines such as TNF-α and IL-1β through the improvement of antioxidant activity [43–46]. Thus, the evidences provided in this study indicate that exercise training attenuates hepatic oxidative damage and inflammation triggered by insulin signaling dysregulation by enhancing the expression of antioxidant enzymes and regulating ROS level. Hence, understanding the precise underlying biological mechanisms mediated during exercise training can aid in the development of proper therapeutic interventions. Hyperinsulinemia is the initiating agent which exists long before type 2 diabetes occurs, and an intervention or treatment during this early stage/hyperinsulinemic period may have a better opportunity to avoid or interrupt the development of diabetes and its complications.

Conclusion

Our study confirms that adhering to regular exercise training from early hyperinsulinemic stage lowered the high insulin levels and improved hepatic insulin sensitivity thereby delaying the onset of diabetic complications. Regular physical training reduces the adverse effects of hyperinsulinemia caused by chronic intake of high carbohydrate and fat diet like improvement in hepatic insulin signaling pathway, liver enzyme and protein expression of oxidative stress enzymes.

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Acknowledgements

The authors thank Mr Sreerenj and faculties of Department of Health Sciences, University of Calicut for allowing the use of department facilities. We also thank the MIMSRF Lab manager Mr Varun and assistant Mrs Mahishiya for their timely help.

Author contributions

AJ: Conceptualization, Methodology, Writing- Reviewing and Editing, PS.: Methodology, Investigation, Validation Writing – Original Draft, KKV: Supervision, Writing – Review & Editing. AN- Standardisation All authors have read and approved the final manuscript.

Funding

This work is supported by funding from DST SERB, Govt. of India (ECR/2015/000158) and MIMS Research Foundation, Calicut, Kerala.

Compliance with ethical standards

Ethics approval and consent to participate

All animal care and procedures were in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals by the Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA), Govt. Of India and was approved by MIMS Research Foundation Ethics Committee.

Conflict of interest

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

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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