Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice

Insulin treatment was confirmed to reduce insulin resistance, but the underlying mechanism remains unknown. Caveolin-1 (Cav-1) is a functional protein of the membrane lipid rafts, known as caveolae, and is widely expressed in mammalian adipose tissue. There is increasing evidence that show the involvement of Cav-1 in the AKT activation, which is responsible for insulin sensitivity. Our aim was to investigate the effect of Cav-1 depletion on insulin sensitivity and AKT activation in glargine-treated type 2 diabetic mice. Mice were exposed to a high-fat diet and subject to intraperitoneal injection of streptozotocin to induce diabetes. Next, glargine was administered to treat T2DM mice for 3 weeks (insulin group). The expression of Cav-1 was then silenced by injecting lentiviral-vectored short hairpin RNA (shRNA) through the tail vein of glargine-treated T2DM mice (CAV1-shRNA group), while scramble virus injection was used as a negative control (Ctrl-shRNA group). The results showed that glargine was able to upregulate the expression of PI3K and activate serine phosphorylation of AKT through the upregulation of Cav-1 expression in paraepididymal adipose tissue of the insulin group. However, glargine treatment could not activate AKT pathway in Cav-1 silenced diabetic mice. These results suggest that Cav-1 is essential for the activation of AKT and improving insulin sensitivity in type 2 diabetic mice during glargine treatment.


Introduction
Insulin resistance plays an important role in the occurrence and development of type 2 diabetes mellitus (T2DM) [1]. Early intensive insulin therapy has been reported to delay or even partially restore the development of type 2 diabetes mellitus by improving insulin resistance (IR) [2,3]. However, the underlying mechanism is still unclear.
Visceral adipose tissue is a vital insulin responsive organ and has been confirmed to be positively correlated with insulin resistance [4]. Thus, improving insulin responsiveness of visceral fat has been regarded as an effective way to alleviate insulin resistance. Caveolin (Cav-1) is a structural component of caveolae and was discovered in 1950s [5,6]. Cav-1 is expressed abundantly in adipose tissue and is involved in many signaling pathways (e.g. insulin signaling) [7]. Caveolin-1 knockout mice develop remarkable insulin resistance [8,9], while upregulation of caveolin-1 could enhance insulin signal transduction and therefore improve glucose uptake after insulin stimulation [10]. Besides, Cav-1 minor allele could predict an association between IR and body fat composition [11]. In our preliminary study, we found a reduction of caveolin-1 mRNA in the omental adipose tissue of overweight patients [12]. Taken together, these results suggest that Cav-1 is responsile for insulin sensitivity. Additionally, previous studies showed that  was involved in the adipocyte differentiation process through the PI3K/AKT pathway and the development of IR [13,14]. Thus, we hypothesized that insulin treatment would upregulate Cav-1 expression in adipose tissue to alleviate insulin resistance. In this study, we used lentivirus to silence Cav-1 in type 2 diabetic mice and investigated the effect of Cav-1 depletion on insulin sensitivity, PI3K p110 expression, and AKT activation after glargine treatment.

Material and Methods
2.1. Cav-1 Knockout Type 2 Diabetic Mouse Model. Eightweek-old male C57BL/6J mice, 20-24 g, were purchased from the SLAC Animal Laboratory Company (Shanghai, China) and fed in the animal center at The Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The animal containment room was conditioned at 20°C -25°C and 50% ± 5% relative humidity with a 12 h light-dark cycle. A standard chow diet (4% fat (wt/wt), Guangdong Medical Laboratory Animal Center) was used to feed the negative control (NC) group (n = 5) and a highfat diet (HFD) (60% fat, D12492, Research Diets) to feed the experimental group (n = 20) (the feeding condition continues till the end of experiment). After 4 weeks of HFD feeding, the proposed diabetic group (n = 20) was injected intraperitoneally with a low dose streptozotocin (STZ, 40 mg/kg) (Sigma, USA) for 3 consecutive days after overnight fasting. Two weeks after the last injection of STZ, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine whether the diabetic mice were successfully set up. The diabetic mice were then randomly randivided into 4 groups (n = 5 for each group): T2DM (without any treatment), insulin (receiving insulin treatment), CAV-1-shRNA (receiving insulin treatment after Cav-1 silencing), and Ctrl-RNA (receiving insulin treatment without Cav-1 silencingd) ( Figure 1). In the CAV-1-shRNA group, Cav-1 was silenced by injection of lentivirus via the tail vein. In the Ctrl-RNA group, the mice were injected vectors through the tail vein. All the insulin-treated groups were injected subcutaneously with glargine, starting with 0.4 u/d and then adjusted to the amount of maintaining their fasting blood glucose at the same level for 2 weeks (14 days). The body weight of mice was measured biweekly, while their fasting blood glucose was monitored weekly during the whole process. At the end of the experiment (on the 15 th day of insulin treatment), all mice were fasted for 8 hours, anaesthetised by ether, and sacrificed for blood and periepididymal adipose tissue collection. All procedures were carried out in accordance with National Institutes of Health guidelines and approved by the Institute Animal Care and Use Committee (IACUC) at the Animal Ethics Committees of the Sun Yat-Sen University.

Intraperitoneal Glucose Tolerance Test and
Intraperitoneal Insulin Tolerance Test. In the intraperitoneal glucose tolerance test (IPGTT), mice were administered with glucose (2 g/kg wt i.p.) after overnight fasting. The intraperitoneal insulin tolerance test (IPITT) was conducted by insulin injection (0.75 units/kg wt i.p.) (Novolin R; Novo Nordisk, Denmark) after 6 hours of fasting (7 am-1 pm). Tail vein blood glucose level was measured at 0, 30, 60, and 120 min by the OptiumXceed glucometer (Abbott Diabetes Care, Inc., Alameda, CA). The methods were confirmed elsewhere [15]. To avoid hypoglycemia, the fasting periods for IPGTT and IPITT were different. For IPGTT, mice were usually fasted overnight. But for IPITT, the fasting period was shorter, usually 6 hours [15,16] or even 4 hours [17]. Here, we adopted overnight fasting for IPGTT and 6 hours fasting for IPITT, respectively.
2.6. Statistical Analysis. Data were presented as mean ± SEM. One way ANOVA test was performed to determine whether there were significant differences between groups. Multiple comparisons between groups were analyzed with LSD analysis of variance. P < 0:05 was considered to be statistically significant.

Glargine
Upregulates the Expression of PI3K p110, AKT Phosphorylation and Caveolin-1 in Periepididymal Adipose Tissue. After development of type 2 diabetes, mice in the insulin group were treated with glargine for 2 weeks. Compared with the mice in the NC group, mice in the T2DM group had lower levels of PI3K p110, a reduction in AKT phosphorylation, and caveolin-1 was downregulated in their periepididymal adipose tissue. However, after 2 weeks of glargine administration, PI3K p110 and p-AKT levels in T2DM groups were increased, and caveolin-1 expression was upregulated (Figure 4).

The Expression of PI3K p110 and AKT Phosphorylation
Could Not Be Upregulated after Caveolin-1 Depletion Even with Treatment of Glargine. In order to address the role of caveolin-1 in glargine-stimulating PI3K p110 expression and AKT activation, CAV-1 was silenced by short hairpin RNA in diabetic mice (CAV-1-shRNA group). Lentivirus vector without CAV-1 sequence was used as RNA inference control (Ctrl-RNA group). Glargine was administrated to the CAV-1-shRNA group, Ctrl-RNA group, and insulin group for 2 weeks. The inhibition of caveolin-1 expression led to a reduction in PI3K p110 and p-AKT expression in

Higher Glargine Doses Were Required to Maintain
Fasting Blood Glucose in CAV-1 Silenced Diabetic Mice. During the course of glargine treatment, the starting dose administered was 0.4 u/d and then adjusted ±0.1 u/d to maintain the fasting blood glucose between 72 mg/dL and 144 mg/dL [18]. Higher doses of glargine were administered, ranging from 0.4 u/d to 1.6 u/d in the CAV-1-shRNA group, while the insulin group received stable doses between 0.4 u/d and 0.8 u/d (Figure 6(a)). On day 1 after glargine treatment, the fasting blood glucose (FBG) between these two groups was markedly different (178:97 ± 61:80 mg/dL vs. 291:60 ± 60:62 mg/dL, P < 0:05).
However, compared with the insulin group, the CAV-1-shRNA group required more glargine to lower blood glucose.
In line with the glargine requirement to maintain the fasting blood glucose, the CAV-1-shRNA group had a higher HOMA-IR than the insulin group (24:802 ± 4:688 vs. 16:615 ± 2:407, P < 0:05). These results suggest that the CAV-1-shRNA group experienced worse insulin resistance than the insulin group.

Discussion
T2DM has become a global concern over the past three decades, accounting for more than 90% of individuals diagnosed with diabetes [19,20]. Attenuation of insulin metabolic action plays a vital role in the development of T2DM [21]. Thus, improving insulin sensitivity has become a recognized way to treat T2DM patients. Hu et al. showed that insulin sensitivity of patients with T2DM had been improved after insulin treatment as demonstrated by lower HOMA-IR values compared with normal glucose tolerance people [2]. But the mechanism is still unknown. In the molecular biology researches of insulin resistance and metabolism, caveolin-1 has been studied since early 1990s [22]. In the current study, we found that caveolin-1 could play a vital role in alleviating insulin resistance during insulin treatment by activating AKT phosphorylation. Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. It involves in many bioprocesses that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction [23]. Caveolin-1, the major scaffolding protein of caveolae (21-24 kDa), can recruit and regulate various signaling proteins through the interation of its scaffolding domain with hydrophobic sequence [24]. It has been demonstrated that Cav-1 can assist Akt signaling in mechanotransduction of vascular smooth muscle cells [25], indicating the close relationship between Cav-1 and AKT signaling. As a classical pathway in insulin signal transduction, any defects in the PI3K p110 and AKT activation along with the downstream molecules will lead to insulin resistance [26][27][28]. Consistent with these studies, our study showed that the expression of PI3K p110 and AKT phosphorylation were lowered in visceral adipose tissue of T2DM mice. Additionally, we found a lower expression of caveolin-1 in periepididymal adipose tissue of diabetic mice when compared to the NC and insulin groups. Similar results (the reduced expression of caveolin-1) have been reported in the omental adipose tissue of individuals who were overweight or had obesity [12,29]. However, the lowered expression of caveolin-1 in diabetic mice was not observed in subcutaneous fat ( Supplementary Fig. 1). This discrepancy may be due to the different function of intraabdominal fat and subcutaneous fat. Intra-abdominal fat is strongly and independently associated with both insulin resistance and poor glycemic control [30], while the subcutaneous adipose tissue is thought to maintain insulin sensitivity [31]. Thus, this suggests that downregulation of caveolin-1 in periepididymal adipose tissue can contribute to the pathogenesis of obesity and insulin resistance.
Schultze et al. revealed that glargine could activate the PI3K/AKT pathway and lower the blood glucose in humans and mice [32]. In accordance with these findings, our results showed an increase in p110 PI3K expression and AKT phosphorylation in periepididymal white adipose tissue of mice after receiving glargine. In addition, it was observed that the expression of caveolin-1 in T2DM was also increased after the glargine treatment. These results suggests that upregulation of caveolin-1 may be involved in activating AKT phosphorylation during insulin treatment. In order to address this speculation, CAV-1 was silenced by lentivirus with hairpin RNA in diabetic mice and then treated with glargine (CAV-1-shRNA group). The results showed that Akt activation could not be improved by glargine treatment when caveolin-1 was silenced. Moreover, the CAV-1-shRNA group expressed lower levels of PI3K p110 expression and p-AKT compared with the Ctrl-shRNA group, which implied the impairment of AKT activation in caveolin-1 absent mice. Taken together, these results suggest that caveolin-1 is indispensable to the insulin-activated AKT phosphorylation.
High-fat diet would interfere in lipid metabolism and exacerbate insulin resistance. Previous studies have demonstrated that caveolin-1 is involved in this process [35]. Caveolin-1 null mice were lean and resistant to dietinduced obesity due to reduced/atrophic fat deposits [9]. Histologically, when fed with HFD, Caveolin-1 knockout mice showed reduced adiposity, decreased expression of leptin and adiponectin compared with wild-type mice [9]. In our study, after feeding HFD for 8 weeks in total, the diameter of adipocytes in the Ctrl-RNA group was larger than the CAV1-shRNA group (Supplementary Fig. 3). This is similar with the previous study that caveolin-1 Even with the atrophy of adipose tissue, CAV-1 null mice showed insulin resistance in our study. The reason might be that exacerbation of hyperphagia driven by low leptin levels leads to the harmful spillover of lipids to other insulin sensitive tissues [9,36], leading to inflammation of these tissues such as muscles and liver. Thus, the lower expression of caveolin-1 could severe insulin resistance by affecting lipid metabolism.
In the future, further investigations are required to enrich our study. Firstly, more accurate methods can be used. For instance, a glucose clamp may be used to assess insulin resistance in diabetic mice before and after insulin treatment. Secondly, apart from silencing caveolin-1, the effect of caveolin-1 overexpression should also be applied. Thirdly, the lentivirus shRNA Cav-1 was not specific for adipose tissue; it was injected by tail vein, so it would silence Cav-1 expression in all the tissues of the mice. Thus, the effect of other insulin sensitive organs such as muscles and liver should be considered. And specific virus could be used to avoid the influence of other tissues. Finally, both in vitro and in vivo studies would be favorable to elucidate more underlying mechanisms, e.g. PI3K inhibitors need to be applied to further investigate caveolin's rolein PI3K/AKT pathway.
In conclusion, our study showed that caveolin-1 depletion could impair insulin sensitivity, PI3K p110 expression, and AKT activation. These findings suggest that CAV-1 has the potential to be a therapeutic target for the treatment of type 2 diabetes, obesity, and other metabolic disorders involving insulin resistance.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflict of interest.

Authors' Contributions
Hangya Peng contributed to the project design, experiment conduction, data analysis, and manuscript writing. Panwei Mu contributed to the data analysis, interpretation, and manuscript writing. Haicheng Li contributed to the project design and experiment conduction. Chuwen Lin contributed to the experiment conduction and data analysis. Shuo Lin, Keyi Lin, and Kunying Liu contributed to the experiment conduction and data analysis. Wen Zeng and Longyi Zeng contributed to the project design, data analysis and interpretation, manuscript revision, financial support, and final approval of the manuscript and were the guarantors of this work. All authors gave the final approval of the version to be published. Hangya Peng and Panwei Mu contributed equally to this work.