Background
Insulin resistance is a complex pathological state of inappropriate cellular response to insulin hormone in insulin dependent cells, and it is a common risk factor in metabolic disorder associated diseases [
1]. Adipose is now recognized as not only an energy-storage tissue, but also an endocrine tissue that can secrete a variety of bioactive substances including adipokines and proinflammatory cytokines [
2]. Adipocytes and adipose tissue dysfunctions are believed to promote insulin resistance and lead to obesity [
3,
4]. Although considerable progress has been made in understanding the molecular mechanisms underlying these individual disorders, satisfactory treatment modalities remain limited.
Abnormal autophagy has been implicated in a variety of diseases, such as obesity, type 2 diabetes, cancer and cardiovascular disease [
5,
6]. The expression of
Atg7 in adipose tissue has a protective effect on insulin sensitivity in high-fat diet induced obesity, indicating autophagy activation contributes to the regulation of fat mass [
7]. Autophagy was activated in adipose tissues of obese individuals and inhibition of autophagy enhanced pro-inflammatory gene expression both in adipocytes and adipose tissue explants, indicating autophagy might inhibit inflammatory gene expression in adipose tissue during obesity [
8,
9]. Recently, autophagic dysfunction has been suggested a potential link to obesity and ER stress. There are three central ER stress signaling molecules in mammalian cells, namely IRE1α, PERK, and ATF6 [
10]. Several strategies have been proposed to target ER stress as a therapeutic approach for pharmacological intervention in obesity and type 2 diabetes. Thus, downregulation of autophagy could be beneficial for adipocytes under ER stress and insulin resistance.
CaMKIV is a multifunctional serine/threonine protein kinase encoded by
CaMKIV gene, and it plays a critical role in process of transcriptional regulation of lymphocytes, neurons and male germ cells [
11‐
13]. Recently, CaMKIV has been identified as a regulator in glucose metabolism and insulin genes expression [
14,
15], as well as in process of autophagy. For instance, CaMKIV not only increased autophagy to limit hepatic damage, but also involved in lipopolysaccharide induced inflammation and acute kidney injury [
16,
17]. In the previous study, we further demonstrated CaMKIV limits metabolic damage through induction of hepatic autophagy by CREB in high-fat diet-induced obese mice [
18]. As a significant regulator of autophagy, mTOR could be upregulated by CaMKIV [
17]. In adipose tissues, autophagy was significantly increased in diabetes compared with non-diabetes, and mTOR expression was decreased in adipose of diabetes cases, indicating autophagy was negatively regulated by mTOR expression in adipose tissues of patients with diabetes [
19].
In the previous study, to upregulate CaMKIV expression, constitutive active form of CaMKIV was usually used in vitro by transfection. However, in recent years, recombinant CaMKIV peptide has been used in several in vivo and in vitro studies [
18,
20]. Exogenous CaMKIV peptides were suspected to transport intracellularly through binding to specific receptors. However, the membrane receptors of CaMKIV still remain unknown. Signaling through the transmembrane receptor Notch is widely used throughout animal development, and it is a major regulator of cell proliferation and differentiation [
21]. It is interesting to note CaMKIV enhanced osteoclast differentiation through up-regulating Notch signaling [
22]. In addition, it can potentiate Notch-dependent transcription by triggering nuclear export of SMRT (silencing mediator for retinoid and thyroid hormone receptor) [
23]. These results gave us a clue that Notch might to be a potential receptor of CaMKIV.
Our previous study has demonstrated that CaMKIV plays an important role in regulating liver insulin sensitivity and plasma inflammation factors in high-fat diet-induced obese mice [
18]. On the other hand, in white adipose tissues, CaMKK2 regulates adiposity and pre-adipocyte differentiation. In brown adipose tissues, CaMKK2 plays a critical role in adaptive thermogenesis [
24]. Furthermore, CaMKIV is a direct downstream substrate of CaMKK2. Hence, we hypothesized CaMKIV might play an important role in regulating the metabolism of adipose tissue.
As a basic leucine zipper type transcription factor, CREB is ubiquitously expressed in organs. Its phosphorylation at Ser 133 is initiated by the recruitment of CaMKII and CaMKIV, interestingly, CaMKII can also phosphorylate CREB at Ser 142 and induce negative regulation [
25,
26]. It has been suggested CREB regulates expression of IRE1a and PERK, which suggested CREB regulates the key components of UPR [
27]. Rapamycin-induced autophagy against oxidative stress, synaptic/neurotransmission dysfunction, and cognitive deficits in the hippocampus of the rat brain through PI3K/Akt1-mTOR-CREB signaling pathway(s), which indicates mTOR/CREB signaling plays a critical role in autophagy function [
28]. Therefore, we propose that CaMKIV could regulate mTOR/CREB signaling to inhibit ER stress and improve insulin sensitivity through reduction of autophagy in adipocytes.
This study was undertaken to test our hypothesis that CaMKIV through decreased autophagy can suppress ER stress and improve insulin resistance by mTOR/CREB signaling. We first tested the insulin sensitivity, ER stress and autophagy function in Tun (tunicamycin)-treated mature 3 T3-L1 cells with or without recombinant CaMKIV. To further identify the mechanism of CaMKIV on insulin resistance, we next analyzed the markers of ER stress, autophagy and insulin sensitivity after blockage mTOR/CREB signaling in Tun-treated adipocytes. Our results provided a reciprocal functional interaction among CaMKIV, ER stress, autophagy and insulin signaling in Tun-treated adipocytes, indicating that CaMKIV regulated autophagy may function as an adaptive role in response to ER stress-induced insulin resistance.
Methods
Antibodies and reagents
The following antibodies were used: Atg7 (Cell Signaling, #2631), p62 (Cell Signaling, #5114), LC3 (Cell Signaling, #4108), CREB (Cell Signaling, #9197), p-CREB (Cell Signaling, #9198), mTOR (Cell Signaling, #2972), p-mTOR (Cell Signaling, #2971), IRS-1 (Cell Signaling, #2382), p-IRS-1 (Cell Signaling, #2381), Akt (Cell Signaling, #4685), p-Akt (Cell Signaling, #4060), p-PERK (Cell Signaling, #3179), PERK (Cell Signaling, #5683), cleaved-ATF-6 (Santa Cruz Biotechnology, #sc-166,659), GAPDH (Santa Cruz Biotechnology, #sc-47,724) and peroxidase goat anti-rabbit IgG (Santa Cruz Biotechnology, #sc-2768). Insulin were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Mouse recombinant CaMKIV were obtained from Sino Biological (Sino Biological Inc. Wayne, PA, USA). Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate Kit (PerkinElmer Inc., Richmond, CA, USA) was used to detect protein expression. Individual protein bands were quantified by ImageJ software. All other chemicals were obtained from standard resource and were of the highest grade available.
Cell culture and treatment
3 T3-L1 were purchased from American Tissue Culture Collection (ATCC, Manassas, Virginia, USA, #ATCC® CL-173™) and maintained in DMEM (Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere with 5% CO
2. The protocol of inducing maturation of 3T3L1 cells was performed as described [
29] and which was mini-modified in our present study. In brief, for adipocytes differentiation, 100% confluent 3 T3-L1 were induced with MDI induction media (0.5 mM 1-methyl-3-isobutylmethylxanthine, 200 nM dexamethasone, 160 nM insulin, and DMEM with 10% FBS) (day 0). Two days later media was changed to 10% FBS/ DMEM with 160 nM insulin. Cells were then fed with this maintenance medium every 2 days. Full differentiation is usually achieved on the 12th day. The mature 3 T3-L1 adipocytes were used in our ongoing experiments. To induce ER stress, mature 3 T3-L1 cells were treated with different concentration (0-5 μg/ml) of Tun for 4 h. For the effects of CaMKIV, cells were treated with 100 ng/ml CaMKIV for 24 h. For blocking mTOR or CREB signaling, cells were transfected with 100 nM mTOR siRNA or 100 nM CREB siRNA for 24 h, respectively. For insulin signaling, cells were stimulated with 10 nM insulin for 10 min. Before each experiment, the medium was replaced by fresh medium.
Electron microscopy analysis
The protocol we followed was described previously [
30]. In brief, we fixed adipocytes in 4% paraformaldehyde/2% glutaraldehyde/0.1 M sodium cacodylate pH 7.3, post-fixed in 1% osmium tetraoxide and embedded in epoxy resin (Epon). Then the ultrathin sections (80 nm) were stained by aqueous uranyl acetate, and lead citrate and examined with JEOL 2000FX transmission electron microscope (JEOL)., The numbers of autophagolysosomal-like vacuoles were counted in each field and normalized by the surface area for quantification of autophagolysosome-like vacuoles.
Small interfering RNAs (siRNAs) and transfection
Small interfering RNA (siRNA) for target genes (Atg7: sc-41,448; CREB: sc-35,111; mTOR: sc-35,410, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or scrambled siRNA (CREB, sc-37,007, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfected cells were cultured in medium containing 10% FBS for 24 h after transfection. The knockdown efficiency was assessed by western blot.
Western blotting and immunoprecipitation
To prepanre cell lysates, RIPA Lysis and Extraction Buffer (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) which contained 10% protease inhibitor (Thermo Scientific, USA) were used to extract cell lysates by incubated on ice for 30 min, and then centrifuged at 14000 x g for 15 min at 4 °C. The protein concentrations were determined by BCA kit (Thermo Scientific, USA). For western blotting, we used the protocol which was performed previously [
31]. In brief, we first mixed supernatants with 4x SDS-PAGE sample loading buffer, and they were denatured at 95 °C for 10 min. Second, the proteins were separated by SDS-PAGE gel, transferred to a polyvinylidene difluoride membranes, incubated with specific primary antibodies at 4 °C overnights, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA). For immunoprecipitation, the lysate was treated using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) according to the protocol. The final precipitated proteins were analyzed via western blotting with the corresponding antibodies.
Statistics
Data were analyzed by the Prism software, version 8.0 (GraphPad Software Inc., San. Diego, CA, US). Characteristics of subjects between 2 groups was performed using Mann Whitney test. Multiple comparisons of quantitative variables among groups were made using Kruskal Wallis test [
32]. Data were presented as mean ± SD. N represents the number of animals used. A
P value of<0.05 or
P value of<0.01 was considered as significantly or highly significantly difference.
Discussion
In this study, we first proved recombinant CaMKIV protein inhibites autophagy and ER stress and improves insulin sensitivity in tunicamycin-treated mature 3 T3-L1 adipocytes. Next, we further identified these protective effects of CaMKIV were nullified by downregulating mTOR or CREB expression, indicating CaMKIV regulates ER stress, abnormal autophagy and insulin sensitivity in Tun-treated 3 T3-L1 cells through mTOR/CREB signaling. In addition, CaMKIV inhibits ER stress and improves insulin sensitivity in Atg7 siRNA transfected cells. This result further demonstrated the protective effect of CaMKIV on autophagy, ER stress and insulin signaling.
Recently, autophagy dysfunction and ER stress are recognized as the important factors of insulin resistance [
33]. In the process of ER stress, ER stress sensors IRE1, PERK and ATF6 are activated, leading to a series of downstream events, including reducing translation and increasing transcription ER chaperones to make sure that normal cell function and viability are maintained [
34]. Autophagy is an evolutionarily conserved lysosomal mechanism that enable cells to conserve and maintain cellular biomass quality and quantity by targeting damaged or unused proteins and even organelles of degradation [
6]. Previous studies have reported ER stress can be triggered by obesity or metabolic factors, such as lipids, glucose and cytokines [
35‐
37], and it was a common factor in high-fat feeding, genetic obesity and elderly [
37,
38]. These results demonstrated autophagic dysfunction and ER stress were the main pathway that response to the pathological factor, including lipotoxicity, inflammation and insulin resistance. In the present study, Tun treatment not only induces ER stress, but also induces autophagic dysfunction and insulin resistance in adipocytes. Our data further provided the evidence that autophagy remarkably associated with ER stress and insulin resistance.
CaMKIV has been identified as a regulator in glucose metabolism and insulin signaling. For instance, its overexpression in skeletal muscle led to systemic improvements in insulin sensitivity and its activation involved in hepatic and adipose insulin action via increases in myokines released from the skeletal muscle [
39]. Moreover, 12-week of CaMKIV injection in obese mice could improve high-fat diet-induced hepatic insulin resistance, further indicating CaMKIV plays an important role in whole-body glucose metabolism and hepatic insulin signaling [
18]. It is well-known that Ca
2+ signaling is a major regulator of CaMKIV in cellular, and disruption of Ca
2+ homeostasis in the ER is well documented to trigger ER stress. According for these finding, we suspected CaMKIV might affect ER stress. Our results demonstrated CaMKIV inhibits ER stress markers, such as PERK and Cleaved-ATF6, indicating CaMKIV plays a critical role in regulating ER function.
Recent evidences implicate CaMKIV involved in autophagy regulation. However, its effect on autophagy activation is opposite in various tissues. John Evankovich and their colleagues [
16] suggested CaMKIV limited organ damage in hepatic ischemia-reperfusion (I/R) injury through induction of autophagy. Compared with wild-type mice, the expressions of LC3II and Beclin 1 were significantly decreased in CaMKIV KO mice after I/R. The in vitro study also demonstrated upregulation of CaMKIV by transfecting with dominant-active mutant CaMKIV-dCT could increase LC3II expression. Another study showed Atg7 and LC3II expressions were significantly induced in pMФ which were transfected with constitutively active CaMKIV (CaMKIV-dCT), indicating CaMKIV increases autophagy activity. Interestingly, elevating CaMKIV activity through the transfection of active CaMKIV-dCT increased mTOR protein concentration, suggesting CaMKIV increases autophagy through inducing mTOR expression [
17]. In our previous study, high-fat diet-induced defective liver autophagy was improved by long-term recombinant CaMKIV protein injection in obese mice [
18]. These results indicated CaMKIV have a pro-autophagic effect in liver. Nevertheless, autophagy was significantly induced in adipose tissue of diabetes compared with non-diabetes, as well as decreased mTOR expressions in adipose tissue of diabetes cases. However, LC3 expression significantly increased after rapamycin (an inhibitor of mTOR) treatment with adipocytes, indicating autophagy was negatively regulated by mTOR expression in adipose tissues [
19]. mTOR typically serves as a negative regulator of autophagy, and as a consequence, initiation of autophagy is largely dependent on release of mTOR inhibition [
40]. Our data suggested CaMKIV incubation significantly increased the p-mTOR expression, suggesting CaMKIV inhibited autophagy associated with p-mTOR expression. Hence, CaMKIV not only can increase autophagy but also can decrease autophagy in different tissues might be due to regulate mTOR signaling pathway.
Recent studies strongly suggested several factors including metabolic stressors, obesity, free fatty acid, and inflammatory cytokines could promote autophagic disorder of adipocytes [
41,
42]. Targeted deletion of the
Atg7 gene in adipose tissue can destroy autophagy pathway, which protects mice from high-fat diet-induced obesity and insulin resistance, suggesting that activation of the autophagy-mediated pathway may be one of the mechanisms of obesity-induced insulin resistance [
7]. In our study,
Atg7 ablation-induced ER stress and impaired insulin sensitivity could be reversed by CaMKIV incubation, suggesting the protective role of CaMKIV in autophagy defective adipocytes.
CREB is a transcription factor that integrates growth factors, Ca
2+, and cyclic AMP-induced signaling [
43]. As a target of the cAMP/PKA pathway, CREB can be activated by Ca
2+/calmodulin-dependent protein kinase and phosphorylated by kinases of the MAPK pathway [
44]. Several groups subsequently showed that CaMKIV phosphorylated CREB at Ser133 in vitro and stimulated CREB transcriptional activity in vivo, which led to the suggestion that CaMKIV was the principal Ca
2+-stimulated CREB kinase [
26,
45]. The mTOR/CREB pathway is an intracellular signaling pathway, and it is important in several normal cellular function and in regulation of autophagy [
46,
47]. Here, we supposed that the activation of mTOR/CREB signaling is required for CaMKIV-mediated ER stress, autophagy, and the restoration of insulin signaling. It is interesting to note activated CREB has been demonstrated in adipose cells under obese conditions, where it promotes insulin resistance by triggering expression of ATF3 and downregulating expression of GLUT4, indicating CREB plays and negative role in obesity induced insulin resistance [
48]. However, in our study, ablation of CREB nullified the protective role of CaMKIV in regulation of autophagy, ER stress and insulin resistance. Our study enhances understanding of the mechanisms by which mTOR/CREB contributes to the regulation of CaMKIV-induced modulation in adipose.
This study demonstrated for the first time that the inhibition of ER stress and improvement of insulin signaling is due to the restored autophagy function which improved by recombinant CaMKIV protein rather than the direct effects of this enzyme. However, our study does have limitations which should be further investigated. First of all, it has not been shown the effects of transfection-mediate overexpression of CaMKIV on ER stress, autophagy and insulin signaling in mature adipocytes. Therefore, we cannot be certain that whether the in vitro treatment by CaMKIV protein and transfection-mediate overexpression of CaMKIV produce the same effects on ER stress, autophagy and insulin signaling in these adipocytes or not. On the other hand, although we proposed the protective effects of CaMKIV in adipocytes might be due to intracellular signaling, the receptors of CaMKIV have not been identified, as well as the binding sites. Hence, our further prospective studies are needed to demonstrate the effects of transfection-mediate overexpression of CaMKIV on ER stress, autophagy and insulin sensitivity in adipocytes, and to find the specific membrane receptors of CaMKIV and the binding sites. Although further studies are required, our results provided therapeutic implications of CaMKIV for modifying insulin signaling and autophagy function under the condition of ER stress in the adipocytes.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.