Insulin resistance is one major features of type 2 diabetes mellitus (T2DM). Deuterohemin-βAla-His-Thr-Val-Glu-Lys (DhHP-6), a novel microperoxidase mimetic designed and synthesized based on microperoxidase 11 (MP-11), can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we showed that oral DhHP-6 could reduce blood glucose and improve insulin resistance. To investigate the mechanisms of how DhHP-6 ameliorates oxidative stress and insulin resistance, we established T2DM mouse models and glucosamine-induced HepG2 cell insulin resistance models. The results suggested that DhHP-6 decreased blood glucose, increased antioxidant enzyme activity, and inhibited glycogen synthesis in T2DM mice. In addition, DhHP-6 improved insulin resistance by activating phosphatidylinositol 3-kinase (PI3K)/AKT, and AMP-activated protein kinase (AMPK) pathway in T2DM mice. Furthermore, DhHP-6 also activated PI3K/AKT and AMPK pathway in glucosamine-induced HepG2 cells. However, LY294002 did not completely inhibit AKT phosphorylation, and partially inhibited AMPK phosphorylation, whilst compound C only partially reduced AMPK phosphorylation, and also partially inhibited AKT phosphorylation, suggesting that AKT and AMPK interact to improve insulin resistance. Thus, these data suggest that DhHP-6 attenuates insulin resistance via the PI3K/AKT and AMPK pathway.

Introduction

Type 2 diabetes mellitus (T2DM) is a common multifactorial metabolic disease. T2DM has now developed into one of the largest public health problems globally [1]. The numbers suffering from diabetes according to the International Diabetes Federation (IDF) report published in 2013 was 387 million, which is expected to rise to 592 million by 2035 [2]. T2DM accounts for 90–95% of all diabetic patients, and is one of the top five leading causes of death globally [3].

Insulin resistance in target tissues, a characteristic feature of T2DM, leads to disorders in gluconeogenesis and glycolysis that lead to hyperglycemia, due to a loss of phosphatidylinositol (PI3K)/AKT signaling [4–6]. Excessive levels of nutrients induce oxidative stress pathways, including mitochondrial oxidative stress and endoplasmic reticulum (ER) stress [7,8]. Accumulating evidence indicates that oxidative stress induced by imbalances between reactive oxygen species (ROS) generation and antioxidant defenses contributes to the pathogenesis of T2DM due to the loss of β-cell function and an impaired sensitivity of peripheral tissues to insulin [9–11]. ROS production in diabetes impairs PI3K/AKT and insulin signaling [4,5]. Oxidative stress promotes the degradation of IRS-1 due to the activation of the ubiquitin-dependent proteolytic pathway [12,13]. Insulin resistance leads to impaired metabolic homeostasis due to dysfunctional AMP-activated protein kinase (AMPK) signaling [14]. AMPK plays a central role in energy balance [15,16] and controls glucose homeostasis by inhibiting the expression and activity of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate kinase (PEPCK), key enzymes of gluconeogenesis [15,17]. Studies have shown that oxidative stress regulates AMPK activity through oxidation of cysteine residues [18,19]. Thus, the supplementation of exogenous microperoxidases that eliminate ROS can improve insulin resistance in T2DM.

NF-E2-related factor 2 (Nrf2) contributes to the regulation of antioxidant signaling and can maintain redox homeostasis [20]. Kelch-like ECH-associated protein 1 (Keap1) binds to- and prevents Nrf2 translocating to the nucleus, thus inhibiting the transcription of antioxidant genes [21,22]. Cysteine residues in Keap1 are modified by ROS and free Nrf2 regulates phase detoxification enzymes [23,24]. AMPK leads to sequestration and lysosomal degradation of Keap1 and thus to the accumulation of Nrf2 [25]. Nrf2 inducers enhance the phosphorylation of AMPK and increase glucose uptake while suppressing glucose production in liver, indicating that the accumulation of Nrf2 enhances AMPK phosphorylation to suppress the onset and/or progression of insulin resistance in the liver [26,27]. AKT activation can increase Nrf2 expression by phosphorylating specific sites to release Nrf2 from Keap1 [21,28]. The absence of Nrf2 leads to a loss of PI3K/AKT signaling [21]. The Nrf2-Keap1 axis, therefore, plays a key role in improving oxidative stress and insulin resistance in T2DM.

Deuterohemin-βAla-His-Thr-Val-Glu-Lys (DhHP-6) is a mimic enzyme of microperoxidase-11 (MP-11) [29] that shows antioxidant properties due to the presence of a deuterohemin group. The linear peptide AHTVEK is highly conserved in MP, the solubility of which is enhanced by APx [29,30]. DhHP-6 extends the lifespan of Caenorhabditis elegans (C. elegans) by scavenging ROS [31,32]. DhHP-6 also ameliorates the hallmarks of Alzheimer's disease (AD) in APPswe/PSEN1dE9 transgenic mouse models [33]. When injected, DhHP-3 (deuterohemin-Ala-His-Lys) can reduce fasting blood glucose (FBG) levels in T2DM rat models [34]. Although we demonstrated that oral DhHP-6 lowers blood glucose and improves insulin resistance by oral glucose tolerance tests (OGTT) and insulin resistance tests (ITT) in T2DM mouse models, the mechanisms governing these effects remained undefined [35].

In this study, to further understand the mechanisms by which DhHP-6 improves hepatic insulin resistance in T2DM, we further examined the effects of DhHP-6 on redox status, glucose metabolism, glycogen synthesis, PI3K/AKT and AMPK pathway in T2DM mice. We also examined the effect of DhHP-6 on HepG2 cell insulin resistance models. DhHP-6 improved antioxidant activity and promoted glycogen synthesis in T2DM mice. Besides, DhHP-6 activated PI3K/AKT and AMPK pathway in T2DM mice. In addition, DhHP-6 inhibited ROS production and restored mitochondrial respiratory dysfunction in glucosamine-induced HepG2 cells. Furthermore, DhHP-6 activated Nrf2-Keap1, PI3K/AKT and AMPK in vitro. Taken together, DhHP-6 attenuates hepatic oxidative stress and insulin resistance via the PI3K/AKT and AMPK pathway.

Materials and methods

Materials

HepG2 cells were cultured in Dulbecco's modified Eagle medium (DMEM), penicillin and streptomycin. Fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz Beit — Haemek, Israel). Primary antibodies included anti-IRS-1 (#2390), anti-phospho-IRS-1 (Ser307) (#2381), anti-Keap1 (#8047), anti-Nrf2 (#12721), anti-phosphoPlus®AMPKα (Thr172) (#8208) (Cell Signaling, Beverly, MA, U.S.A.); anti-AKT (sc-81434), anti-p-AKT1/2/3 (sc-81433), anti-GSK-3β (sc-377213), anti-p-GSK-3β (sc-373800), anti-PEPCK (sc-377136) (Santa Cruz Biotechnology, Inc, Texas, U.S.A.); anti-phospho-IRS-1 (Tyr632)/HRP (bs-8707R-HRP), and mouse anti-beta-Actin Monoclonal Antibodies (bsm-33139M) (Bioss, Beijing, China). Secondary antibodies included anti-rabbit IgG, HRP-conjugated antibodies (#7074) (Cell Signaling, Beverly, MA, U.S.A.), and rabbit anti-mouse IgG H&L (HRP) (ab6728) (Abcam, Cambridge, U.K.).

T2DM mouse model and DhHP-6 treatment

In the present paper, we further examined the effect of DhHP-6 on insulin resistance in T2DM mice. The animal samples were collected in our previous experiment. Our previous animal experiment procedures are as followed [35,36]: After a week of adjustable feeding in Core Facilities for Life Science, Jilin University, male C57BL/6L mice (weight, 20 ± 2 g) were randomly divided into two groups: group 1: normal diet (ND group); group 2: fed a high-fat diet for 4 weeks (HFD group). Mice in the HFD group were intraperitoneally injected with STZ (100 mg/kg body weight). When the FBG levels of the HFD group were ≥11.2 mmol/l and the plasma insulin concentration was significantly higher than that in the control group, mice were considered as T2DM. Besides, we also justify the T2DM mice model by oral glucose tolerance and ITT before DhHP-6 treatment.

T2DM mice were randomly divided into three groups (n = 8 in each group): and eight normal mice in the ND group were used as controls. The groups were treated as follows: (1) control group administered physiological saline; (2) Type 2 diabetes model group administered physiological saline; (3) low-dose group administered DhHP-6 (20 mg/kg body weight); (4) high-dose group administered DhHP-6 (60 mg/kg body weight). All groups were orally administered equivalent drug volumes for 4 weeks. All animal experimental procedures conformed to the Animal Ethical Standards and Use Committee at Jilin University (The Animal Care Committee of Jilin University (License No.: 20160518)).

Serum sample preparation and determination

At the end of the experimental protocol, blood samples were collected from the eye socket and centrifuged at 1000 g for 15 min for serum extraction. Serum G6Pase, fructose-bisphosphatase (FBPase) and Hexokinase (HK) activity were determined using mouse insulin ELISA kits (Bioss Biotechnology Co., Ltd, Beijing, China). Serum glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) were determined using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China).

Histochemistry

At the end of the experiment, mice were sacrificed by neck brace and the livers were immediately removed and fixed in 4% paraformaldehyde (Solarbio & Technology Co., Ltd, Beijing, China). After dehydration in an ethanol gradient, tissues were paraffin embedded and sectioned (5 μm thick). Sections were stained in periodic acid–Schiff (PAS) dye and mounted for microscopic examination using an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan).

Hepg2 cell culture and treatment

Human hepatocellular carcinoma cells (HepG2) were cultured in DMEM containing 10% FBS, 1% penicillin–streptomycin, and 2 mM l-glutamine at 37°C in a humidified atmosphere (5% CO2). Glucosamine-induced HepG2 cells were considered to be the cell insulin resistance model [36–39]. Glucosamine affects the regulation of ROS production, mitochondrial function and glucose absorption, and then induces insulin resistance [37,38]. To establish the insulin resistance model, HepG2 cells were incubated with 18 mM glucosamine for 18 h

Cell viability

Cell viability was assessed through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. HepG2 cells were seeded into 96-well plates at a density of 2 × 104 cells/well and cultured overnight. Insulin-resistant HepG2 cells were established and treated with various concentrations of DhHP-6 for 24 h. MTT reagent (10 μl) was then added to each well for 4 h at 37°C. Media was then removed and 150 μl of DMSO was added to each well to dissolve the formazan crystals. OD values were measured using a microplate reader at 570 nm.

Glycogen content assay

HepG2 cells were seeded into six-well plates at a density of 4 × 105 cells/well overnight and the insulin resistance model was established. HepG2 cells were incubated with varying concentration of DhHP-6 in DMEM for 24 h and glycogen content was analyzed using commercially available kits (Solarbio & Technology Co., Ltd, Beijing, China).

Glucose uptake assay

HepG2 cells were seeded into six-well plates at a density of 4 × 105 cells/well overnight and the insulin resistance model was established. HepG2 cells were incubated with varying concentration of DhHP-6 in DMEM for 24 h. Cells were then treated with 100 nM insulin in serum and phenol-red free DMEM for 15 min and glucose content in the supernatants and original DMEM were measured at 570 nm using enzyme standards from glucose diagnostic kits (Solarbio & Technology Co., Ltd, Beijing, China). Glucose content in the cells was subtracted from blank wells to measure glucose uptake [37,40]. We also examined glucose production in glucosamine-induced HepG2 cells in glucose production buffer, and the glucose content was undetectable.

Reactive oxygen species (ROS) assays

HepG2 cells were incubated with varying concentrations of DhHP-6 in DMEM for 24 h and treated with 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (Solarbio & Technology Co., Ltd, Beijing, China) for 30 min at 37°C. Cells were then washed three times in DMEM, and fluorescence intensities were analyzed at excitation and emission wavelengths of 488 and 535 nm using an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) [34].

Mitochondrial respiration

Oxygen consumption rates (OCR) were analyzed using an XF-96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA). Cells were seeded at a density of 5000 cells/well in 96-well plates and insulin resistance models were established, HepG2 cells were incubated with varying concentrations of DhHP-6 in DMEM for 24 h and OCR was measured in basal medium and after the sequential addition of 2.4 µM oligomycin, 0.3 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.6 µM antimycin A (AA)/rotenone (Rot). Basal respiration, spare respiratory capacity, proton leak and ATP production were determined [41–45]. Briefly, the OCR after AA/Rot was considered as the non-mitochondrial OCR. Basal OCR was calculated as the OCR prior to oligomycin minus non-mitochondrial OCR. ATP production was calculated as the basal OCR minus the OCR prior to FCCP treatment. Maximal OCR was calculated as the OCR after AA/ Rot minus non-mitochondrial OCR. Spare capacity was calculated as the maximal OCR after FCCP minus basal OCR. Proton leak was calculated as the OCR prior to FCCP minus the non-mitochondrial OCR.

Western blot analysis

Total proteins (20 μg) extracted from cell and liver tissue lysates were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked with skimmed milk (5% in tris-buffered saline containing 0.5% Tween-20) at room temperature for 2 h and probed with primary antibodies overnight at 4°C. Membranes were washed and labeled with secondary antibodies (horseradish peroxidase [HRP]-conjugated anti-rabbit immunoglobulin G (IgG), anti-mouse IgG) for 2 h at room temperature. Bands were detected using the West Pico ECL Substrate (Solarbio & Technology Co., Ltd, Beijing, China), and visualized using the Tanon 5200 Gel Imaging System (Biotanon Co., Ltd, Shanghai, China). Band-density quantifications were normalized to β-actin.

Statistical analysis

Data are the mean ± standard deviation (SD). Statistical analysis was performed using a one-way ANOVA with LSD post-hoc test. Differences were considered statistically significant if p < 0.05.

Results

DhHP-6 restores redox status and activates the Nrf2-Keap1 axis in T2DM mice

DhHP-6 can scavenge ROS in vivo [34]. To examine the effects of DhHP-6 on oxidative stress in T2DM mice, endogenous enzymes (including GSH, SOD, CAT) that improve oxidative stress by catalyzing antioxidant reactions were examined. Compared with the control group, the serum levels of GSH, SOD, CAT (Figure 1A–C) in the control group were higher than those of the T2DM model group, which were restored after treatment with low- and high-doses of DhHP-6. To further estimate oxidative stress levels, serum levels of MDA, a product of lipid peroxidation, was determined. MDA in the T2DM model group increased compared with that of the control group (Figure 1D). DhHP-6 at concentrations of 20 and 60 mg/kg reduced MDA levels in T2DM mice. GSH, SOD, CAT are downstream targets of Nrf2 [24]. Under stress conditions, Nrf2 was activated through uncoupling from its native repressor Kelch-like ECH-associated protein 1 (Keap1) [28]. The expression of Keap1 and Nrf2 were, therefore, examined in T2DM mice. Keap1 levels increased, whilst Nrf2 levels decreased in T2DM mice, which was reversed by DhHP-6 in a dose-dependent manner (Figure 1E,F). These data suggested that DhHP-6 restored redox status by activating the Nrf2-Keap1 system in T2DM mice.

DhHP-6 improves antioxidant status and activates the Nrf2-Keap1 axis in T2DM mice.

Figure 1.
DhHP-6 improves antioxidant status and activates the Nrf2-Keap1 axis in T2DM mice.

(AD) SOD, GSH, CAT and MDA in serum were measured. (E) Nrf2 and Keap1 expression were examined by western blot analysis. The levels of Nrf2 (F) and Keap1 (G) was analyzed using Image J. All data are the means ± SD, n = 8. ***p < 0.001 vs. control group. #p < 0.05, ###p < 0.001 vs. model group.

Figure 1.
DhHP-6 improves antioxidant status and activates the Nrf2-Keap1 axis in T2DM mice.

(AD) SOD, GSH, CAT and MDA in serum were measured. (E) Nrf2 and Keap1 expression were examined by western blot analysis. The levels of Nrf2 (F) and Keap1 (G) was analyzed using Image J. All data are the means ± SD, n = 8. ***p < 0.001 vs. control group. #p < 0.05, ###p < 0.001 vs. model group.

DhHP-6 improves glucose metabolism in T2DM mice

G6Pase and FBPase are two key enzymes of gluconeogenesis that are associated with elevated blood glucose. HK is the key to glycolysis and reduces blood glucose levels [46]. To investigate the effects of DhHP-6 on hepatic gluconeogenesis, the activity of these enzymes were assessed. Compared with the control group, the expression of FBPase and G6pase (Figure 2A,B) in T2DM mice significantly increased, which were restored after treatment with DhHP-6 at 20 and 60 mg/kg. The expression of HK (Figure 2C) decreased in T2DM mice, whilst treatment with DhHP-6 increased the expression of HK. These results suggest that DhHP-6 improves glucose metabolism by regulating enzymes linked to gluconeogenesis and glycolysis.

Effects of DhHP-6 on glucose metabolism in T2DM mice.

Figure 2.
Effects of DhHP-6 on glucose metabolism in T2DM mice.

(AC) FBPase, G6Pase and HK contents in liver homogenates were analyzed by ELISA. Data are the mean ± SD, n = 8. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

Figure 2.
Effects of DhHP-6 on glucose metabolism in T2DM mice.

(AC) FBPase, G6Pase and HK contents in liver homogenates were analyzed by ELISA. Data are the mean ± SD, n = 8. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 promotes glycogen synthesis in T2DM mice

To further study the effects of DhHP-6 on glycogen storage in T2DM mice, liver glycogen levels were determined through PAS staining. In the liver sections, glycogen was stained red and cell nuclei were stained blue. Compared with the control group, liver cells in the T2DM mice showed low levels of glycogen storage and micro- and macrovesicular lipid infiltration in the cytoplasm (Figure 3A,B). In contrast, the liver cells of DhHP-6 treated groups, particularly at concentrations of 60 mg/kg exhibited lower levels of vesicular lipid infiltration and higher levels of glycogen storage compared with T2DM mice, consistent with its effects on glucose metabolism. These results demonstrate that DhHP-6 enhances glycogen synthesis in T2DM mice.

DhHP-6 ameliorates glycogen synthesis in T2DM mice.

Figure 3.
DhHP-6 ameliorates glycogen synthesis in T2DM mice.

(A) Glycogen content in hepatic tissue analyzed by PAS staining. (B) Glycogen content was analyzed using Image J. Data are the mean ± SD, n = 8. ***p < 0.001 vs. control group. ###p < 0.01 vs. model group.

Figure 3.
DhHP-6 ameliorates glycogen synthesis in T2DM mice.

(A) Glycogen content in hepatic tissue analyzed by PAS staining. (B) Glycogen content was analyzed using Image J. Data are the mean ± SD, n = 8. ***p < 0.001 vs. control group. ###p < 0.01 vs. model group.

DhHP-6 ameliorates hepatic insulin signaling in T2DM mice

Hepatic insulin resistance results from a loss of insulin signaling [47]. To investigate the effects of DhHP-6 supplementation on the impairment of insulin signaling in T2DM mice, we examined the phosphorylation of IRS-1, AKT and GSK-3β. In T2DM mice, hepatic IRS-1 phosphorylation at Ser307 (Figure 4A,B) increased, but IRS-1 phosphorylation at Tyr632 decreased (Figure 4A,C). AKT phosphorylation at Ser473 (Figure 4A,D) and GSK-3β phosphorylation at Ser9 (Figure 4A,E) also decreased, suggesting that insulin signaling was severely impaired. When treated with both 20 and 60 mg/kg body weight DhHP-6, insulin signaling recovered in a dose-dependent manner. These data suggest that DhHP-6 improved hepatic insulin signaling and reversed insulin resistance in T2DM mice.

DhHP-6 restores the impairment in insulin signaling in T2DM mice.

Figure 4.
DhHP-6 restores the impairment in insulin signaling in T2DM mice.

T2DM mice were treated with/without 20, 60 mg/kg body weight DhHP-6 for 4 weeks. (A) Effects of DhHP-6 on insulin signaling determined by western blot. Levels of IRS-1 phosphorylation at Tyr632 (B) and IRS-1 phosphorylation at Ser307 (C), AKT phosphorylation at Ser473 (D) and GSK-3β phosphorylation at Ser9 (E). Data are the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

Figure 4.
DhHP-6 restores the impairment in insulin signaling in T2DM mice.

T2DM mice were treated with/without 20, 60 mg/kg body weight DhHP-6 for 4 weeks. (A) Effects of DhHP-6 on insulin signaling determined by western blot. Levels of IRS-1 phosphorylation at Tyr632 (B) and IRS-1 phosphorylation at Ser307 (C), AKT phosphorylation at Ser473 (D) and GSK-3β phosphorylation at Ser9 (E). Data are the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 prevents gluconeogenesis by reversing the suppression of AMPK phosphorylation inT2DM mice

AMPK activation through its phosphorylation at threonine 172 regulates energy metabolism by promoting glucose absorption and inhibiting hepatic gluconeogenesis through the down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). AMPK phosphorylation also stimulates insulin signaling by phosphorylating serine 473 in AKT [48,49]. In T2DM mice, AMPK phosphorylation was markedly reduced (Figure 5A,B), but could be restored by treatment with 20 and 60 mg/kg DhHP-6. Meanwhile, the expression of G6Pase (Figure 5A,C) and PEPCK (Figure 5A,D) were elevated, and could be reduced by treatment with both 20 and 60 mg/kg DhHP-6. These results indicate that DhHP-6 inhibits hepatic gluconeogenesis partly through its effects on AMPK signaling.

DhHP-6 activates AMPK signaling in T2DM mice.

Figure 5.
DhHP-6 activates AMPK signaling in T2DM mice.

T2DM mice were treated with/without 20, 60 mg/kg DhHP-6 for 4 weeks. (A) Effects of DhHP-6 on AMPK signaling determined by western blot. Levels of AMPK phosphorylation at Thr172 (B) and G6Pase (C), PEPCK (D). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. #p < 0.05, ###p < 0.001 vs. model group.

Figure 5.
DhHP-6 activates AMPK signaling in T2DM mice.

T2DM mice were treated with/without 20, 60 mg/kg DhHP-6 for 4 weeks. (A) Effects of DhHP-6 on AMPK signaling determined by western blot. Levels of AMPK phosphorylation at Thr172 (B) and G6Pase (C), PEPCK (D). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. #p < 0.05, ###p < 0.001 vs. model group.

DhHP-6 improves cell viability, glucose uptake and glycogen synthesis in glucosamine-induced HepG2 cells insulin resistance model

To investigate the effects of DhHP-6 on insulin resistance, insulin-resistant HepG2 cell models were established [37]. As shown in Figure 6A, the cytotoxicity of DhHP-6 to HepG2 cells was first assessed. DhHP-6 promoted HepG2 cell proliferation in a concentration-dependent manner at concentrations ≤128 μM. The viability of HepG2 cells (Figure 6B), glucose uptake (Figure 6C) and glycogen synthesis (Figure 6D) significantly decreased after treatment with glucosamine for 18 h, consistent with insulin resistance. DhHP-6 also prevented glucosamine induced cytotoxicity (Figure 6E) and promoted glucose uptake (Figure 6F) and glycogen synthesis (Figure 6G) in glucosamine-induced HepG2 cells. These results suggest that DhHP-6 ameliorates insulin resistance in glucosamine-induced HepG2 cells insulin resistance model.

Establishment of HepG2 cells insulin resistance model and the effects of DhHP-6 on cell viability and glucose uptake in glucosamine-induced HepG2 cells.

Figure 6.
Establishment of HepG2 cells insulin resistance model and the effects of DhHP-6 on cell viability and glucose uptake in glucosamine-induced HepG2 cells.

(A) Viability of HepG2 cells assessed by MTT assays after treatment with various concentrations of DhHP-6 for 24 h. Cell viability (B) of HepG2 cells assessed via MTT, glucose uptake (C) and glycogen synthesis assays (D) in the presence of varying concentrations of glucosamine for 18 h. Effects of DhHP-6 on cell viability (E), glucose uptake (F) and glycogen synthesis (G) following DhHP-6 treatment in HepG2 cell insulin resistance models. Data are the mean ± SD, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

Figure 6.
Establishment of HepG2 cells insulin resistance model and the effects of DhHP-6 on cell viability and glucose uptake in glucosamine-induced HepG2 cells.

(A) Viability of HepG2 cells assessed by MTT assays after treatment with various concentrations of DhHP-6 for 24 h. Cell viability (B) of HepG2 cells assessed via MTT, glucose uptake (C) and glycogen synthesis assays (D) in the presence of varying concentrations of glucosamine for 18 h. Effects of DhHP-6 on cell viability (E), glucose uptake (F) and glycogen synthesis (G) following DhHP-6 treatment in HepG2 cell insulin resistance models. Data are the mean ± SD, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 ameliorates the loss of cellular redox status in glucosamine-induced HepG2 cells insulin resistance model

Hepatic insulin resistance results from an impairment in insulin signaling, leading to excessive ROS production [38]. To investigate whether DhHP-6 eliminates excessive ROS in glucosamine-induced HepG2 cells insulin resistance model, ROS production was determined using DCF-DA labeling. As shown in Figure 7A,B, glucosamine treatment markedly increased ROS production compared with the control group. DhHP-6 treatment also significantly lowered ROS levels at concentrations of 32 and 128 μM DhHP-6. These results suggest that DhHP-6 supplementation prevents ROS production.

DhHP-6 eliminates excessive ROS in glucosamine-induced HepG2 cells insulin resistance cells.

Figure 7.
DhHP-6 eliminates excessive ROS in glucosamine-induced HepG2 cells insulin resistance cells.

Cells were treated with the indicated concentrations of DhHP-6 for 24 h after the establishment of the insulin resistance cell model. (A) ROS production was examined by DCH-DA staining. (B) The relative fluorescence intensity was analyzed using Image J. Data are the means ± SD from three independent experiments. ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01 vs. model group.

Figure 7.
DhHP-6 eliminates excessive ROS in glucosamine-induced HepG2 cells insulin resistance cells.

Cells were treated with the indicated concentrations of DhHP-6 for 24 h after the establishment of the insulin resistance cell model. (A) ROS production was examined by DCH-DA staining. (B) The relative fluorescence intensity was analyzed using Image J. Data are the means ± SD from three independent experiments. ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01 vs. model group.

DhHP-6 improves mitochondrial respiration dysfunction in glucosamine-induced HepG2 cells insulin resistance model

To further study the effects of DhHP-6 on mitochondrial function in glucosamine-induced HepG2 cells insulin resistance model, we examined mitochondrial respiration using an XF-96 extracellular flux analyzer. Compared with the control group, basal respiration, spare respiratory capacity (Figure 8A,B), proton leak and ATP production (Figure 8A,C) were reduced in glucosamine-induced HepG2 cells, but restored after treatment with 32 and 128 μM DhHP-6. These results further confirmed that DhHP-6 ameliorates mitochondrial dysfunction.

DhHP-6 restores mitochondrial respiration dysfunction in glucosamine-induced HepG2 cells insulin resistance model.

Figure 8.
DhHP-6 restores mitochondrial respiration dysfunction in glucosamine-induced HepG2 cells insulin resistance model.

HepG2 cells were treated with different concentration of DhHP-6 for 24 h after the establishment of the insulin resistance cell model. (A) Oxygen consumption rates were examined using an XF-96 extracellular flux analyzer. Basal respiration and spare respiratory capacity (B), proton leak and ATP production (C) were analyzed according to the oxygen consumption rate. Data are the means ± SD, n = 6. **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

Figure 8.
DhHP-6 restores mitochondrial respiration dysfunction in glucosamine-induced HepG2 cells insulin resistance model.

HepG2 cells were treated with different concentration of DhHP-6 for 24 h after the establishment of the insulin resistance cell model. (A) Oxygen consumption rates were examined using an XF-96 extracellular flux analyzer. Basal respiration and spare respiratory capacity (B), proton leak and ATP production (C) were analyzed according to the oxygen consumption rate. Data are the means ± SD, n = 6. **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 activates insulin signaling in glucosamine-induced HepG2 cells insulin resistance model

To study the effects of DhHP-6 on insulin signaling in glucosamine-induced HepG2 cells insulin resistance model, we examined components of the insulin signaling pathway. As shown in Figure 9A, glucosamine supplementation led to a significant reduction of hepatic IRS-1 phosphorylation at Tyr632 (Figure 9B) and a marked increase in IRS-1 phosphorylation at Ser307 (Figure 9C), AKT phosphorylation at Ser473 (Figure 9D) and GSK-3β phosphorylation at Ser9 (Figure 9E), which recovered after treatment with 32 and 128 μM DhHP-6 in a concentration-dependent manner. Moreover, we investigated the effects of DhHP-6 on the Nrf2-Keap1 system and found that glucosamine administration reduced Nrf2 expression and increased Keap1 expression (Figure 9A,F,G). These effects could be reversed by DhHP-6 treatment. These data suggest that DhHP-6 improves insulin signaling in glucosamine-induced HepG2 cells insulin resistance model.

DhHP-6 promotes AKT phosphorylation, GSK-3β phosphorylation, Nrf2 and inhibits Keap1 in glucosamine-induced HepG2 cells insulin resistance model.

Figure 9.
DhHP-6 promotes AKT phosphorylation, GSK-3β phosphorylation, Nrf2 and inhibits Keap1 in glucosamine-induced HepG2 cells insulin resistance model.

(A) Effects of DhHP-6 on insulin signaling was determined by western blot. Levels of IRS-1 phosphorylation at Tyr632 (B) and IRS-1 phosphorylation at Ser307 (C), AKT phosphorylation at Ser473 (D), GSK-3β phosphorylation at Ser9 (E), Nrf2 (F) and Kepa1 (G). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

Figure 9.
DhHP-6 promotes AKT phosphorylation, GSK-3β phosphorylation, Nrf2 and inhibits Keap1 in glucosamine-induced HepG2 cells insulin resistance model.

(A) Effects of DhHP-6 on insulin signaling was determined by western blot. Levels of IRS-1 phosphorylation at Tyr632 (B) and IRS-1 phosphorylation at Ser307 (C), AKT phosphorylation at Ser473 (D), GSK-3β phosphorylation at Ser9 (E), Nrf2 (F) and Kepa1 (G). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 restores the impairment of insulin signaling in glucosamine-induced HepG2 cells insulin resistance model through the activation of PI3K/AKT signaling

AKT is a key therapeutic target for insulin resistance. In the liver, AKT phosphorylation increases glycogen synthesis [37]. Akt also facilitates the separation of Nrf2 from Keap1 [21,28]. Glucosamine administration led to an impairment in insulin signaling in HepG2 cells compared with the control group, yet DhHP-6 treatment increased AKT and GSK-3β phosphorylation (Figure 10A), suggesting that DhHP-6 activates PI3K/AKT signaling. Using LY294002, a specific inhibitor of PI3K, we further investigated whether DhHP-6 enhanced glycogen synthesis through the activation of insulin signaling. As is shown in Figure 10, DhHP-6 enhanced AKT phosphorylation to levels 2.10-fold higher than the glucosamine group. However, in LY294002 treated cells, DhHP-6 enhanced AKT phosphorylation by 1.43-fold compared with the glucosamine group (Figure 10A,B). In normal cells, DhHP-6 enhanced IRS-1 phosphorylation at Tyr632 and GSK-3β phosphorylation at Ser9 by 1.76-fold and 1.79-fold, respectively, but inhibited IRS-1 phosphorylation at Ser307 (2.23-fold decrease) compared with the glucosamine group. However, in LY294002 treated cells, DhHP-6 only modestly enhanced IRS-1 phosphorylation at Tyr632 (1.43-fold increase) and GSK-3β phosphorylation at Ser9 (1.60-fold) and inhibited IRS-1 phosphorylation at Ser307 (1.76-fold decrease) compared with the glucosamine group (Figure 10A,C–E). Taken together, these data indicated that other pathways exist through which DhHP-6 improves insulin resistance. Interestingly, in normal cells, DhHP-6 enhances AMPK phosphorylation by 1.83-fold, but inhibits G6Pase and PEPCK by 1.45-fold and 1.42-fold compared with the glucosamine group. However, in LY294002 pretreated cells, DhHP-6-treated groups showed higher levels of AMPK phosphorylation (1.56-fold increase) and inhibited G6Pase (1.36-fold decrease) and PEPCK (1.32-fold decrease) compared with that of the glucosamine group (Figure 10A,F–H). These results suggest that AKT inhibition also inhibits AMPK activation. DhHP-6 also increased Nrf2 expression by 1.86-fold and decreased Keap1 expression by 1.59-fold compared with the glucosamine group. In the presence of LY294002, the changes in Nrf2 and Keap1 expression (1.24-fold and 1.23-fold) were less pronounced (Figure 10A,I,J). These results suggest that DhHP-6 restores insulin resistance and oxidative stress partly through the activation of PI3K/AKT signaling in HepG2 cells insulin resistance model.

DhHP-6 inhibits gluconeogenesis and promotes glycogen synthesis in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the PI3K/AKT/Nrf2-Keap1 pathway.

Figure 10.
DhHP-6 inhibits gluconeogenesis and promotes glycogen synthesis in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the PI3K/AKT/Nrf2-Keap1 pathway.

(A) Effects of DhHP-6 on insulin signaling determined by western blot analysis in insulin resistance cell models (A). Levels of AKT phosphorylation at Ser473 (B), IRS-1 phosphorylation at Tyr632 (C), IRS-1 phosphorylation at Ser307 (D), GSK-3β phosphorylation at Ser9 (E), AMPK phosphorylation at Thr172 (F), G6Pase expression (G), PEPCK expression (H), Nrf2 expression (I) and Kepa1 expression (J) presented as the mean ± SD from three independent experiments.

Figure 10.
DhHP-6 inhibits gluconeogenesis and promotes glycogen synthesis in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the PI3K/AKT/Nrf2-Keap1 pathway.

(A) Effects of DhHP-6 on insulin signaling determined by western blot analysis in insulin resistance cell models (A). Levels of AKT phosphorylation at Ser473 (B), IRS-1 phosphorylation at Tyr632 (C), IRS-1 phosphorylation at Ser307 (D), GSK-3β phosphorylation at Ser9 (E), AMPK phosphorylation at Thr172 (F), G6Pase expression (G), PEPCK expression (H), Nrf2 expression (I) and Kepa1 expression (J) presented as the mean ± SD from three independent experiments.

DhHP-6 activates AMPK signaling in glucosamine-induced HepG2 cells insulin resistance model

AMPK activation inhibits gluconeogenesis in the liver. To investigate whether DhHP-6 ameliorates AMPK signaling to prevent insulin resistance, we examined the signaling components of the AMPK pathway. AMPK phosphorylation at Thr172 (Figure 11A,B) significantly decreased, and could be reversed by DhHP-6 treatment. G6Pase (Figure 11A,C) and PEPCK (Figure 11A,D), two key enzymes of gluconeogenesis, increased in glucosamine-induced HepG2 cells but were suppressed by DhHP-6 treatment in a concentration-dependent manner. These results suggest that DhHP-6 treatment promotes AMPK signaling in glucosamine-induced HepG2 cells insulin resistance model.

Figure 11.

Effects of DhHP-6 on AMPK signaling in glucosamine-induced HepG2 cells insulin resistance model (A). AMPK phosphorylation at Thr172 (B), G6Pase expression (C) and PEPCK expression (D). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

Figure 11.

Effects of DhHP-6 on AMPK signaling in glucosamine-induced HepG2 cells insulin resistance model (A). AMPK phosphorylation at Thr172 (B), G6Pase expression (C) and PEPCK expression (D). Data are the mean ± SD from three independent experiments. ***p < 0.001 vs. control group. ##p < 0.01, ###p < 0.001 vs. model group.

DhHP-6 ameliorates insulin resistance in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the AMPK/Nrf2-Keap1 pathway

AMPK activation inhibits gluconeogenesis through the suppression of G6Pase and PEPCK [49]. To further study the effects of DhHP-6 on insulin resistance induced by glucosamine in HepG2 cells, we examined proteins related to gluconeogenesis and the Nrf2-Keap1 system in the presence/absence of Compound C, a specific inhibitor of AMPK. As shown in Figure 12A,B, in normal cells, glucosamine supplementation resulted in a loss of AMPK phosphorylation at Thr172, which was reversed by DhHP-6 (2.27-fold increase). However, in Compound C treated cells, AMPK phosphorylation at Thr172 only modestly increased (1.50-fold higher levels) in the presence of DhHP-6. Glucosamine supplementation led to increased levels of G6Pase (Figure 12A,C) and PEPCK (Figure 12A,D) by 1.61-fold and 2.30-fold, which were repressed by DhHP-6 (1.31-fold and 1.52-fold, respectively). Notably, Compound C supplementation inhibited insulin signaling. As shown in Figure 12A,E–H, in normal cells, glucosamine supplementation decreased IRS-1 phosphorylation at Tyr632, AKT phosphorylation at Ser473, and GSK-3β phosphorylation at Ser9 by 2.04-fold, 2.05-fold and 1.78-fold, but increased IRS-1 phosphorylation at Ser307 by 1.65-fold. However, in Compound C treated cells, DhHP-6-treatment enhanced IRS-1 phosphorylation at Tyr632, AKT phosphorylation at Ser473, and GSK-3β phosphorylation at Ser9 by 1.30-fold, 1.45-fold and 1.25-fold, and inhibited IRS-1 phosphorylation at Ser307 by 1.40-fold. Additionally, in normal cells, glucosamine supplementation significantly increased Keap1 and inhibited Nrf2 (Figure 12A,I,J), both of which were restored by DhHP-6 (1.74-fold and 2.30-fold change in expression, respectively, compared with the glucosamine group). However, in Compound C treated cells, DhHP-6 only modestly restored Keap1 and Nrf2 expression (1.20-fold and 1.52-fold change in expression, respectively). These results indicate that DhHP-6 supplementation inhibits gluconeogenesis and decreases glucosamine-induced insulin resistance partly through the activation of the AMPK signaling.

DhHP-6 improves insulin resistance in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the AMPK/Nrf2-Keap1 axis.

Figure 12.
DhHP-6 improves insulin resistance in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the AMPK/Nrf2-Keap1 axis.

(A) Effects of DhHP-6 on insulin signaling determined by western blot in insulin resistance cell models. Levels of AMPK phosphorylation at Thr172 (B), G6Pase (C), PEPCK (D), IRS-1 phosphorylation at Tyr632 (E), IRS-1 phosphorylation at Ser307 (F), AKT phosphorylation at Ser473 (G), GSK-3β phosphorylation at Ser9 (H), Nrf2 (I) and Kepa1 (J). Data are the mean ± SD from three independent experiments.

Figure 12.
DhHP-6 improves insulin resistance in glucosamine-induced HepG2 cells insulin resistance model partly through the activation of the AMPK/Nrf2-Keap1 axis.

(A) Effects of DhHP-6 on insulin signaling determined by western blot in insulin resistance cell models. Levels of AMPK phosphorylation at Thr172 (B), G6Pase (C), PEPCK (D), IRS-1 phosphorylation at Tyr632 (E), IRS-1 phosphorylation at Ser307 (F), AKT phosphorylation at Ser473 (G), GSK-3β phosphorylation at Ser9 (H), Nrf2 (I) and Kepa1 (J). Data are the mean ± SD from three independent experiments.

Discussion

DhHP-6, a MP-11 mimetic, can scavenge ROS in vivo because of the porphyrin ring at its N terminus. In our previous studies, we found that orally administered DhHP-6 reduced FBG levels and improved insulin resistance in T2DM mouse models [35]. In this study, we report that: (1) DhHP-6 reduces the expression of enzymes linked to hepatic gluconeogenesis, increases glycogen synthesis, and ameliorates insulin resistance in T2DM mice; (2) DhHP-6 activates the Keap1-Nrf2 system and improves redox status in T2DM mice; (3) DhHP-6 activates AKT/PI3K and AMPK signaling in T2DM mice and glucosamine-induced HepG2 cells; (4) DhHP-6 ameliorates insulin resistance in IR HepG2 cells partly through the AMPK/AKT/PI3K/Nrf2-Keap1 axis.

T2DM is characterized by hyperglycemia and insulin resistance [50]. In accordance with our previous studies, we found that oral DhHP-6 not only decreased FBG levels, but ameliorated insulin resistance in T2DM mice [35]. Hyperglycemia is induced by the elevation of gluconeogenesis and the loss of glycogen synthesis. In this study, DhHP-6 promoted glycogen synthesis and inhibited the expression of enzymes linked to gluconeogenesis in T2DM. Exogenous DhHP-6 supplementation restored the imbalanced redox status in T2DM mice. DhHP-6 supplementation activated the Nrf2-Keap1 system by enhancing Nrf2 and inhibiting Keap1 expression. Oral DhHP-6, therefore, ameliorated the symptoms of T2DM, including hyperglycemia, insulin resistance and imbalanced redox status.

Insulin regulates glucose homeostasis through its effects on gluconeogenesis and glycogen synthesis in the liver [51]. Upon the binding and activation of insulin receptors, insulin receptor substrates (IRS-1) are activated through the phosphorylation of specific tyrosine residues (Tyr632), leading to the activation of PI3K/AKT signaling. However, Serine (Ser307) phosphorylation at IRS-1 prevents IRS-1 from coupling with the insulin receptor and inhibits IRS-1 phosphorylation at Tyr632 [36,51]. AKT stimulation results in the inhibition of glycogen synthase kinase-3β (GSK-3β) through phosphorylation, which dephosphorylates and activates glycogen synthase (GS), promoting glycogen synthesis. Oxidative stress induces insulin resistance by degrading IRS-1 to impair insulin signaling [52,53]. We found that DhHP-6 inhibited ROS production and restored mitochondrial respiratory dysfunction in T2DM mice and glucosamine-induced HepG2 cells. In T2DM mice, IRS-1 phosphorylation at Tyr632, AKT phosphorylation at Ser473 and GSK-3β phosphorylation at Ser9 were reduced, whilst IRS-1 phosphorylation at Ser307 increased, consistent with data from glucosamine-induced HepG2 cells. Our current results showed that DhHP-6 supplementation increased IRS-1 phosphorylation at Tyr632, AKT phosphorylation at Ser473 and GSK-3β phosphorylation at Ser9, but decreased IRS-1 phosphorylation at Ser307 in T2DM mice and glucosamine-induced HepG2 cells. Akt phosphorylation also increased Nrf2 expression by releasing Nrf2 from Keap1 [54,55]. In this study, glucosamine supplementation increased Keap1 expression and inhibited Nrf2 expression, which were restored by DhHP-6 in T2DM mice and glucosamine-induced HepG2 cells. DhHP-6, therefore, inhibited oxidative stress and ameliorated the impairment of insulin signaling in T2DM.

LY294002, a specific Phosphotidylinositol-3-Kinase (PI3K) inhibitor, has been widely used to study insulin signaling. Compared with normal cells, IRS-1 phosphorylation, AKT phosphorylation, and GSK phosphorylation were more modestly reversed by DhHP-6 in the presence of LY294002. These results indicate that other pathways for DhHP-6 to improve insulin resistance exist outside of the PI3K/AKT pathway. In addition, LY294002 partly inhibited AMPK phosphorylation, but promoted G6Pase and PEPCK expression, suggesting that the inactivation of AKT inhibited AMPK signaling. In LY294002 treated cells, Nrf2 expression and Keap1 levels were restored by DhHP-6 but not completely repressed. These results were also suggestive that the accumulation of Nrf2 inhibits Keap1 expression. Taken together, these data indicate that DhHP-6 inhibits gluconeogenesis and improves insulin resistance partly through the activation of the PI3K/AKT pathway and Nrf2-Keap1 system.

T2DM is accompanied by disturbances in glucose metabolism, including gluconeogenesis and glycogen synthesis [36]. AMPK plays a crucial role in regulating cellular energy homeostasis and energy balance by promoting catabolic pathways and inhibiting anabolic pathways [48,56,57]. GS is phosphorylated by AMPK to inhibit glycogen synthesis in the liver [58]. AMPK activation by phosphorylation at threonine 172 (Thr172) enhances energy expenditure and reduces circulating glucose and lipids levels, highlighting its potential for the treatment of T2DM [59]. AMPK activation inhibits gluconeogenesis by inhibiting the expression and activity of G6Pase and PEPCK, both key enzymes of gluconeogenesis [60]. AMPK activation also activates insulin signaling [36]. In this study, AMPK phosphorylation at Thr172 decreased, yet G6Pase and PEPCK expression increased in T2DM mice and glucosamine-induced HepG2 cells. DhHP-6 supplementation increased AMPK phosphorylation at Thr172 and inhibited G6Pase and PEPCK expression. Taken together, these data indicate that DhHP-6 improves the disturbances in glucose metabolism in T2DM.

Compound C is a selective AMPK inhibitor. In Compound C treated cells, the restoration of AMPK phosphorylation, G6Pase and PEPCK expression by DhHP-6 were partly inhibited. Combined with our previous results, Compound C did not completely inhibit AMPK activation by DhHP-6 as AKT activation promoted AMPK phosphorylation. The effects of DhHP-6 on IRS-1 phosphorylation, AKT phosphorylation, and GSK phosphorylation were also partly inhibited in Compound C treated cells, which results from AMPK dephosphorylation. AMPK phosphorylation degrades Keap1 and releases Nrf2 [25]. In Compound C treated cells, Nrf2 expression and Keap1 could be restored by DhHP-6 and were not completely suppressed. These results suggested that the activation of AKT and AMPK induced increases in Nrf2 and decreased Keap1 expression. Taken together, these data indicated that DhHP-6 inhibits gluconeogenesis and improves insulin resistance, partly through the activation of AMPK and the Nrf2-Keap1 system.

Conclusions

In conclusion, the present study suggested that DhHP-6 reduces FBG levels and improves insulin resistance in T2DM mice. DhHP-6 also restored redox homeostasis by increasing the activity of enzymes linked to anti-oxidation and promoted glycogen synthesis in T2DM mice. Moreover, DhHP-6 inhibited ROS production and restored mitochondrial respiration in glucosamine-induced HepG2 cells. Furthermore, DhHP-6 enhanced AKT and AMPK activation, which in turn inhibited gluconeogenesis and promoted glycogen synthesis. DhHP-6 also reduced Keap1 expression and enhanced Nrf2 expression. DhHP-6 ameliorated insulin resistance through the activation of the PI3K/AKT and AMPK pathway. These findings elucidate the mechanisms of how DhHP-6 attenuates insulin resistance in T2DM and further highlight exogenous peroxidase supplementation as a new strategy for the treatment of T2DM.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This research was supported by the Science and Technology Development Program of Jilin Province, China (No. 20150520157 JH), China Postdoctoral Science Foundation Grant (No. 2015M581398) and Special Project for Health from Jilin Province, China (No. 2018SCZWSZX-037).

Author Contributions

Conceived and designed the experiments, K.W. and L.W.; methodology, K.W.; preformed the experiments, K.W., Y.S., and Y.L.; analyzed the data, K.W. and Y.L.; wrote the paper, K.W.; revised the paper, L.W.

Abbreviations

     
  • C. elegans

    Caenorhabditis elegans

  •  
  • AD

    Alzheimer's disease

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • CAT

    catalase

  •  
  • DhHP-3

    Deuterohemin-Ala-His-Lys

  •  
  • DhHP-6

    Deuterohemin-βAla-His-Thr-Val-Glu-Lys

  •  
  • ER

    endoplasmic reticulum

  •  
  • FBG

    fasting blood glucose

  •  
  • FBPase

    fructose-bisphosphatase

  •  
  • G6Pase

    glucose-6-phosphatase

  •  
  • GS

    glycogen synthase

  •  
  • GSH

    glutathione

  •  
  • GSK-3β

    glycogen synthase kinase-3β

  •  
  • HK

    Hexokinase

  •  
  • IDF

    International Diabetes Federation

  •  
  • IRS

    insulin receptor substrates

  •  
  • ITT

    insulin tolerance test

  •  
  • Keap1

    Kelch-like ECH-associated protein 1

  •  
  • MDA

    malondialdehyde

  •  
  • MP-11

    microperoxidase-11

  •  
  • Nrf2

    NF-E2-related factor 2

  •  
  • OCR

    oxygen consumption rate

  •  
  • PEPCK

    phosphoenolpyruvate kinase

  •  
  • PI3K

    phosphatidylinositol 3-kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase

  •  
  • T2DM

    type 2 diabetes mellitus

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