lncRNA Hnscr Regulates Lipid Metabolism by Mediating Adipocyte Lipolysis

Obesity is a process of fat accumulation due to the imbalance between energy intake and consumption. Long noncoding RNA (lncRNA) Hnscr is crucial for metabolic regulation, but its roles in lipid metabolism during obesity are still unknown. In this article, we found that the expression of Hnscr gradually decreased in adipose tissues of diet-induced obese mice. Furthermore, the deletion of Hnscr promoted an increase in body weight and adipose tissue weight by upregulating the expression of lipogenesis genes and downregulating lipolysis genes in inguinal white adipose tissue (iWAT) and brown adipose tissue. In vitro knockdown of Hnscr in adipocytes resulted in reduced lipolysis of adipocytes. Overexpression of Hnscr by adenovirus or drug mimics showed the opposite. Mechanistically, Hnscr regulated adipose lipid metabolism by mediating the cyclic adenosine monophosphate/protein kinase A signaling pathway. This study identifies the initial characterization of Hnscr as a critical modifier that regulates lipid metabolism, suggesting that lncRNA Hnscr is a potential target for treating obesity.

The worldwide obesity pandemic has dramatically increased the incidence of type 2 diabetes, cardiovascular disease, and other related complications (1,2).Obesity is caused by excess energy intake over energy expenditure in adipocytes, stored in white adipose tissue as triglycerides (3).Lipolysis and lipogenesis maintain a dynamic balance in adipocytes (4,5).Studies have shown that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are essential in triglyceride catabolism (6,7).The activity of lipases largely depends on their interaction with cofactors.HSL is a downstream target of protein kinase A (PKA).Several studies have shown that HSL can be upregulated by activating PKA phosphorylation, thereby promoting lipolysis and browning (8)(9)(10).Different phosphorylation sites also regulate the activity of ATGL lipase (11).For instance, β-adrenergic activation can lead to PKA-mediated phosphorylation of ATGL Ser(406) to modestly increase ATGL-mediated lipolysis (12).Although some research has been conducted on lipases, much remains to be said about the function of these lipases in vivo and their relative contribution to adipocyte lipolysis.
Long noncoding RNAs (lncRNAs), defined as nonproteincoding RNA transcripts longer than 200 nucleotides, are emerging as crucial regulators of different cellular processes (13).
The cyclic adenosine monophosphate (cAMP)/PKA pathway is the most well-known mechanism that mediates lipolysis (21).Increased levels of cAMP can lead to the activation of cAMP-dependent PKA.HSL is phosphorylated by PKA, resulting in the catalytic breakdown of triglycerides and diglycerides, producing free fatty acid and glycerol (21).In addition, cAMP/PKA mediates PGC-1α activation by activating ATGL, promoting oxidative metabolism in cell and animal models (22).Our previous study also indicated that RCN2 stimulates lipolysis by binding to NRP-2 and ITGB1 receptor complexes and activating the cAMP/PKA signaling pathway (23).Nevertheless, the role of lncRNA in this pathway is unclear and requires further investigation.
Our previous studies discovered that lncRNA Hnscr (Gm31629) is crucial to glucose metabolism (24).However, the role of Hnscr in regulating lipid metabolism has yet to be elucidated.In this article, we focus on the relationship between Hnscr and adipose tissue lipid metabolism.Decreased Hnscr expression in inguinal white adipose tissue (iWAT) and brown adipose tissue (BAT) was observed in high-fat diet (HFD)-induced obese mice.In in vivo and in vitro experiments, Hnscr reduction promotes adipogenesis and reduces lipolysis in adipocytes.In comparison, overexpression of Hnscr or supplementation with theaflavin 3-gallate (TF2A) stimulated adipocyte lipolysis.Mechanistically we found that Hnscr regulated lipid metabolism by mediating the cAMP/PKA signaling pathway.Taken together, our findings highlight Hnscr as a target for treating obesity by alleviating lipid metabolism disorders.
All mice were kept in a C57BL/6J background, maintained in the specific pathogen-free (SPF) class at the Experimental Animal Research Center of Central South University, with a constant temperature of 22 to 24 °C, constant humidity of 60% to 75%, 12-hour dark/light cycle, 4 or 5 mice per cage, free access to food and water, and regular change of bedding.All animal care protocols and experiments were reviewed and approved by the Animal Care and Use Committee of the Laboratory Animal Research Center, Xiangya School of Medicine, Central South University.The study complied with all relevant ethical regulations for animal research.

Cell Treatment
Gm31629-targeted small interfering RNAs (siRNAs) were purchased.The siRNA was transfected using lipofectamine RNAiMAX (Invitrogen) according to the instructions.Knockdown efficiency was detected by qPCR 2 days after transfection.Adenovirus control (Ad-Scramble) and adenovirus Hnscr (Ad-Hnscr) were generated from Obio Co (Shanghai, China).A 12-well plate was seeded with 3T3-L1 cells at 5 × 104 cells/well overnight (70-80% confluency), then transfected with siRNA or adenovirus at an multiplicity of infection (MOI) of 7 for 2 days before induction of adipocyte differentiation.Cells were treated with TF2A (80 μmol) for 72 hours before induction of adipocyte differentiation.
After 2 to 3 rounds of induction, the fluid was replaced with growth medium.Then cells were treated with H-89 (25 μM; Beyotime) for 2 hours.Cells were collected for further analysis.

Rectal Temperature and Infrared Imaging
The mice were placed in a temperature-controlled chamber (MMM Friocell, Germany) at 4 °C for 4 hours.The rectal temperature was measured with a rectal probe at a specified time after cold exposure.Body temperatures were read before and 2 hours after cold exposure using infrared thermography (T1010, FLIR).Infrared images were analyzed with FLIR-Tools-Software (version 6.4).

Quantitative Real-Time PCR
Real-time PCR analysis was conducted as previously described (28,29).The SYBR Green PCR Master Mix was purchased from Accurate Biotechnology (China).The primer pairs used for qRT-PCR are listed (Table 1).The relative mRNA levels of target genes were given by the 2 −ΔΔCt method using β-actin as an internal control.and eosin (H&E) staining.Adipocyte diameters were quantified using ImageJ V1.42q (National Institutes of Health, Bethesda, MD).

Adipocytes Oil Red O Staining
Primary iWAT adipocytes or 3T3-L1 cells that induce differentiation maturation were collected, the medium was discarded, and cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature.The 4% paraformaldehyde was discarded and cells were immersed in 60% isopropanol for 5 minutes.Then the 60% isopropanol was discarded and cells were stained with Oil Red O.

Glycerin Measurement
Glycerin was measured with a glycerin kit according to the manufacturer's instructions.The glycerin kit was purchased from Elabscience Company (Wuhan, China).

Statistical Analysis
All data are expressed as means ± SEM.The statistical significance of the differences between various treatments or groups was measured by either Student's t-test or analysis of variance (ANOVA) followed by the Bonferroni post-test.Data analyses were performed using GraphPad Prism 7.0.P < .05 was considered statistically significant.

Identification of Adipose lncRNA Hnscr as a Metabolic Signature
Our previous study found that lncRNA Hnscr could regulate systematic aging (25) and system insulin resistance (24).Therefore, we wondered whether lncRNA Hnscr played a vital role in lipid metabolism in adipose tissues.
Firstly, we found that the expression level of Hnscr was decreased in iWAT and BAT in diet-induced obese mice (Fig. 1A).Furthermore, we detected the expression level of Hnscr in mature adipocytes and SVFs from iWAT of WT mice.The results showed that both mature adipocytes and SVFs expressed Hnscr, and mature adipocytes expressed higher levels of Hnscr than SVFs (Fig. 1B).To investigate the effect of Hnscr on lipolysis, we successfully constructed Hnscr-deficient (Hnscr-null) mice using TetraOne technology (Fig. 1C).Then the WT mice and Hnscr-null mice were fed NCD or HFD.For the NCD group, although the body weight displayed no significant difference between the WT mice and Hnscr-null mice (Fig. 1D and 1E), the weights of iWAT and epididymal white adipose tissue (eWAT) were increased in Hnscr-null mice (Fig. 1F and 1G).
In line with our expectation, the phenomena were more pronounced in the HFD groups.At 3 months of HFD feeding, Hnscr-null mice gained weight more rapidly than WT mice (Fig. 1H).We also examined the food intake of the 2 groups of mice and found that there was no significant difference (Fig. 1I).After 3 months of HFD intervention, Hnscr-deficient mice were fatter in appearance than WT mice (Fig. 1J).Although eWAT and BAT weights were not significantly different in Hnscr-null mice, iWAT weights were significantly increased (Fig. 1K and 1L).Together, the expression of Hnscr in adipose tissues decreased after HFD feeding, and Hnscr deletion correlated with body weight and fat weight, highlighting Hnscr as a metabolic feature of adipose tissue.

Hnscr Knockout Promotes Lipogenesis and Decreases Lipolysis in iWAT
White adipose tissue is the largest triacylglycerol reservoir and free fatty acid supplier to other tissues (30).It is noteworthy that in obese people, subcutaneous lipolysis is reduced (31,32).To verify the role of Hnscr in white adipose tissue, we extracted iWAT from Hnscr-null mice and WT mice fed with HFD.Hnscr expression was significantly lower in the iWAT of Hnscr-null mice (Fig. 2A).H&E staining demonstrated the iWAT of Hnscr-null mice contained a higher proportion of large adipocytes (Fig. 2B and 2C).RT-qPCR analysis revealed increased lipogenesis and fat uptake-related genes (Fig. 2D).Despite enrichment in some fat β-oxidation-related genes (Fig. 2D), the expression of browning and lipolysis-related genes decreased (Fig. 2E and 2F).
Western blot (WB) analysis indicated that the expression of UCP1 decreased in iWAT of Hnscr-null mice (Fig. 2G and  2H).We thus asked if Hnscr knockout alters lipid metabolism in iWAT by evaluating the lipolysis enzyme ATGL and HSL phosphorylation that stimulate triglyceride hydrolysis.WB analysis showed that in the iWAT of Hnscr-null mice, the protein levels of ATGL and phosphorylation of HSL were reduced (Fig. 2I and 2J).The results show that the downregulation of Hnscr increases lipogenesis and decreases lipolysis in iWAT.

Hnscr Knockout Decreases Lipolysis in BAT and Impairs Thermogenesis
BAT is considered to be an important site of adaptive thermogenesis.The thermogenic activity of brown adipocytes can help resist obesity and metabolic diseases such as type 2 diabetes and dyslipidemia (33).Active BAT is present in adults, but its activity is impaired in obese patients (34).Therefore, we explored the role of Hnscr deletion in BAT.Compared with WT mice, H&E staining showed an increase in average adipocyte size due to a large amount of lipid accumulation in Hnscr-null mice (Fig. 3A).Similar to iWAT, there was a significant increase in genes associated with lipogenesis, accompanied by a trend toward increased fat uptake and decreased fat β-oxidation (Fig. 3B).UCP1 and lipolysis genes decreased in Hnscr-null mice in BAT (Fig. 3C and 3D).WB analysis revealed downregulation of UCP1 and lipolysisrelated proteins in Hnscr-null mice (Fig. 3E-3H).Our results suggest that Hnscr knockout may promote fatty acid synthesis while reducing basal lipolysis in BAT during conditions of nutrient excess.
To determine the role of Hnscr in adaptive thermogenesis, mice were exposed to cold (4 °C).The rectal temperature of the mice exposed to cold (Fig. 3I) and infrared thermal imaging (under room temperature or 4 °C for 2 hours) (Fig. 3J and 3K) showed that Hnscr-null mice had a lower core body temperature than WT mice.These data demonstrate that Hnscr inhibition decreases lipolysis in BAT and impairs thermogenesis.

Hnscr Knockdown in Adipocytes Decreases Lipolysis
To verify that differences in fat weight, cell size, and gene expression are a direct effect of Hnscr in adipose tissue, we transfected the Hnscr targeted siRNAs (si-Hnscr) and control siRNAs in primary adipocytes.The results show that si-Hnscr significantly inhibited the level of Hnscr in cells (Fig. 4A).Meanwhile, RT-qPCR analysis displayed that higher lipogenesis, fat uptake, and β-oxidation (Fig. 4B) coexisted with lower UCP1 and lipolysis (Fig. 4C and 4D) mRNA levels after Hnscr knockdown.Consistently, si-Hnscr treatment also decreased the expression of ATGL and p-HSL (Fig. 4E and 4F).Moreover, lipid measurement of cells also revealed that knockdown of Hnscr in adipocytes decreased glycerol level (Fig. 4G).
Taken together, we concluded that Hnscr deficiency inhibits adipocytes lipolysis.

Overexpression of Hnscr Facilitates Lipolysis of Adipocytes
To explore the effect of Hnscr in adipocytes, primary adipocytes were isolated and transfected with an Hnscr-expressing adenovirus (Ad-Hnscr).As expected, the expression of Hnscr was dramatically increased compared with the control group (Fig. 5A).Overexpression of Hnscr downregulated mRNA levels of genes involved in lipogenesis in differentiated primary adipocytes (Fig. 5B).Moreover, the levels of β-oxidation, and thermogenic and lipolysis-related genes were elevated in the Hnscr overexpression group (Fig. 5B-5D).WB analysis confirmed the protein level that Hnscr could promote lipolysis (Fig. 5E and 5F).These data indicate that overexpression of Hnscr facilitates lipolysis of primary adipocytes.3T3-L1 preadipocytes are clonally isolated sublineages from 3T3 cells (Swiss albino) (35,36).The 3T3-L1 cell line can be stable for passage and has reasonable specificity for differentiation to adipocytes, so it has become an internationally recognized cell model for studying adipose metabolism (37,38).Therefore, we transfected it with a Hnscrexpressing adenovirus in 3T3-L1 cells (Fig. 5G).Consistent with the situation in primary adipocytes, overexpression of Hnscr in differentiated 3T3-L1 cells produced a trend toward downregulation of mRNA levels of genes involved in lipogenesis and fat uptake.(Fig.5H), accompanied by upregulating β-oxidation, thermogenic, and lipolysis genes (Fig. 5H and 5J).WB analysis also indicated that the levels of lipolysis-related proteins increased (Fig. 5K and 5L).Cell lipid measurement

TF2A Treatment In Vitro Promotes Lipolysis of Adipocytes
Previously, we found that TF2A, a small, naturally occurring compound, could mimic the activity of Hnscr (25).To confirm the role of Hnscr in regulating lipid metabolism, mice primary adipocytes and 3T3-L1 cells were incubated with TF2A.Oil Red O staining results indicated that administration of TF2A inhibited lipid accumulation in adipocytes (Fig. 6A).
The levels of lipogenesis genes in the TF2A groups were also decreased (Fig. 6B and Fig. 6H).Although increased expression of uptake genes and reduced expression of β-oxidation genes (Fig. 6B and Fig. 6H) were found in the TF2A groups, lipolysis and browning-related genes were significantly upregulated (Fig. 6C and 6D and Fig. 6I and 6J).Further verification by WB experiments showed that the lipolytic capacity of primary adipocytes and 3T3-L1 cells was augmented in the TF2A groups (Fig. 6E and F and Fig. 6K and 6L).Cells lipid measurement revealed that treatment with TF2F in adipocytes increased the glycerol level (Fig. 6G).In summary, the Hnscr mimic, TF2A, exerts a beneficial effect on lipid metabolism of adipose tissue by promoting lipolysis, indicating its therapeutic potential.

Hnscr Activates the cAMP/PKA Signaling Pathway to Promote Lipolysis in Adipocytes
The cAMP/PKA signaling pathway is a well-established mechanism for activating lipolysis in adipose tissue (21).In our previous study, the cAMP/PKA signaling generally activated lipolysis by stimulating HSL phosphorylation in peripheral white adipose tissues (23).Therefore, we investigated whether Hnscr promotes adipocyte lipolysis by activating the cAMP/ PKA signaling pathway.WB analysis indicated Hnscr overexpression promoted protein levels of cAMP and phosphorylation of PKA in both primary adipocytes and 3T3-L1 cells (Fig. 7A-7D).Furthermore, the expression levels of cAMP and phosphorylated forms of PKA were also significantly increased in the TF2A treatment groups (Fig. 7E-7H).We pretreated adipocytes overexpressing Hnscr with H-89, a pharmacological PKA inhibitor.The results showed significantly decreased lipolysis-related proteins (Fig. 7I).To confirm our results, we detected the level of cAMP/PKA signaling in vivo.Compared with WT mice, the protein levels of cAMP and phosphorylation PKA were significantly decreased in iWAT and BAT of Hnscr-null mice (Fig. 7J-7M).Therefore, these findings clarify that Hnscr regulates adipose tissue lipolysis by activating the cAMP/PKA signaling pathway (Fig. 7N).

Discussion
It has been shown that HFD-induced obesity significantly decreases the proportion of mature adipocytes, and substantially increases the size of mature fat cells (adipocyte hypertrophy) and adipose tissue weight (39).One of the most important observations in this study was the finding of decreased levels of lipolysis in adipose tissues of Hnscr-null mice, which was particularly evident after excess nutrition.We first found downregulation of lncRNA Hnscr expression in iWAT and BAT of the HFD-induced obese mice.Since Hnscr has been shown to mediate systematic glucose metabolism in previous studies (24), we constructed Hnscr-null mice to investigate its role in lipid metabolism.Past studies have shown that a critical feature of obesity is excess storage of triglycerides in adipose tissue through adipocyte hyperplasia (increased number) or hypertrophy (increased volume), or even both.It is generally believed that adipocyte hypertrophy occurs before adipocyte hyperplasia and is the primary mechanism of fat mass expansion (40).In this study, H&E staining showed that loss of Hnscr resulted in mice adipocyte hypertrophy.In iWAT and BAT, Hnscr deletion led to increased lipogenesis and fat uptake in adipose tissues, but the expression of thermogenic genes such as UCP1 and lipolytic genes was downregulated.
Lipolysis is described as the metabolic process of catabolism of triacylglycerols stored in cellular lipid droplets (41).Abnormal lipolysis may be associated with lipodystrophy, hyperlipidemia, or obesity (42,43).ATGL and HSL are responsible for more than 90% of triglyceride hydrolysis in adipose tissue.It is found that ATGL is the essential lipase for the hydrolysis of triglycerides to diglycerides, which HSL catalyzes into monoglycerides and glycerol (6,7).We explored the role of Hnscr in in vitro experiments and found that overexpression of Hnscr in adipocytes inhibited lipogenesis and promoted lipolysis.Furthermore, our results also showed that although the intervention of adipocytes with TF2A resulted in increased expression of genes related to lipogenesis and uptake, there were significantly increased levels of browning and lipolysis.
The cAMP/PKA signaling pathway is a recognized mechanism for activating lipolysis in adipose tissue (23).The results confirmed that Hnscr regulated adipose lipid metabolism by mediating the cAMP/PKA signaling pathway.Overexpression of Hnscr in adipocytes increased the protein levels of cAMP/ PKA signaling.Moreover, TF2A can mimic the action of Hnscr and promote cAMP/PKA activation.We also verified in Hnscr-null mice that Hnscr knockout downregulated the cAMP/PKA signaling activity in adipose tissues.Starting from the discovery of decreased Hnscr expression in iWAT and BAT of obese mice, we constructed Hnscr-null mice to explore the role of Hnscr in adipose tissue.Meanwhile, we also found that overexpression of Hnscr promoted lipolysis in adipocytes.Mechanistically, Hnscr regulated lipid metabolism by activating the cAMP/PKA signaling pathway.We also intervened in adipocytes with TF2A, a compound that could mimic the activity of Hnscr to promote lipolysis (25).Future works should identify the potential PKA phosphorylation sites on Hnscr and their impact on its function.The reasons for the contradictory relationship between TF2A in fat uptake and lipolysis were also studied.This study revealed that Hnscr could regulate lipid metabolism in obese mice by mediating adipocyte lipolysis, which could become a new therapeutic target for obesity.