The combined effects of high-intensity interval training and time-restricted feeding on the AKT/FOXO1/PEPCK pathway in diabetic rats

Abstract

High-intensity interval training (HIIT) and time-restricted feeding (TRF) have shown promise for improving glucose regulation by increasing insulin sensitivity, enhancing glucose uptake, reducing glucose production. Therefore, this study investigates the combined effects of HIIT and TRF on the AKT/FOXO1/PEPCK signaling pathway in the liver tissue of type 2 diabetic rats. 42 male Wistar rats (4–5 weeks of age) were included in the study. The animals were randomly divided into two groups: (1) Standard diet (SD, non-Diabetic (Non-D, n = 7) (2) High-fat diet (HFD, n = 35) for 4 weeks. To induce diabetes, 35 mg/kg of streptozotocin (STZ) was injected intraperitoneally (IP). Animals with blood glucose levels of > 250 mg/dL were considered as diabetic. Diabetic rats were randomly divided into 5 groups (n = 7): (1) Diabetes-exercise (D-EX), (2) Diabetes-TRF (D-TRF), (3) Diabetes-combined TRF and exercise (D-TRF&EX), (4) Diabetes no treatment (D-NT), (5) Diabetes with metformin (D-MET). Interventions (HIIT and TRF) were performed for 10 weeks. Rats in the Non-D group did not exercise and did not receive metformin or TRF. Periodic Acid-Schiff (PAS) staining was used to histologically analyze the liver tissue. Levels of blood glucose, insulin resistance (IR), FOXO1 protein, PEPCK, and area under the curve (AUC) following the IPGTT test, were significantly decreased in treatment groups compared to the D-NT group (p < 0.05). The AKT protein levels (p < 0.01), glycogen content (p < 0.05), and insulin sensitivity (p < 0.001) increased in the treatment groups as compared with the D-NT group. Microscopic examination of the liver tissue in general showed a better tissue arrangement in both treatment groups than in the D-NT group. Combining HIIT and TRF may be effective for improving blood glucose regulation, insulin sensitivity, in type 2 diabetes, as compared to TRF or HIIT interventions alone.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-96703-2.

Introduction

Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disease that is closely related to the obesity epidemic. Insulin resistance (IR) and impaired insulin secretion are the main deficits in T2DM, which occur subsequent to decreased insulin production and the failure of pancreatic β-cells, leading to a decrease in glucose transfer to muscle and fat cells^1,2. In obesity, the predominant use of lipids for energy reduces glucose absorption and the rate of glycogen synthesis, and disrupts insulin sensitivity³.

In T2DM, there are often disturbances in glycogenesis and glycogenolysis, and insulin acts directly by binding to hepatic insulin receptors and thereby activating insulin signaling pathways in the liver. Hepatic IR leads to increased activity of the hepatic gluconeogenesis pathway (hepatic glucose production), and fasting hyperglycemia. Therefore, understanding the mechanisms by which insulin regulates this process (hepatic IR and gluconeogenesis pathway) is very important⁴.

Among the many molecules involved in intracellular insulin signal processes in the liver are the genes for protein kinase B (PKB, also known as AKT2), and insulin receptor substrates (IRS)^5,6. IRS are cytoplasmic receptors important in insulin signaling. After these proteins are activated, several signaling cascades are activated, leading to many downstream factors and ultimately, insulin signaling to a branching series of intracellular pathways⁷. Phosphorylation of IRS proteins is activated by insulin receptor phosphatidylinositol- 3-kinase (PI3 K) and downstream protein kinases. IRS- 1 and IRS- 2 are widely distributed, and previous research has shown that mice that are deficient in IRS- 2 have hepatic insulin resistance with some other defects⁸.

Insulin reduces liver glucose production via direct and indirect effects on the liver. Insulin acts directly by binding to hepatic insulin receptors and thereby activating insulin signaling pathways in the liver⁹. Phosphoenolpyruvate carboxykinase (PEPCK) is key liver enzyme that plays a role in hepatic gluconeogenesis. The expression of this enzyme is controlled by forkhead box protein O1 (FOXO1), and AKT can prevent the expression of PEPCK by inhibiting FOXO1¹⁰. As a result of this phosphorylation, the IRS1/PI3 K/AKT signaling pathway is activated, which plays an important role in controlling the metabolic actions of insulin (glucose, lipid, and protein metabolism), hepatic gluconeogenesis, and glycogen synthesis¹¹.

There are treatment methods including medications and surgeries to treat T2DM, but most of these treatments are associated with side effects. In recent years, healthy lifestyles, including adequate exercise and weight reducing dietary interventions, have been used as alternative approaches for mitigating and or reversing the effects of T2DM. One of the most frequent dietary intervention methods is caloric restriction, and recently, time-restricted feeding (TRF) has become a popular approach to achieving caloric restriction. TRF restricts eating to a specific time window (typically 8 h or less each day), thereby reducing overall caloric intake. TRF has been shown to be an effective approach, with promising metabolic effects including improved insulin sensitivity and increased lipid oxidation. Previous research also indicates that TRF improves hepatic gluconeogenic activity and protects against hyperglycemia. Therefore, the TRF method is thought to be a suitable method for the treatment of T2DM, improving hepatic IR by regulating the gluconeogenic signaling pathway through a significant reduction in the expression of key PEPCK genes¹².

Physical activity, especially aerobic exercise, can effectively reduce IR, hyperglycemia, and the accumulation of liver fat tissue in people with T2DM¹³. Exercise has the capacity to increase blood flow, capillary density, muscle mass, glycogen storage capacity due to increased glycogen synthase, and muscle glucose transport protein (GLUT4) content. Additionally, exercise can improve the rate of glucose uptake by exercising muscles, thereby improving insulin sensitivity and increasing gene expression or activities of different proteins involved in the insulin signaling cascade¹⁴. Aerobic exercise improves insulin signaling in liver tissue and suppresses hepatic glucose production by affecting the gluconeogenesis pathway including PEPCK¹⁵. More recently, studies have shown that high-intensity interval training (HIIT) is an option for shortening the time required for exercise, while still obtaining optimal results. HIIT includes several repetitions of intense intervals with periods of active or passive rest^16,17. Interval training (IT) is characterized by brief, repeated work intervals interspersed with periods of recovery (e.g., low-intensity exercise), with some recent evidence suggesting that IT may be more enjoyable for some participants than continuous moderate-intensity exercise¹⁸. One study showed that HIIT resulted in a greater reduction in fasting blood glucose and improvements in lipid profiles as compared to endurance training in T2DM rats¹⁹. HIIT also improved insulin sensitivity and reduced hyperglycemia. HIIT significantly reduced hepatic glucose production, reduced fatty liver, and improved the overall metabolic status of the rats^20,21. These results support the potential of HIIT to regulate liver function and improve metabolic health in diabetic conditions.

Therefore, in the current study, the effects of TRF combined with HIIT on the AKT/FOXO1/PEPCK pathway was investigated in the liver tissue of rats with T2DM.

Materials and methods

Animals

This study was conducted in accordance with the principles of animal welfare and ethics, and was approved by the Institutional Review Committee (IR.KASHANU.REC.1402.023) at University of Kashan. All methods are reported in accordance with ARRIVE guidelines. This study was conducted on 42 male Wistar rats, 4–5 weeks old and weighing approximately 200 ± 20 gr. The animals were purchased from the Pasteur Institute of Iran. Rats were housed in a controlled room temperature of 22 ± 2 °C and a relative humidity of 50 ± 5%, with a light/dark cycle of 12:12 h. Rats had free access to water and the pellet diet.

Diabetes induction & experimental groups

After the adaptation of the rats (the process of acclimating or habituating the rats to a new environment), in the first stage, the animals were randomly divided into two groups: (1) Standard diet (SD) (n = 7) (2) High-fat diet (HFD, n= 35), SD was the non-Diabetic group and no therapeutic intervention was provided (Non-D). The HFD group diet consisted of 60% kcals from fat, 20% kcals from carbohydrates, and 20% kcals from protein. The diet was provided for 4 weeks to induce T2DM. Supplementary Table 1 shows the ingredients of HFD. At the end of 4 weeks, rats in the HFD group were injected intraperitoneally (IP) with 35 mg/kg streptozotocin (STZ) (Sigma Aldrich, Hamburg, Germany, CAS NO 18883 - 66- 4) after 5 h of fasting²². The induction of diabetes was confirmed through the glucose tolerance tests (GTT), where fasting blood glucose (FBG) level was greater than 250 mg/dL²³. After complete stabilization of diabetes (one week) while the rats were still receiving the HFD, diabetic rats were then randomly divided into 5 groups (n = 7 per group): (1) Diabetes-exercise (D-EX), (2) Diabetes-TRF (D-TRF), (3) Diabetes- TRF and combined exercise (D-TRF&EX), (4) Diabetes no treatment (D-NT), (5) Diabetes with metformin treatment (D-MET). Two groups included HIIT training with exercised rats, received the TRF intervention (D-TRF, and D-TRF&EX) in addition to the HIIT training for 10 weeks. The control group (Non-D) did not receive any intervention (Fig. 1 A).

Fig. 1.

Experimental design. (A) 42 male Wistar rats were randomly allocated into 2 groups: standard diet (SD) and high-fat diet (HFD). After 4 weeks, rats in the HFD group were injected with STZ. The rats with FBG > 250 mg/dl were randomly divided into diabetic groups. After 10 weeks of intervention, the liver tissue of rats was extracted. (B) Diagram representing the availability of macronutrients during the light and dark periods for each group. Zeitgeber time 0 was set as the beginning of the light period. The following groups had ad libitum access to food and water without time limits: non-Diabetes (Non-D), diabetes-EX(D-EX), non-treatment diabetes(D-NT), and diabetes-MET(D-MET). NT, no treatment; EX, exercise; TRF, time-restricted feeding; AL, ad libitum.

Intraperitoneal glucose tolerance test (IPGTT)

After 12 h of fasting, a blood sample was collected from the tail vein (0 min). Next, 20% glucose solution (2 g/kg) was injected IP, and blood sampling was performed 30, 60, and 120 min after the injection. Blood glucose concentration was measured using the glucose oxidase method with a Glucose-B test kit (Wako, Osaka, Japan) (Accu-Chek Instant S Blood Glucose Glucometer Kit). An increase in the area under the curve (AUC) was used to detect the induction of diabetes²⁴. This test is possible to perform with a glucometer and is used for small animals such as rats. We considered the limitations of using the glucometer and continuously calibrated it according to the instructions.

Exercise protocol

Adaptation

In the treadmill exercise groups, animals underwent a period of adaptation to the treadmill. In the first week, each rat was placed on the treadmill for 15 min/day with an intensity of 10–15 m/min with a zero-degree incline. This adaptation period allowed the rats to achieve their maximum speed during the maximum speed (Vmax) test²⁵.

Vmax Estimation test

The maximum running speed was estimated on a treadmill set at a 5-degree incline. Rats were placed on a treadmill and allowed to acclimatize over a 5-minute period at a speed of 6–10 m/min. Then the speed was gradually increased by 3 m/min every 3 min, until they were unable or unwilling to continue. Maximum running speed was defined as the maximum speed for a set running speed²⁶. This procedure was performed for each rat.

HIIT protocol

The HIIT protocol was carried out for 10 weeks, 5 sessions per week, and included 8 intervals of activity for 3-minutes at an intensity of 85–90% of Vmax (22 m/min) on a treadmill with a 5-degree incline. Active rest intervals (2 min) followed each work interval, at an intensity of 30–40% max speed (8 m/min). At the beginning and end of each training session, warm-up and cool-down periods were performed at 30–40% of max speed (8 m/min)²⁷. The exercise protocol was incremental, with the speeds increasing at week five of the study. The details of the 10-weeks exercise intervention protocol are shown in Table 1.

Table 1.

High-intensity interval training (HIIT) protocol.

Week	Duration of Warm up (minute)	Intensity of warm up (Vo2 max)	Velocity of treadmill in warm-up (m/min)	Repetition	Ratio time practice to rest (minute)	Intensity of time practice (Vo2 max %)	Velocity of treadmill in practice (m/min)	Intensity of active recovery (Vo2 max)	Velocity of treadmill in active recovery	Duration of Cool down (minute)	Intensity of Cool down (Vo2 max)	Velocity of treadmill in cool-down	Incline (degree)	Total duration (minute)
Initial assessment of Vmax
1 th	5	30–40%	8	15	3:2	85–90%	22	30–40%	8	5	30–40%	8	5	48
2 th	5	30–40%	8	15	3:2	85–90%	22	30–40%	8	5	30–40%	8	5	48
3 th	5	30–40%	8	15	3:2	85–90%	22	30–40%	8	5	30–40%	8	5	48
4 th	5	30–40%	8	15	3:2	85–90%	22	30–40%	8	5	30–40%	8	5	48
5 th	5	30–40%	8	15	3:2	85–90%	22	30–40%	8	5	30–40%	8	5	48
Secondary assessment of Vmax
6 th	5	30–40%	10	15	4:2	85–90%	26	30–40%	10	5	30–40%	10	5	56
7 th	5	30–40%	10	15	4:2	85–90%	26	30–40%	10	5	30–40%	10	5	56
8 th	5	30–40%	10	15	4:2	85–90%	26	30–40%	10	5	30–40%	10	5	56
9 th	5	30–40%	10	15	4:2	85–90%	26	30–40%	10	5	30–40%	10	5	56
10 th	5	30–40%	10	15	4:2	85–90%	26	30–40%	10	5	30–40%	10	5	56

Time-restricted feeding

Zeitgeber time (ZT) 0 was set as lights-on time and ZT12 as lights-off time. D-TRF and D-TRF&EX groups underwent a TRF protocol. Between ZT1 (1 h after lights-on) and ZT9 (3 h before lights-off), rats were allowed free access to food. Daily food access was regulated by moving rats between cages with food and water and cages with water only. This diet was the same as before diabetes induction. To control for rat movement between cages, rats released for free feeding were also simultaneously transferred between feeding cages. Weekly food consumption was measured by monitoring the weight of food residue²⁸ (Fig. 1B). To reduce stress in the animals, access to food was controlled with a regular pattern and the cage and experimental environment was also kept constant. Gentle methods for animal handling (smooth and slow movements) were used, observing the appropriate number of rats.

Metformin

In diabetic rats treated with metformin, metformin (300 mg/kg) was dissolved in drinking water and administered orally. Metformin concentrations were re-adjusted according to water consumption after an evaluation period of approximately 1 week²⁹.

Preparation of liver tissue

48 h after completing the research protocol, the rats were anesthetized using IP injection of ketamine (50 mg/kg) and Xylazine (10 mg/kg), and liver tissue was extracted. Liver tissue was subsequently washed in ice-cold isotonic saline. Following saline washing, 50 to 100 mg of tissue was stored in a high-quality plastic microtube (without DNase/RNase), and immediately placed in a nitrogen tank and stored at − 80 °C. Generally, sampling was performed from the left and right middle lobes.

Biochemical analyses

FBG (spectrophotometry using commercial glucose kits) and insulin (ELISA) were performed in biochemical laboratories using standard methods. Insulin sensitivity (IS) was calculated using the homeostasis model assessment for insulin resistance (HOMA-IR) indices. Insulin and blood glucose concentrations determined in fasting conditions were used to determine indices. FBG and insulin concentrations were obtained using biochemical analyses. HOMA-IR in diabetic rats are presented in Fig. 2.

Fig. 2.

Serum glucose, insulin levels, and HOMA-IR in all groups. A) Serum glucose. B) Serum insulin. C) HOMA-IR.

HOMA-IR = fasting insulin (microU/L) x fasting glucose (nmol/L)/22.5³⁰.

Western blot

The liver tissue was homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer. This solution is very useful due to its strong ability to disrupt cell membranes and denature proteins and is designed to extract proteins from cells and tissues. This solution usually contains a basic buffer (tris-HCL and NaCl), detergents, protease and phosphatase inhibitors, and EDTA or EGTA. The protein content after centrifugation (12,000 rpm, at 4 °C for 15 min) was dissected and the total protein concentration was evaluated based on the Lowry method. Then the protein samples were diluted in the loading buffer and heated at 95 °C for 5 min, and then subjected to SDS-PAGE 10% acrylamide at 120 V voltage to separate proteins. Then, the proteins were transferred to a nitrocellulose membrane at 100 V for 1–2 h. Next, nitrocellulose membranes were washed in Tris-buffered saline (pH 7.2, containing 0.1% Tween 20), blocked with blocking buffer (5% milk and 0.5% BSA) for 1 h, and overnight at 4 °C. They were incubated with AKT (Elabsciences, E-AB- 63467), FOXO1 (Elabsciences, E-AB- 70144), and PEPCK (SANTA CRUZ, sc- 271029) antibodies. After removing the unbound antibodies, the membranes were incubated with the secondary antibody for 1 h RT. Then, the blots were detected using the advanced chemiluminescence detection kit (Clarity Western ECL Substrate, Cat N: 1705061) were observed. Protein intensity was visualized and analyzed using an amplified laser densitometer with onboard software (RADA, Model 1200, IRAN)³¹.

Study of lipids (Sudan black Staining)

For the study of lipids, we adapted Oil-Red-O (Pajohesh Asia, Iran) and Sudan Black B (Pajohesh Asia, Iran) techniques. In this procedure, the tissue specimens were fixed in 10% neutral formaldehyde solution, sectioned with a cryostat microtome (Bright 00361, England), and stained using separate techniques for each. The atretic follicles with 20% positive lipid-stained cells were scored as one (1+), 50% positive lipid-stained cells were considered as two (2+), 60% positive lipid stained cells were considered as three (3+), and 100% positive lipid-stained cells were considered as four (4+)³².

PAS staining

For the study of carbohydrates for the PAS, we adopted a paraffin sectioning procedure and stained using a special technique for carbohydrates. After fixing the liver tissue in 10% formalin, paraffin blocks were prepared. Thin sections of 4–5 μm were made from the block and placed on slides and incubated at 60 °C. Then the slides were immersed in xylene (2–3 times) and dehydrated with reducing alcohols of different percentages. The slides were first placed in periodic acid solution and then in shift reagent solution. After washing, the slides were stained with hematoxylin. Atretic follicles with 20% positive carbohydrate-stained cells were scored as one (1+), 50% positive carbohydrate-stained cells were considered as two (2+), 60% positive carbohydrate-stained cells were considered as three (3+), and 100% positive carbohydrate-stained cells were considered as four (4+)³².

Statistical analysis

Values are presented as means ± standard deviations (SD). The Shapiro-Wilk test was used to check the normality of the data distribution. Two-way analysis of variance (ANOVA) with Tukey post hoc comparisons were conducted to determine the effects of HIIT, TRF, metformin, and interactions between HIIT and TRF (D-TRF&EX). All statistical analyses were conducted using IBM SPSS (version 26; IBM Corp., Armonk, N.Y., USA), and significant differences were set at p < 0.05 for all analyses.

Results

Test of normality

The table below shows that the Shapiro-Wilk results for the data distribution were normal (p > 0.05) (Supplementary Table 2).

Table 2.

Two-way ANOVA for protein content of AKT, FOXO1, and PEPCK.

	Main effect, p-value, Effect size				Interaction, p-value, Effect size
Parameters	Exercise		TRF		Exercise × diet
	Effect size	p-value	Effect size	p-value	Effect size	p-value
Glucose (Serum-mg/dl)	0.60	< 0.001	0.48	< 0.001	0.008	0.69
Insulin (Serum-microU/L)	0.34	0.004	0.28	0.011	0.001	0.94
HOMA-IR	0.70	< 0.001	0.61	< 0.001	0.001	0.91
AKT/β-Actin protein	0.21	0.17	0.25	0.13	0.19	0.19
FOXO1/β-Actin protein	0.39	0.051	0.58	0.01	0.001	0.95
PEPCK/β-Actin protein	0.88	< 0.001	0.81	< 0.001	0.69	0.003

Effects of high-fat diet and STZ on serum blood glucose/insulin/HOMA-IR in male Wistar rats

All rats had normal blood glucose levels at the start of the induction period. FBG levels in the diabetic control group (D-NT) significantly increased as compared with the healthy control group (Non-D) (p < 0.001). FBG levels in the diabetic metformin group (D-MET) increased significantly as compared with the control group (D-NT) (p < 0.001). There was a significant difference in the FBG of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercised groups (effect size = 0.60, p < 0.001), and a significant difference in FBG in the rats treated with TRF (D-TRF and D-TRF&EX) compared to the non-TRF groups (effect size = 0.48, p < 0.001). However, there were no significant interactions between EX and TRF (effect size = 0.008, p = 0.69) (Fig. 2 A; Table 2).

Insulin levels in the diabetic control group (D-NT) significantly increased as compared with healthy control group (Non-D) (p = 0.027). Insulin levels in the diabetic metformin group (D-MET) increased significantly as compared with control group (D-NT) (p < 0.001). There was significant difference in Insulin of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercised groups (effect size = 0.34, p = 0.004), and there was significant difference in Insulin in the rats treated with TRF (D-TRF and D-TRF&EX) compared to the non-TRF groups (effect size = 0.28, p = 0.011). However, there were no interactions between exercise and TRF (effect size = 0.001, p = 0.94) (Fig. 2B; Table 2).

HOMA-IR levels in the diabetic control group (D-NT) significantly increased as compared with healthy control group (Non-D) (p < 0.001). HOMA-IR levels in the diabetic metformin group (D-MET) increased significantly as compared with control group (D-NT) (p < 0.001). There was significant difference in HOMA-IR of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercised groups (effect size = 0.70, p < 0.001), and there was significant difference in HOMA-IR in the rats treated with TRF (D-TRF and D-TRF&EX) compared to the non-TRF groups (effect size = 0.61, p < 0.001). However, there were no interactions between exercise and TRF (effect size = 0.001, p = 0.91) (Fig. 2 C; Table 2).

Intraperitoneal glucose tolerance tests (IPGTT) in the experimental groups

The IPGTT tests were performed for Non-D and D-NT groups (after STZ injection and before the start of treatment for the groups). IPGTT results, and the corresponding areas under the curve (AUC) for all study groups are depicted in (Fig. 3). Blood glucose levels in the D-NT group were increased significantly as compared to the Non-D group (p < 0.001), confirming induction of diabetes.

Fig. 3.

Blood glucose monitored throughout the treatment of standard and HFD groups. (A) intraperitoneal glucose tolerance test (IPGTT) in standard diet and HFD rats after 4 weeks of intervention. (B) total area under the curve (AUC) for the IPGTT was significantly higher in the D-NT rats group compared with the Non-D rats. **p < 0.01 and ***p < 0.001, compared to Non-D rats. All data are shown as mean ± SD.

Effects of HIIT, TRF, and metformin on the liver signaling pathway AKT, FOXO1, PEPCK Western blotting

AKT decreased significantly in the D-NT group (p = 0.001) compared to Non-D rats, and increased significantly in D-MET rats compared with D-NT rats (p = 0.007). There was no significant difference in AKT content in the liver tissue of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercised groups (effect size = 0.21, p = 0.17) (Non-D, D-NT and D-MET), and there was no significant difference in AKT in the rats treated with TRF (D-TRF and D-TRF&EX) compared to the non-TRF groups (effect size = 0.25, p = 0.13). Additionally, there were no interactions between exercise and TRF (effect size = 0.19, p = 0.19) (Fig. 4 A, and 4B) (Table 2).

Fig. 4.

The effects of HIIT, TRF, and Metformin on the liver signaling pathway AKT, FOXO1, PEPCK, Western blotting relative to β-Actin. (A) Western blot analysis of the proteins. (B) Western blot analysis of liver AKT levels in T2DM rats. (C) Western blot analysis of liver FOXO1 levels in type T2DM rats. (D) Western blot analysis of liver PEPCK levels in T2DM rats. All data are shown as mean ± SD, n = 7 per group. AKT, protein kinase B; FOXO1, forkhead box protein O1; PEPCK, phosphoenolpyruvate carboxykinase; D, diabetes; EX, exercise; TRF, time-restricted feeding.

FOXO1 was significantly increased in the D-NT rats compared to non-Diabetic rats (p = 0.01), and was significantly decreased in D-MET rats compared to D-NT rats (p < 0.001). There was no significant difference in FOXO1 content in the liver tissue of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercise groups (effect size = 0.39, p = 0.05) (Non-D, D-NT and D-MET), and there was a significant decrease in FOXO1 in the D-TRF rats compared to the non-TRF groups (effect size = 0.58, p = 0.01) with a moderate effect. There were no interactions between exercise and TRF (effect size = 0.001, p = 0.95) (Fig. 4 A, and 4 C) (Table 2).

PEPCK increased significantly in the D-NT rats as compared to the non-Diabetic rats (p < 0.001), and decreased significantly (p = 0.008) in the D-MET rats compared to D-NT rats. A significant decrease in the PEPCK content in the liver tissue of the exercised rats (D-EX and D-TRF&EX) compared to the non-exercised groups (effect size = 0.88, p < 0.001) (Non-D, D-NT and D-MET), with a large effect size and there was a significant decrease in PEPCK in the D-TRF compared to the non-TRF groups (effect size = 0.81, p < 0.001). Additionally, there was a significant interaction between exercise and TRF (effect size = 0.69, p = 0.003). Details of all results related to AKT/FOXO1/PEPCK protein content are shown in (Fig. 4 A, and 4D) (Table 2).

Changes in liver glycogen profiles (PAS)

Changes related to liver glycogen content for all groups are shown in Fig. 4. Injection of STZ and induction of diabetes, significantly decreased liver glycogen storage in the D-NT group compared to the Non-D group. Treatment of rats with exercise and TRF (D-EX, D-TRF and D-TRF&EX) led increased glycogen storage compared to the D-NT rats. While the glycogen reserves remained significantly higher in the rats of the (D-TRF&EX) group. Finally, analysis of liver sections showed large areas of cytosolic liver glycogen in treatment groups. Details are given in (Fig. 5 A, and 5B).

Fig. 5.

Low and high magnification photomicrographs for Periodic Acid Schiff (PAS), and Sudan-Black-B (SBB) staining of hepatic cross-sections. A) Intensive intracytoplasmic carbohydrate storage is presented close to the portal area (PAS staining, thin arrows) and faint black reactions (representing intacytoplasmic lipid foci) are detectable in the SBB-stained sections. Faint PAS reactions and micro and macro steatosis (black reactions in SBB staining, presented with white head arrows). Better PAS reactions and lower SBB-positive reactions were revealed. Note the cross-section of the D-TRF&EX representing lipid foci accumulation in the hepatocyte’s cytoplasm, representing a similar phenotype to the D-NT group. B) The histopathological changes in the cross-sections from diabetes groups versus the cross-sections from the D-NT group: note the intensive congestion (arrowhead) with mild inflammatory cells infiltration and fibrosis in the perivascular congestion area in the first left-hand-side section. Intensive mononuclear immune cells infiltration (ICI), apoptotic hepatocytes (arrowheads) are presented in the up right-hand- side photo. Hepatocytes focal and zone necrosis (N) with micro steatosis is presented in the left-hand-side photo. Note: severe sinusoidal congestion (headarrows) and distributed immune cells infiltration in the photo in the right-hand-side below.

Sudan Black-B (SBB) staining

Sudan Black-B (SBB) staining showed faint black reactions in the hepatocytes of the cross-sections from the Non-D group. Most faint reactions were confined to the cytoplasm of hepatocytes distributed in the portal area. However, in the (D-NT) and (D-TRF&EX) groups, the hepatic acinus—the region between a central vein and a nearby portal triad—was clearly demarcated, showing more intense staining in the zones closer to the central vein. The D-EX, D-MET, and D-TRF groups exhibited similar phenotypes to the Non-D group. Details are given in (Fig. 5 A, and 5B).

Descriptions

Histopathological analyses revealed normal hepatocytes, bile ducts, sinusoids, central veins, and hepatocyte orientation in the cross-sections of the Non-D. In contrast, the cross-sections from the diabetes-only (D-NT) group showed severe mononuclear immune cell infiltration, ballooning of hepatocytes, and both focal and zonal necrosis. Additionally, microvascular and macro vascular steatosis were observed in the hepatocytes of the diabetic livers. Cross-sections from diabetes-induced groups exhibited varying degrees of lymphocyte infiltration (both local and zonal), periportal and per sinusoidal fibrosis, sinusoidal dilation, and congestion. All these histopathological changes were reduced in the cross-sections of the treated groups.

Periodic Acid-Schiff (PAS) staining revealed intact intracytoplasmic carbohydrate storage in the hepatocytes near the central vein in cross-sections from the control and exercise-only groups. In contrast, the hepatocytes from the D-EX and D-NT groups exhibited a significant reduction in intracytoplasmic carbohydrate storage, particularly close to the central vein. The D-TRF&EX group showed vacuoles and steatosis in the PAS staining. The D-MET and D-TRF groups demonstrated increased PAS reactions (indicating intracytoplasmic carbohydrate storage) compared to the D-NT rats.

Sudan Black-B (SBB) staining showed faint black reactions in the hepatocytes of the cross-sections from the Non-D. Most faint reactions were confined to the cytoplasm of hepatocytes distributed in the portal area. However, in the diabetes-only (D-NT) and D-TRF&EX groups, the hepatic acinus the region between a central vein and a nearby portal triad—was clearly demarcated, showing more intense staining in the zones closer to the central vein. The D-EX, D-MET, and D-TRF groups exhibited similar phenotypes to the Non-D.

Discussion

The present study investigated the role of different HIIT and TRF interventions on the liver tissue of STZ-induced diabetic Wistar rats. The AKT/FOXO1/PEPCK pathway is critical in glucose homeostasis, primarily regulating hepatic gluconeogenesis and insulin signaling. According to the current results, HFD-fed rats had metabolic disturbances, including increased fasting blood glucose (FBG), insulin, abnormal glucose tolerance, and increased HOMA-IR. In addition, HFD-fed rats had impaired hepatic glycogen synthesis, which was characterized by increased gluconeogenesis through increased protein expression (FOXO1/and PEPCK) and decreased AKT expression^33,34.

In line with the present study, several previous animal studies have shown that intense endurance training on a treadmill significantly improved FBG levels and insulin resistance (IR) index compared to a D-NT group^35–37. Considering that in the treatment groups of the current study, insulin levels also increased, it may be that improvements in blood glucose levels are related to the secretion of insulin. Because in the D-NT group, the insulin level is disturbed^38,39. In our study, according to the glycogen level obtained, it can be hypothesized that the treatments stimulate glucose absorption, especially via the liver, and this increase in glucose absorption causes a decrease in blood glucose levels. In insulin target cells, multiple genes are involved in insulin signaling pathways, including protein kinase B (PKB/AKT2), insulin receptor substrate 2 (IRS2), FOXO1 and PEPCK. Based on the current results, with HFD feeding, the expression of FOXO1 and PEPCK proteins increased, and the expression of AKT2 decreased in diabetic groups⁴⁰. In contrast, in the intervention groups, glycogen synthesis was maintained. In HIIT and TRF treated groups, following changes in insulin levels, the expression of gluconeogenesis regulators (FOXO1, and PEPCK) decreased, which could indicate excess glucose storage in the form of glycogen in the liver. The decrease in blood glucose could be due to the increase in AKT pathway activity and suppression of PEPCK expression, which was also confirmed by Western blot results.

HIIT affects the AKT/FOXO1/PEPCK pathway through several complex mechanisms. HIIT increases protein kinase B (AKT) activation and FOXO1 phosphorylation, which inhibits FOXO1 activity and reduces the expression of PEPCK, a key enzyme in hepatic gluconeogenesis. These changes contribute to reduced hepatic glucose production and improved glycemic control⁴¹. In addition, HIIT increases insulin sensitivity and facilitates glucose uptake by increasing glucose transporter type 4 (GLUT4) in skeletal muscle⁴². HIIT can also facilitate better functioning of insulin signaling pathways by reducing oxidative stress and inflammation. Thus, by affecting these pathways, HIIT improves glucose regulation and reduces IR in diabetes⁴³.

In a study on the efficacy of TRF against hepatic gluconeogenesis activity in obese rats, it was shown that TRF reversed glucose intolerance, hyperglycemia, and IR induced by HFD in obese rats. In rats, where highly controlled feeding and exercise are possible, TRF protects against hyperglycemia and improves hepatic gluconeogenic activity. As a result, TRF may be an effective strategy against hyperglycemia caused by obesity, diabetes, and IR⁴⁴. In the current study, in the D-TRF group with access to the same HFD, significant weight loss was observed, followed by an improvement in IR which is consistent with the previous study. Several studies have confirmed the reducing effects of exercise training on blood glucose, IR, as well as the increase in insulin in diabetic patients^45–47.

HIIT also led to improvements in diabetic conditions in the current study, which is consistent with previously published studies^48,49. The current results showed that in rats of TRF&EX group, the effects of HFD feeding on the insulin signaling pathway were mitigated. TRF&EX combined, has several advantages over TRF or EX alone, and can reduce the harmful effects of HFD on liver metabolism and glycemic homeostasis in rats. TRF has been shown to increase insulin sensitivity in diabetic rats, allowing cells to respond better to insulin and take up more glucose from the bloodstream, thereby lowering blood glucose levels. However, TRF and HIIT combined (D-TRF&EX) increased liver glycogen content in STZ diabetic rats⁵⁰. The amount of glycogen in the liver is a good indicator to clarify the effectiveness of treatment with a hypoglycemic agent or strategy. The secretion and activity of insulin leads to the accumulation of tissue glycogen⁵¹.

The present study showed a significant decrease in the expression of FOXO1, and PEPCK proteins in the liver of diabetic rats as compared to the D-NT group following HIIT and TRF interventions. Decreases in glucose and IR in diabetic rats in response to HIIT and TRF in the present study may be attributed to decreases in the expression of FOXO1, and PEPCK proteins in the liver tissue. The present study shows that HIIT and TRF interventions reduced fasting hyperglycemia and improved insulin by reducing the expression of PGC- 1α and PEPCK proteins, indicating an important signaling pathway that can control glucose synthesis in the liver. With phosphorylation, AKT decreases PGC- 1α and subsequently PEPCK, and plays an important role in suppressing hepatic gluconeogenesis. These results are in line with other studies^52–54. Targeting the components in the gluconeogenic pathway seems to improve hyperglycemia. Although the benefits of exercise and fasting on metabolism are significant, effects are short-term. The combined effect of HIIT and TRF is superior to either HIIT or TRF alone, and therefore should be considered when determining optimal adjuvant therapies for T2DM.

As previously stated, in this study we used HOMA-IR and QUICKI to evaluate insulin sensitivity. The present study suggests that HIIT may improve insulin sensitivity in the liver tissue of diabetic rats by enhancing the AKT/FOXO1 pathway and possibly reducing gluconeogenesis activity. The improvement in QUICKI in this study seems to emphasize the importance of combining HIIT and TRF in regulating metabolism. The exact mechanisms underlying this effect are unclear, but may include improvements in hepatic glucose uptake, reduced inflammation, and increased mitochondrial function^20,21. The results of our study are consistent with previous studies^55,56.

The effect of TRF on body weight and physiological functions has been extensively studied in rodent models, which have shown considerable therapeutic effects for TRF⁵⁷. There are many possible explanations for the effects of TRF on the AKT/FOXO1/PEPCK signaling pathway in the liver of diabetic rats: (1) The AKT/FOXO1/PEPCK pathway is regulated by the circadian clock and the maximum expression of these genes occurs during the day. TRF may help maintain or restore the normal circadian regulation of this pathway, leading to improved glucose metabolism⁵⁸; (2) Limiting the amount of feeding time may reduce oxidative stress in the liver⁵⁹; (3) Autophagy may be increased and cell homeostasis maintained when oxidative stress is reduced⁶⁰; (4) Modulation of epigenetic markers (histone changes and DNA methylation) may occur following TRF⁶¹; (5) Changes in the intestinal microbiome may improve glucose metabolism⁶²; (6) Inhibition of the mTOR signaling pathway may occur during TRF⁶³(7) TRF may increase the activity of AMPK⁶⁴; (8) TRF may reduce inflammation in the liver⁶⁵; and (9) TRF may result in negative energy balance, leading to changes in liver glucose and lipid metabolism⁶⁶. These mechanisms are not mutually exclusive, and it is likely that multiple mechanisms are involved in the effects of TRF on the AKT/FOXO1/PEPCK signaling pathway. Despite the observed effects, further investigation is required to elucidate the underlying mechanisms. It is also important to consider that individual variability may play a significant role, as TRF may influence distinct mechanisms in each person. This could depend on factors such as age, biological sex, dietary habits, and overall health status⁶⁷.

In the present study, we also had a group for treatment with metformin. A study on diabetic rats in the past showed that the level of TG, blood glucose and HbA1c in the diabetes + exercise + metformin group was lower compared to the diabetic group. (Effect of High Intensity Interval Training with Metformin on Lipid Profiles and HbA1c in Diabetic Rats), which is consistent with the results of our study. The findings of the study showed that diabetes caused by HFD increases the expression of FOXO1 and HIIT can inhibit these increases, but the reducing effect of metformin on the expression of this gene was not significant and the combined effect of HIIT and metformin on the expression of this gene was greater than the effect there was no HIIT person. These results indicated that HIIT (but not metformin) may prevent the down regulation of FOXO1 induced by T2DM and that metformin could not influence the effect of exercise on this atrophied gene. In one study, exercise compared to metformin alone showed more improvement in blood glucose control, liver diacylglycerol content, liver mitochondrial palmitate oxidation, citrate synthase activity, and attenuation of liver fatty acid markers.

It seems that other factors may explain the effects of HIIT and TRF on metabolic health and function. HIIT and TRF can induce epigenetic changes that help to fine-tune the AKT/FOXO1/PEPCK pathway⁶⁸. These changes may include DNA methylation and histone modifications that affect the activation or inhibition of these pathways. HIIT, due to its high intensity, may increase free radical production, which, if properly managed, can help promote cellular adaptation and improve metabolic function. This oxidative stress can affect the activation of the AKT/FOXO1 pathway and the regulation of PEPCK. TRF is usually associated with changes in the timing of food intake, which can help to better regulate circadian rhythms and metabolic activity. These changes can lead to the regulation of signaling pathways such as AKT/FOXO1/PEPCK at the molecular level, which leads to improved glycemic control and metabolic function in diabetic rats^58,69. HIIT and TRF may influence systemic inflammation by modulating key cytokines such as TNF-α and IL- 6^70,71. HIIT has been shown to reduce chronic inflammation by suppressing TNF-α and increasing anti-inflammatory cytokines, while TRF may regulate inflammation by aligning metabolic processes with circadian rhythms^72,73. HIIT and TRF can affect lipid profiles by reducing triglycerides and improving cholesterol balance. HIIT enhances lipid oxidation and increases HDL levels, while TRF optimizes lipid metabolism by modulating insulin sensitivity and hepatic lipid turnover⁷⁴.

Therefore, the results showed that these interventions simultaneously had significant effects on metabolic indices and related signaling pathways. The combination of these two interventions (HIIT and TRF) likely had synergistic effects on improving metabolic parameters and molecular pathways. However, further studies are recommended to confirm these results and to more precisely understand the mechanisms involved.

Future studies should explore the role of epigenetic mechanisms in mediating the effects of HIIT and TRF on hepatic glucose metabolism. Investigating histone modifications and DNA methylation patterns associated with the AKT/FOXO1/PEPCK pathway could provide deeper insights into the long-term metabolic adaptations induced by these interventions. Additionally, transcriptomic and proteomic analyses could help identify novel gene expression changes that contribute to improved insulin sensitivity and glucose regulation.

Another promising area of research is the effect of TRF and HIIT on gut microbiota composition and function. Since the gut microbiome plays a crucial role in metabolic homeostasis, studying changes in microbial diversity and metabolite production following these interventions could reveal potential microbiota-mediated mechanisms underlying the observed metabolic improvements. Metagenomic and metabolomic studies could further elucidate how TRF and HIIT influence gut-liver axis interactions in diabetic models. Moreover, while our study provides valuable insights using a rodent model, translating these findings to humans is critical. Future clinical studies should examine the effects of HIIT and TRF in individuals with T2DM to assess their efficacy, safety, and feasibility in real-world settings. Investigating the role of biological factors such as age, sex, genetic predisposition, and baseline metabolic status could help personalize these interventions for better clinical outcomes.

Additionally, mechanistic studies exploring how HIIT and TRF interact with other key metabolic pathways, such as the AMPK/mTOR and PGC- 1α signaling pathways, could provide a more comprehensive understanding of their therapeutic potential. Examining inflammatory markers and oxidative stress responses will also be essential for determining the broader physiological benefits of these interventions beyond glucose metabolism.

Finally, given the potential circadian regulation of the AKT/FOXO1/PEPCK pathway, future research should assess the optimal timing of TRF and HIIT to optimize metabolic benefits. Chrononutrition studies investigating how meal timing and exercise scheduling influence hepatic glucose metabolism could offer new strategies for diabetes management.

Conclusion

The present study demonstrated that HIIT and TRF interventions positively affect glucose homeostasis by modulating the AKT/FOXO1/PEPCK pathway in the liver tissue of STZ-induced diabetic Wistar rats. The combination of these interventions resulted in significant improvements in insulin sensitivity, glycogen synthesis, and the suppression of hepatic gluconeogenesis. Our findings highlight the potential for integrating HIIT and TRF as an effective non-pharmacological strategy to manage diabetes and metabolic disorders. However, several key areas require further investigation to fully elucidate the mechanisms and broader implications of these findings.

While our study highlights the benefits of HIIT and TRF on glucose regulation, further research is needed to elucidate the underlying mechanisms and validate these findings in human populations. Integrating epigenetic, microbiome, and clinical research will be crucial in advancing our understanding of how these interventions can be effectively applied to prevent and manage metabolic diseases.

Electronic supplementary material

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Author contributions

F. SH.: Experimental design, collection and/or assembly of data, data analysis, interpretation and manuscript writing.F. K.: Experimental design, interpretation and manuscript writing and final approval of manuscript.M. GH.: Manuscript writing and final approval of manuscript.K. G.: Manuscript writing and final approval of manuscript.S. KR.: Manuscript writing and final approval of manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

None of the authors has any conflicts of interest to disclose and all authors support submission to this journal.