Introduction

Adipose tissue is not only a site of energy transactions, but also generates various cytokines (adipokines) that maintain metabolic homeostasis [1, 2]. Obesity-induced excessive visceral fat accumulation is associated with insulin resistance (IR), systemic oxidative stress and chronic inflammation [3, 4]. IR appears to promote obesity-driven type 2 diabetes mellitus (T2DM) and leads to impairments of the action of insulin [1]. Adipose tissue is a target tissue in terms of insulin-mediated glucose uptake, and thus, defects in insulin signaling involving glucose uptake are strongly related to IR in patients with obesity-driven T2DM [6, 7]. The growing evidences have indicated that chronic low-grade inflammation and oxidative stress in white adipose tissue (WAT) can induce the inhibition of insulin signal transduction and IR [8]. The elevated levels of plasma free fatty acids (FFAs) such as palmitic acid (PA) in patients or animals with obesity are thought to play an important role in IR [9]. In addition, elevated FFAs levels lead to a variety of fat-derived toxic metabolites, which in turn induce defects in insulin-mediated PI3K/Akt pathway and glucose uptake [9, 10].

The endogenous protection strategies are currently developed to prevent adipocytes low-grade inflammation, insulin resistance and metabolic disturbances [11, 16]. Adrenomedullin (ADM) is an endogenous active peptide with ubiquitous distribution and acts as a circulating hormone or a local paracrine mediator with multiple physiological and pathological activities [17], including vasodilatation, angiogenesis, bronchodilatation, diuresis promotion, food intake, hormone regulation, gastrointestinal modulation, immune regulation, tumor progression, diabetes, heart failure, sepsis and so on [17,18,19,20,21,22]. However, few studies have explored the specific role of ADM in obesity. ADM shares structural similarities with calcitonin gene-related peptide, amylin and intermedin and possesses beneficial properties in preventing chronic diseases affecting cellular activities via oxidative and inflammatory pathways [23,24,25]. More importantly, the protein expressions of ADM and its receptor system including calcitonin receptor-like receptor (CRLR) combined with a specific receptor activity-modifying protein 2 (RAMP2) or RAMP3 have been detected in adipocytes and adipose tissues [26, 27]. It has been found that ADM can regulate the physiological and physiological functions in adipocytes and is considered as an endogenous adipokine [28]. For instance, it has been found that ADM knockout mice exhibited obesity, hypertension and IR, and oxidative stress-induced alterations were associated with the insulin sensitivity accompanied with impairment of insulin signaling in aged mice. However, these abnormalities can be reversed by treatment with ADM [29]. Researchers also found that ADM is an antiadipogenic factor in preadipocyte cell lines, and insulin has an inhibitory effect on ADM expression in isolated human adipocyte cells [30], but there is higher ADM expression in white adipose tissue (WAT) in patients with diabetes or obesity accompanying IR [31,32,33]. So it is necessary to demonstrate the role of ADM in IR, especially in WAT under obesity condition. As well known, WAT is a source of proinflammatory cytokines, causing oxidative stress and promoting adipose tissue dysfunction and insulin resistance [34]. Our previous study showed that ADM and its receptor system were upregulated in visceral WAT, and ADM application effectively attenuated inflammation in visceral WAT of obese rats [35]. So it can be speculated that ADM may be an endogenous protective adipokine against IR in adipocytes. Therefore, we aimed to investigate whether ADM modulates insulin pathway and has the roles in insulin signaling, inflammation and oxidative stress in PA-treated adipocytes or in vWAT of obese rats induced by high-fat diet.

Materials and methods

3T3-L1 cell line culture

Mouse 3T3-L1 preadipocytes from China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei, China) were cultured in medium containing Dulbecco's modified essential medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin (100 U)/streptomycin (100 mg/mL). The cell cultures were incubated in an incubator with humidified atmosphere containing 5% CO2 at 37 °C. Fully differentiated 3T3‐L1 adipocytes were used for experiments. Preadipocytes differentiation was induced by 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 2 μM rosiglitazone and 10 μg/mL insulin for 3 days, followed by fresh medium every 3 days until cells were fully differentiated into mature adipocytes verified by Oil Red O staining [25]. Differentiated 3T3-L1 cells were pretreated ADM (10 nM) for 30 min, subsequently exposed to 200 μM PA for further 24 h and finally treated with 100 nM insulin for 30 min. ADM22-52 (10–6 M, ADM receptor antagonist) or A6730 (10 μM, Akt activation inhibitor, iAkt) was added 10 min before ADM application, respectively.

Cell viability assay

The differentiated 3T3-L1 adipocyte suspension (100 μL) was added into a 96-well plate and cultured for 24 h at 37 °C in a cell incubator. The Cell Counting Kit-8 (CCK8) cell cytotoxicity test was used to determine the effect of ADM (10 nM), ADM22-52 (10–6 M), PA (200 nM) and A6730 (Akt inhibitor, 10 μM) on cell viability for 24 h. The test substances (10 μL) were administrated into the plate, respectively. After incubation, the CCK solution (10 μL) was added into each well and incubated for 3 h. Finally, the absorbance was determined by a microplate reader (ELX800; BioTek, Winooski, Vermont) at 450 nm.

Oil red O staining

Differentiated 3T3-L1 adipocytes were identified with Oil Red O staining. Cells were fixed in 4% formalin and then washed with phosphate-buffered saline (PBS). The 0.6% Oil Red O solution (Oil Red O dye in isopropyl alcohol) was used to bind intracellular lipids. Stained lipid droplets were observed with an inverted microscope as shown in Fig. 1a.

Fig. 1
figure 1

Differentiated 3T3-L1 adipocytes were identified using Oil red O staining (a). The effects of adrenomedullin (ADM, 10 nM) and palmitic acid (PA, 200 μM) alone, or chemicals combination for 24 h on cell viability (b) in the differentiated 3T3-L1 adipocytes. Western blotting showed that PA’ effects on the endogenous protein expressions of ADM, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2) and RAMP3 in the differentiated 3T3-L1 adipocytes (c–f) exposed to 200 μM PA for 24 h. ADM, CRLR, RAMP2 and RAMP3 protein levels were normalized to GAPDH protein expression. Original magnification 100 × (a); n = 3 to 5. Each value indicates mean ± SEM. *P < 0.05 vs. control group

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Intracellular ROS level assay

Dihydroethidium (DHE, Beyotime, China) fluorescence staining in adipocytes was used to evaluate intracellular ROS production. Cells (3 × 105 cells/mL) in 6-well plates were incubated with DHE (10 μM) in PBS for 30 min in a dark and humidified container at 37 °C. After washed twice with cold PBS, the fluorescence was examined with a fluorescence microscope (DP70, Olympus Optical, Tokyo, Japan) under excitation at 518 nm and emission at 605 nm.

Western blotting

Adipocytes or adipose tissue was homogenized, and the proteins were dissolved in the protein extraction reagent (#78510; Thermo Fisher Scientific, IL). Equal amounts of protein extracts from adipocytes or adipose tissue were separated by polyacrylamide gel electrophoresis (PAGE) and then were electrotransferred to polyvinylidene difluoride membranes. Primary antibodies were used against ADM, CRLR, RAMP2, RAMP3, TNFα, IL-1β, IL6, CAT, GPx1, SOD2, PI3K p85, phosphorylated-Akt (pAkt), total-Akt (T-Akt), GLUT-4 or GAPDH overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG were used as secondary antibodies. The immunoblots were visualized with the electrochemiluminescence (ECL) Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). Protein band intensities were normalized with total-Akt or GAPDH levels. The signals were quantified using Odyssey Imaging System (LI-COR Biosciences, Lincoln, Nebraska). The amount of detected protein was quantified by the Image J software and was expressed as the ratio to GAPDH protein.

Animals

Male Sprague–Dawley rats (200–220 g) were used for animal experiments. Rats were randomly divided into two groups and were housed in a temperature- and humidity-controlled room with a 12-h light–dark cycle. They were allowed access to rat chow and tap water ad libitum. The control group received a normal diet (12% of kilocalories from fat, Trophic Animal Feed Hightech Co. Ltd., Nantong, China), and the obese group received a high-fat diet (45% of kilocalories from fat, Trophic Animal Feed Hightech Co. Ltd., Nantong, China) for 12 weeks. The experiments were approved by the Animal Experimental Ethics Committee of the Animal Core Facility of Nanjing Medical University (1911016, September 14, 2020) and complied with the Guidelines for the Care and Use of Laboratory Animals (NIH publication, 8th edition, 2011). The criterion for the obese rats was that the body weight in the high-fat diet group was 20% more than that of the mean weight of control group after 12-week feeding. Obese rats were further randomized into two groups (n = 8/group) and continued to be fed a high-fat diet fed for 4 weeks. The obese rat was treated with intraperitoneal injection of ADM (7.2 μg/kg/day, n = 8) or an equal volume of saline for the remaining rats (n = 8). At the end of treatment, visceral fat (inguinal, epididymal, mesenteric and perirenal) was collected after rats were anesthetized with sodium pentobarbital via intraperitoneal injection. Finally, the visceral fat was kept at −80 °C for further analysis. A scheme of the in vivo experimental design is shown in Fig. 5a.

Homeostasis model assessment–insulin resistance (HOMA-IR) and oral glucose tolerance test (OGTT)

All rats were fasted overnight for more than 12 h, and about 1.5 mL of blood was collected from the tail vein at the end of 12 and 16 weeks, respectively. The plasma insulin level was detected by using the enzyme-linked immunoassay (Elisa) method with a kit (RayBiotech, Norcross, GA, USA). The oral glucose tolerance test (OGTT) was performed on the day after 16 weeks. The glucose (2 g/kg of BW) was applied by gavage. The blood was collected from the tail vein every 0, 30, 60 and 120 min after oral glucose supplementation, and the glucose levels were measured with a handheld glucometer. The area under the glucose curve (AUC) was calculated by the trapezoidal rule [36]. Insulin resistance was assessed by calculating the HOMA-IR (homeostasis model assessment of insulin resistance) index using the following formula: HOMA-IR = [fasting glucose (mmol/L) × fasting insulin (mIU/L)]/22.5.

Reagents and antibodies

ADM (molecular formula: C242H381N77O75S5) was obtained from Bachem (Bubendorff, Switzerland). PA, Akt inhibitor A6730 and ADM receptor antagonist ADM22-52 were from Anaspec (Fremont, California). DMEM, FBS, 0.25% trypsin–EDTA, streptomycin/penicillin and trypsin were obtained from Thermo Fisher Scientific (Pudong New District, Shanghai, China). The ADM, CRLR, RAMP2/3 and TNFα antibodies were from Affinity Biosciences (Pottstown, PA, USA). The IL-1β, IL-6, Catalase, GPx1, SOD2, GLUT-4 and GAPDH antibodies were obtained from Proteintech (SANYING, Wuhan, China). The PI3K p85, phosphorylated-Akt and total-Akt were from Cell Signaling Technology (Shanghai, China). IBMX, dexamethasone, rosiglitazone and insulin were from Sigma-Aldrich.

Statistics

The GraphPad Prism version 8.00 (San Diego, California) software was used to analyze the data. Differences in the mean values between two groups were assessed by unpaired t test. One-way ANOVA was used for data analysis of more than two groups followed by Bonferroni post hoc analysis. All data illustrated are expressed as mean ± S.E. A p-value of < 0.05 was considered statistically significant. The experiments were conducted at least three times to verify reproducibility.

Results

Effects of chemicals on cellular viability and endogenous expressions of ADM and its receptor system in adipocytes

The differentiated 3T3-L1 adipocytes were treated with different chemicals including PA (200 μM), ADM (10 nM), ADM22-52 (10–6 M) and A6730 (Akt activation inhibitor, 10 μM) for 24 h. As shown in Fig. 1b, the chemicals administration had no significant impact on cellular viability in the differentiated 3T3-L1 adipocytes in each group. We also found that the endogenous expressions of ADM and its receptor system including CRLR, RAMP2 and RAMP3 were increased under PA (200 μM) stimulation for 24 h (Fig. 1c–f), which suggested that ADM and its receptor system may play an important role in regulating adipocytes functions such as IR.

ADM attenuated PA-induced inflammation and oxidative stress in adipocytes

Inflammation and oxidative stress are critical elements in relationship to the pathophysiology of IR [6]. To assess the role of ADM in IR, we first used the differentiated 3T3-L1 adipocytes to investigate ADM treatment on inflammation and oxidative stress in vitro. We detected the protein expressions of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-6 to assess ADM’s action on inflammation. As shown in Fig. 2a–c, these increased inflammatory cytokines in PA-treated adipocytes were down-regulated in ADM-treated adipocytes. To explore the function of ADM against oxidative stress in adipocytes, we measured the ROS levels and the protein expressions of the key anti-oxidant enzymes including SOD2, GPx1 and CAT. The results showed that the protein expression levels of SOD2, GPx1 and CAT in the PA group were significantly down-regulated compared with the control group, whereas their levels in the ADM group showed obvious up-regulation compared with the PA group (Fig. 2e–g). Furthermore, DHE staining showed that the ROS production in PA group was increased, which was attenuated by ADM treatment in adipocytes (Fig. 2d). Together, these results confirmed the anti-inflammatory and anti-oxidative effects of ADM in adipocytes treated by PA.

Fig. 2
figure 2

Effects of ADM (10 nM) on inflammation and oxidative stress stimulated by PA (200 μM). The protein expression of pro-inflammatory cytokines including tumor necrosis factorα (TNFα), interleukin-1β (IL-1β) and IL-6 levels (a–c), anti-oxidant enzymes including manganese superoxide dismutase (SOD2), glutathione peroxidase (GPx1) and catalase (CAT) levels (e–g) and reactive oxygen species (ROS) levels detection by dihydroethidium (DHE) staining (d) were determined in the differentiated 3T3-L1 adipocytes pretreated or not with ADM (10 nM) for 30 min, subsequently exposed to 200 μM PA for further 24 h. The protein expressions of cell lysates were analyzed by the Western blotting method. GAPDH was used as an internal control for Western blotting analysis. Each value indicates mean ± SEM; n = 3 to 5. *P < 0.05 vs. control group. #P < 0.05 vs. PA group. C: control

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ADM improved PA-induced IR in adipocytes

High levels of circulating cytokines or FFAs cause drastic changes in blocking the PI3K/Akt pathway with subsequent IR [37, 38]. Akt activation plays an essential role in the metabolic effects of insulin, including the increase in the translocation of glucose carriers such as GLUT-4 [38]. In order to evaluate the effect of ADM on insulin signaling pathway perturbed by PA, the protein levels of PI3K (p85) and the phosphorylation of Akt (pAkt) were analyzed in 3T3-L1 adipocytes by Western blot. Adipocytes exposure to PA decreased PI3K (p85) and pAkt protein levels when compared to the insulin group, and PA inhibited insulin-induced increases in PI3K (p85) and pAkt protein levels, whereas pretreatment with ADM improved their levels. Moreover, ADM receptor antagonist ADM22-52 effectively blocked ADM’s action (Fig. 3a, b). In fact, PA caused the interruption of insulin pathway, which was improved by cell pretreatment with ADM. Furthermore, the increased expression in PI3K (p85) and pAkt levels in ADM pretreatment group was more obviously than insulin pretreatment group, and this effect could be effectively inhibited by ADM receptor antagonist and Akt inhibitor A6730 (Fig. 3e, f). Indeed, even in the insulin-free condition, ADM still exhibited the ability to independently activate the PI3K/Akt pathway, and this effect of ADM on PI3K/Akt activation was in a time- and dose-dependent manner (Fig. 3c, d).

Fig. 3
figure 3

Effects of ADM on PI3K (p85) and pAkt (Ser473) (a, b, e, f) phosphorylation modulated by PA or insulin. The effects of ADM on the pAkt protein expressions at different of time points (0, 5 min, 10 min, 30 min, 60 min and 24 h) and at different concentrations (10–10 M, 10–9 M, 10–8 M and 10–7 M) were explored, respectively (c, d). Each value indicates mean ± SEM; n = 3 to 5.*P < 0.05 vs. control group, #P < 0.05 vs. Insulin group, $P < 0.05 vs. PA + Insulin group and &P < 0.05 vs. ADM + PA + Insulin group (a, b); *P < 0.05 vs. 0 min and #P < 0.05 vs. 5 min (c); *P < 0.05 vs. 10–9 M (d); *P < 0.05 vs. PA group, #P < 0.05 vs. ADM + PA group (e, f). C: control

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ADM facilitated insulin-mediated glucose transporter-4 translocation in adipocytes

It has been demonstrated that GLUT-4 is very important for contributing to the effect of insulin to stimulate glucose transport in adipocytes. Insulin can stimulate GLUT-4 translocation from endosomal storage vesicles to the cell surface [39]. To confirm the effect of ADM on GLUT-4 modulation, whole-cell protein, membrane protein and cytoplasmic protein of GLUT-4 levels were investigated, respectively. In this study, there was no significant change in the whole-cell protein of GLUT-4 level in adipocytes after exposure to PA or ADM alone (Fig. 4a). However, exposure of adipocytes to PA decreased GLUT-4 membrane protein levels demonstrating reduced insulin-sensitivity, and PA inhibited insulin-induced increase in GLUT-4 membrane protein levels, but ADM pretreatment improved the GLUT-4 membrane protein expression. This effect of ADM was also blocked by ADM receptor antagonist ADM22-52. Meanwhile, the alteration of cytoplasmic protein of GLUT-4 levels in each group also proved the trends of membrane protein of GLUT-4 levels (Fig. 4b, c).

Fig. 4
figure 4

Effect of ADM on glucose transporter-4 (GLUT-4) protein expression in differentiated 3T3-L1 cells. The total (T-GLUT-4), membrane (M-GLUT-4) and cytoplasmic (C-GLUT-4) protein expressions of GLUT-4 were determined, respectively. Each value indicates mean ± SEM; n = 3 to 5. *P < 0.05 vs. control group, #P < 0.05 vs. Insulin group, $P < 0.05 vs. PA + insulin group and &P < 0.05 vs. ADM + PA + insulin group (b-c). C: control

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ADM attenuated inflammation and oxidative stress, and improved insulin sensitivity in adipose tissue of obese rats

Obese rats were injected intraperitoneally with saline or ADM (7.2 μg/kg/day) for 4 weeks. The vWAT was collected for experiment. The results showed that HFD fed rats increased the plasma levels of glucose and insulin, HOMA-IR and AUC compared to the control rats (Table 1, Fig. 6a–c). ADM treatment had significant effects on reducing insulin level, glucose tolerance and AUC (Table 1, Fig. 6a–c), but did not caused a significant decrease in plasma glucose level. In vWAT, the increased protein expressions of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, and decreased anti-oxidant enzymes including SOD2, GPx1 and CAT in the obese group were significantly improved in the obese rats after ADM treatment (Fig. 5b–g). We also found that ADM treatment in obese rats could increase the level of whole cell protein expression of GLUT-4 and the activation of PI3K/Akt pathway resulting in significant increases in the protein expression levels of PI3K p85 and pAkt in vWAT when compared to obese rats (Fig. 6d–f).

Table 1 Glucose and insulin level was measured in rats after 12 or 16 weeks of diet
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Fig. 5
figure 5

A scheme of the in vivo experimental design (a) and the effects of ADM (7.2 μg /kg/day) chronic administration for 4 weeks on inflammation and oxidative stress in visceral white adipose tissue (vWAT) of rats with obesity induced by high-fat diet. The protein expressions of pro-inflammatory cytokines including TNFα, IL-1β and IL-6 levels (b–d) and anti-oxidant enzymes including SOD2, GPx1 and CAT (e–g) were analyzed by Western blotting method. Each value indicates mean ± SEM. n = 3 to 5. *P < 0.05 vs. Control + saline group, #P < 0.05 vs. OB + saline group

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Fig. 6
figure 6

Effects of ADM (7.2 μg/kg/day) chronic administration for 4 weeks on HOMA-IR (a), the oral glucose tolerance test (OGTT), namely the glucose curve (b) and the total area under the glucose curve (c) in obese rats. The protein expressions of PI3K (p85) (d), pAkt (Ser473) (e) and total GLUT-4 levels (f) in visceral white adipose tissue (vWAT) of rats with obesity induced by high-fat diet. Each value indicates mean ± SEM. n = 3 to 5. *P < 0.05 vs. control + saline group, #P < 0.05 vs. OB + saline group

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Discussion

Diminished adipocyte inflammation and oxidative stress, and enhanced insulin signaling could be effective for improvement in adipocytes function [34, 40]. Our results demonstrated that ADM not only inhibited inflammation and oxidative stress but also increased insulin sensitivity, promoting GLUT-4 translocation through the regulation of PI3K/Akt pathway. Moreover, ADM could also exert beneficial effects on IR in vWAT of rats with obesity induced by high-fat diet. We also found that PA stimulated the endogenous protein expressions of ADM and its receptor system. These results demonstrate the potential protective effects of ADM on adipocytes function involving obesity-related IR.

Obesity is an important health problem and is associated with many chronic diseases such as T2DM and cardiovascular diseases [41, 42]. FFAs, mainly from lipolysis processes of adipose tissue, are the main causes of the onset of inflammation, oxidative stress and IR in the adipose tissue [9, 10]. High levels of intracellular FFAs such as PA promote the greater expression of inflammatory adipokines and ROS generation that cause the alteration of the insulin sensitivity [9, 10]. It has been reported that increased pro-ADM levels in obesity may reflect disease severity such as cardiovascular diseases and IR related to obesity [43,44,45]. So it can be speculated that the increased ADM levels may have a protective role against IR. In the present study, ADM and its receptor system protein levels were increased in the differentiated adipocytes under PA stimulation; this may represent a compensatory mechanism against inflammation, oxidative stress or IR in adipocytes. In recent decades, numerous studies have shown that ADM is able to exert its protective roles involving anti-inflammatory or anti-oxidant mechanisms in many diseases such as cardiovascular or renal diseases, lung diseases and intestinal diseases [24, 46,47,48]. However, there are only a few studies to explore specific ADM’s action on metabolic diseases such as diabetes or obesity-related diseases, and most of the underlying mechanisms are still unclear [49,50,51]. In our previous study, we have found that ADM attenuated systemic inflammation in obese rats, and the anti-inflammatory effect was also found in vWAT of obese rats [35]. But we only investigated the ADM’s action on inflammation. The IR, OGTT and oxidative stress in vWAT were not be explored. To further understand the role of ADM in adipose tissue, we continued to clarify the anti-inflammatory effect and investigate anti-oxidant and insulin-sensitizing effects of ADM and elucidated the potential molecular mechanisms involved in these beneficial effects by using an in vitro experimental model based on murine 3T3-L1 adipocytes. FFAs such as PA, mainly from lipolysis processes of WAT, greatly promote the onset of inflammation, oxidative stress and IR in obesity [9, 10]. We used the high level of PA similar to the obese status, but not LPS in previous study, to stimulate the adipocytes for exploring the ADM’s action on PA-induced inflammation, oxidative stress and IR. It would make us convinced about the effects of ADM on adipose tissue in obesity.

IR is strongly associated with the pathogenesis of T2DM and obesity and results from a sustained low-grade inflammatory status and oxidative stress [6, 9]. Pro-inflammatory cytokines increase the adipose triglyceride lipase and hormone-sensitive lipase (HSL) activity, which increases FFAs release, generating a vicious cycle [52]. In this study, ADM was able to diminish the production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in 3T3-L1 adipocytes stimulated by PA. Here, ADM-suppressed adipocyte inflammatory status may improve insulin signaling. It has also been demonstrated the role of oxidative stress in obesity and IR [8]. Numerous studies indicate that an increase in ROS significantly affects WAT biology and leads to an inflammatory profile and IR, which could contribute to obesity-associated diabetes [53]. Our results showed that ADM suppressed the increased production of ROS and recovered the expression of anti-oxidatant enzymes SOD2, GPx1 and CAT caused by PA. Therefore, ADM improving inflammation and oxidative stress may protect adipocytes function. The effects of ADM in vitro experiment were similar with in vivo experimental animal model.

Insulin binds to its receptor and promotes a tyrosine phosphorylation of IRS, which in turn induces the activation of PI3K/Akt signaling, the subsequent GLUT-4 translocation and glucose uptake [6]. Because the adipocytes dysfunction is associated with IR, we evaluated the insulin pathway. We confirmed that IR was induced by PA in 3T3-L1 adipocytes, as observed by inhibition of PI3K/pAkt axis and GLUT-4 membrane protein expression. Besides, for the first time, we demonstrated that pretreatment with ADM can restored PI3K/pAkt pathway and GLUT-4 membrane protein levels altered by PA in adipocytes, and these roles were more effective than insulin. Furthermore, in vivo studies showed that ADM improved IR by reducing HOMA-IR. The effects of ADM on increasing insulin sensitivity by affecting PI3K/Akt axis and GLUT-4 levels were also found in vWAT. It has been reported that ADM plays a protective role through the activation of the PI3K/Akt pathway [54]. In this study, ADM improved the activation of the PI3K/Akt pathway and GLUT-4 expression that may be related to the inhibition of inflammation and oxidative stress caused by PA. Moreover, ADM itself could also activated PI3K/Akt pathway, so ADM exerted insulin-mimicking activity by activating PI3K/Akt signaling pathway, therefore favoring GLUT-4 translocation, so supporting an insulin sensitizer role for ADM. However, another study found that ADM could not improve IR, but promoted lipolysis in vWAT in a diabetic mouse model induced by high-fat–high-sucrose (HFHS) diet for 8 weeks, which may be associated with the reduced expression of insulin signaling factors including insulin receptor and Glut4, and adipogenic factor Pck1 [55]. These findings are inconsistent with our results, perhaps because of differences in animal models. But this model did not show the high levels of insulin and glucose, which are related to IR. In any case, the role of ADM in WAT in obesity deserves further investigation.

In summary, ADM attenuated inflammation and oxidative stress. Additionally, IR was prevented via modulation of the insulin/PI3K/Akt/GLUT-4 pathway. We demonstrated, for the first time, that ADM alleviated IR in adipocytes. Furthermore, animal model investigations also confirmed the effects observed in vitro. Taken together, our results support the notion that ADM can improve insulin sensitivity in adipocytes, and it may have positive effects on inflammation, oxidative stress and IR in obesity.