Crocin Improves Insulin Sensitivity and Ameliorates Adiposity by Regulating AMPK-CDK5-PPARγ Signaling

Crocin is a carotenoid compound which possesses multiple biological activities. Our and other laboratory's previous findings show that crocin alleviates obesity and type 2 diabetes-related complications. We have found that crocin activates AMP-activated protein kinase (AMPK) signaling and inhibition of AMPK suppresses crocin-induced protective effects. However, the causal role of AMPK activation in the biological role of crocin is still not verified. In the present study, we showed that crocin markedly inhibits the changes of glucose metabolic parameters and serum lipid profiles in wild type diabetic mice. In AMPKα KO diabetic mice, those protective effects of crocin against glucose and lipid metabolic dysfunction were abolished. These results demonstrated AMPK activation was responsible for the beneficial effects of crocin on metabolic dysfunction. Moreover, we have shown that the antiobese effect of crocin has been abolished by the deficiency of AMPKα. We also showed that crocin induced a significant decrease of CDK5 protein level in wild type diabetic mice, while this effect was abolished in AMPKα KO diabetic mice. The regulation of downstream targets of CDK5/PPARγ by crocin was abolished by the deficiency of AMPK. In conclusion, our study verified that activation of AMPK is involved in crocin-induced protective effects against glucose and lipid metabolic dysfunction. Activation of AMPK downregulates the protein level of CDK5, followed by the decrease of PPARγ phosphorylation, leading to the inhibition of adipose formation and metabolic dysfunction. Our study provides new insights into the mechanism of protective effects of crocin and interaction of AMPK and CDK5/PPARγ signaling.


Introduction
The prevalence of obesity and type 2 diabetes (T2D) have become major health problems worldwide [1,2]. With the increasing number of obese individuals associated with the aging of population, the prevalence of T2D and other associated complications is increasing at an unexpected rate. However, there is still no curative pharmacological treatment for obesity and type 2 diabetes.
Crocin is a water-soluble carotenoid compound and a main active constituent found in the stigmas of Crocus sativus, commonly known as saffron [3,4]. Crocin was used flavoring and coloring agent in food manufacture [5]. Crocin has been found to possess multiple pharmacological effects, including antioxidant, antihyperlipidemic, anti-inflammatory, anticancer, antiarthritic, hepatoprotective, and cardio-protective effects [6][7][8]. Our and other laboratory's findings show that crocin alleviates obesity and type 2 diabetesrelated complications [9][10][11][12][13]. We have found that crocin activates AMP-activated protein kinase (AMPK) signaling and inhibition of AMPK significantly suppresses crocininduced protective effects against metabolic disorders [9,10]. However, the causal role of AMPK activation in the biological role of crocin is still not verified, and the AMPK-associated downstream signaling pathway mediating the beneficial effect of crocin is still not known.
Peroxisome proliferator-activated receptor gamma (PPARγ) is a pivotal nuclear receptor and transcription factor that plays substantial roles in the regulation of glucose and lipid metabolism through the regulation of gene expression [14,15]. The chemical ligands have been developed into therapeutic drugs for the treatment of type 2 diabetes. In addition to the ligand activation, PPARγ can also be regulated through diverse posttranslational modifications [16]. In recent years, it has been found that PPARγ serine 245 (or S273 in PPARγ isoform 2) can be phosphorylated by cyclin-dependent kinase 5 (CDK5), and this posttranslational modification is related with insulin resistance in obese individuals [17]. However, no evidence has shown whether crocin could regulate CDK5/PPARγ signaling.
In the present study, we designed experiments to evaluate the causal role of AMPK in the protective effects of crocin against metabolic disorders and to investigate the possible effects of crocin on CDK5/PPARγ signaling.

Animal Experiments.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology and were conducted in accordance with ARRIVE and NIH guidelines for animal welfare. Male mice with global knockout of AMPKα2 gene (AMPKα2-KO) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Wild-type (WT) mice with C57BL/6 J background were used as control.
All mice were housed in the University of Louisville Research Resources Center with a constant temperature at 22°C and a 12-h light/dark cycle. Mice had free access to tap water. The model of T2D was established by high-fat diet (HFD; 60.3 kcal% fat, TD. 09766; Research Diets, Teklad Custom, Envigo) feeding for three months to induce insulin resistance, followed by a single intraperitoneal injection of 100 mg/kg streptozotocin (STZ; Sigma-Aldrich) in 0.1 mol/L of citrate acid buffer (pH 4.5) to induce partially impaired βcell function. The dose of STZ was used according to previous reports [18]. The survival rates after STZ injection in wild type and AMPKα2-KO mice were 93.3% and 86.7%, respectively. 7 days post-STZ injection, mice with hyperglycemia (3-h fasting blood glucose levels ≥250 mg/dL) were defined as diabetic. The diabetic mice were daily orally administrated with 20 mg/kg crocin for 12 weeks. After the experiment, all mice were fasted for 12 h and anesthetized using 2% isoflurane and sacrificed. Blood samples were collected, and tissue samples were snap-frozen in liquid nitrogen or collected for paraffin embedding.

Histopathology of Adipose
Tissue. Histopathology of adipose tissue was observed using H&E staining. In brief, adipose tissue was fixed in 10 formaldehyde, embedded in paraffin, and then 5 μm sections were cut and stained with H&E. Capture of images was performed using a light microscope (Olympus, Japan).

Glucose and Insulin
Tolerance. 11 weeks after the treatment of crocin, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine the ability of the body to respond to glucose or insulin load. Before the IPGTT and IPITT, mice were fasted for 12 h or 6 h, respectively. Blood glucose level was measured 0, 30, 60, 90, and 120 min after the administration of glucose and insulin using an Accu-Chek glucometer (Roche, Basel, Switzerland) via tail vein blood.
2.5. Real-Time Quantitative PCR. Total RNA from adipose tissues was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) as per the manufacturer's instructions. RNA quantity and quality were confirmed using Nanodrop (Thermo Fischer Scientific, USA). After treating with DNase, reverse transcription into cDNA was performed using Superscript II (Life Technologies, Foster City, CA). Real-time RT-PCR reaction was prepared using SYBR Green PCR Master Mix (Takara, Tokyo, Japan) in an ABI StepOne-Plus Real-time PCR System. Expression of glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used to normalize the expression of target genes used in the study.

Statistical Analysis.
Results are expressed as the mean ± SD. Significant differences among groups were assessed using one-way ANOVA followed by Dunnett's test. Statistical significance was defined as P < 0:05. BioMed Research International protective effects of crocin in the context of obesity and type 2 diabetes, we established obese and type 2 diabetic animal models using AMPKα global KO mice. As illustrated in Figure 1(a), the treatment of crocin significantly reduced the body weight in wild type diabetic mice, while this effect was abolished in AMPKα KO diabetic mice. We then determined the effect of crocin on general biochemical profiles in both wild type and AMPKα KO diabetic mice. We showed that in wild type diabetic mice, crocin significantly reduced the serum levels of triglycerides (TG), nonesterified fatty acids (NEFAs), total cholesterol (TC), and aspartate aminotransferase (AST) and alanine transaminase (ALT) (Figures 1(b)-1(f)). In contrast, in AMPKα KO diabetic mice, crocin did not show any inhibitory effects on serum levels of TG, NEFAs, TC, AST, and ALT (Figures 1(b)-1(f)).

Deficiency of AMPKα Abolished Crocin-Induced
Protective Effects on Glucose Metabolic Activity in Type 2 Diabetic Mice. We further determined the effect of crocin on glucose metabolism in both wild type and AMPKα KO diabetic mice. As shown in Figure 2(a), fasting blood glucose began to reduce after 6-week treatment of crocin, and at the end of the experiment, fasting blood glucose in wild type diabetic mice was markedly inhibited by crocin. However, no marked change of fasting blood glucose in AMPKα KO diabetic mice was observed after the treatment of crocin (Figure 2(a)). Moreover, crocin remarkably reduced the level of insulin in wild type diabetic mice, while this effect was abolished in AMPKα KO diabetic mice (Figure 2(b)). We also used IPGTT and IPITT to evaluate the ability of the body to respond to glucose and insulin. As shown in Figures 2(c) and 2(d), the inhibitory effect on the increase of area under the curve of IPGTT and IPITT by crocin was abolished when AMPKα was deficient.

Deficiency of AMPKα Abolished Crocin-Induced
Protective Effects on Adiposity in Type 2 Diabetic Mice. Considering the important role of obesity in glucose metabolic  , we showed that in wild type diabetic mice, crocin markedly reduced the perirenal and epidydimal adipose mass, while this effect of crocin on adipose tissue was abolished in AMPKα KO diabetic mice. Moreover, crocin reduced the size of adipocyte in wild type diabetic mice, which effect was not observed in AMPKα KO diabetic mice (Figures 3(c)  and 3(d)).

Deficiency of AMPKα Abolished Crocin-Induced
Effects on CDK5/PPARγ in Type 2 Diabetic Mice. To investigate the possible mechanism of AMPK-mediated protective effects of crocin against obesity and type 2 diabetes, we evaluated the changes of CDK5/PPARγ signaling. Previous liter-ature has demonstrated that CDK5 can phosphorylate PPARγ and thus influence the downstream target genes expression, leading to the regulation of lipid metabolism [19]. We showed that in wild type diabetic mice, crocin significantly reduced the protein level of CDK5 and phosphorylated PPARγ (S273) (Figure 4(a)). However, this effect of crocin on the protein level of CDK5 and phosphorylation of PPARγ (S273) was abolished by the deficiency of AMPKα (Figure 4(a)). Furthermore, we showed that in wild type diabetic mice, the mRNA expression of target genes of CDK5/PPARγ signaling, including adipsin, adiponectin, Txnip, Nr1d2, Ddx17, Rybp, and Nr3c1, was significantly increased by crocin (Figures 4(b)-4(h)). However, in AMPKα KO diabetic mice, no significant effect of crocin on the expression of those genes was observed (Figures 4(b)-4(h)).

Discussion
Our previous results have shown that crocin ameliorates glucose and lipid metabolic dysfunction in vivo and in vitro [9,10]. Using pharmacological inhibitors in mice and genetic manipulation in cells, we have found that AMPK activation plays a substantial role in the beneficial effect of crocin [9,10]. However, the causal role of AMPK in the biological and pharmacological activities of crocin is still not clear, and the downstream signaling of AMPK is still not completely understood.
In the present study, our goal is to define the causal role of AMPK activation in the protective effects of crocin against metabolic disorders using AMPKα KO mice. We used highfat diet and STZ to induce obese and type 2 diabetic models. Our results showed that crocin markedly inhibits the changes of glucose metabolic parameters and serum lipid profiles in wild type diabetic mice. In AMPKα KO mice, those protective effects of crocin against glucose and lipid metabolic dys-function were abolished. These results demonstrated AMPK activation was responsible for the beneficial effects of crocin on metabolic dysfunction. Moreover, we have shown that the antiobese effect of crocin has been abolished by the deficiency of AMPKα. Activation of AMPK is at the center of the proposed mechanism of metformin's action, which is the first-line drug for the treatment of type 2 diabetes [20]. AMPK is a heterotrimeric protein with α, β, and γ subunits. AMPK activation has been verified to enhance cellular glucose uptake and inhibit intracellular glucose production [21]. AMPK activity is reduced under both obese and type 2 diabetic conditions [22,23]. It has been suggested that AMPK plays an important role in the regulation of adipose dynamics through increasing basal lipolysis restraining βadrenergic-induced lipolysis [24]. All these results suggest that AMPK is a pivotal target of crocin which mediates the protective effect on glucose and lipid metabolic dysfunction.
PPARγ is an important transcription factor that plays substantial roles in the regulation of glucose and lipid

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BioMed Research International metabolism which is also a target of pharmacological therapy of diabetes [14,15]. PPARγ is critical for the adipogenesis through regulating a battery of genes responsible for triglyc-eride synthesis [25]. However, the interaction between PPARγ and AMPK is still not clear. In this study, we found that crocin significantly inhibited the phosphorylation of    Figure 4: Deficiency of AMPKα abolished crocin-induced effects on CDK5/PPARγ in type 2 diabetic mice. Type 2 diabetes was induced in wild type and AMPKα mice using high-fat diet incorporated with STZ injection. The type 2 diabetic mice were treated with crocin or vehicle. (a) CDK5 expression and phosphorylation of PPARγ in adipose tissue were determined using western blot (n = 6). (b-h) mRNA expression of CDK5/PPARγ targets was determined using RT-qPCR (n = 6). * P < 0:05, * * P < 0:01, * * * P < 0:005, * * * * P < 0:001. 6 BioMed Research International PPARγ in wild type diabetic mice, while this effect was abolished in AMPKα KO diabetic mice. The data suggest that AMPK activation is crucial for crocin-induced inhibition of the phosphorylation of PPARγ. This finding provides a link between the regulation of AMPK and PPARγ. In the literature, CDK5 is reported to regulate posttranslational modifications of PPARγ [16]. Phosphorylation of PPARγ at serine 273 by CDK5 has been shown to stimulate diabetogenic gene expression in adipose tissues [17]. The antidiabetic PPARγ ligand drugs, such as the thiazolidinediones and PPARγ partial/nonagonists, can inhibit this modification, leading to the therapeutic improvement [26]. In our study, we showed that crocin resulted in a significant decrease of CDK5 protein level in wild type diabetic mice, while this effect was abolished in AMPKα KO diabetic mice. The regulation of downstream targets of CDK5/PPARγ by crocin was abolished by the deficiency of AMPK. The results suggest that the downregulation of CDK5 might be involved in the regulation of PPARγ induced by crocin, and this effect is mediated by the activation of AMPK.
In this study, we confirmed the beneficial effects of crocin on liver injury, adiposity, obesity, and insulin resistance. These results suggest that crocin could have the potential to prevent diabetes-associated liver injury. Crocin is also a promising agent in preventing obesity and insulin resistance. Based on the crucial roles of phosphorylation of PPARγ and PPARγ signaling in the regulation of insulin signaling and adipocyte differentiation, we propose that AMPK-mediated inhibition of CDK5 and thus repression of PPARγ phosphorylation and activation is a pivotal step for crocin-induced beneficial effects. We have used AMPKα KO mice to confirm the causal role of AMPK activation in the protective effects of crocin. However, further studies are still needed to verify whether CDK5/PPARγ signaling is the unique downstream pathway and essential for crocin-induced protective effects.
In conclusion, our study verified that activation of AMPK is involved in crocin-induced protective effects against glucose and lipid metabolic dysfunction. Activation of AMPK downregulates the protein level of CDK5, followed by the decrease of PPARγ phosphorylation, leading to the inhibition of adipose formation and metabolic dysfunction. Our study provides new insights into the mechanism of protective effects of crocin and the interaction of AMPK and CDK5/PPARγ signaling.

Data Availability
The data will be available on request.

Conflicts of Interest
The authors declare no conflict of interest.