Result

3.4.1 TN is secreted from human adipocytes and expressed in the adipose tissues of the mice.

To identify protein factors secreted from adipocytes, we analyzed the culture medium of human primary adipocytes by 2-D PAGE. As expected, there is at least 80 spots found on the gels (Fig. 3-1), among which three prominent ones are further characterized by mass spectrometry (LC-MS/MS) and found to be TN (with pI = 5.3 and, M.W. = 22 kDa, Fig. 3-1), retinal binding protein 4 (RBP4) and cysteine sulfinic acid decarboxylase (data not indicated), respectively. We later confirmed that they are indeed expressed in adipocytes and were previouly considered to be serum proteins ^85,86 with RBP4 defined as an adipocytokine, which regulates insulin signaling pathways and plays a role in the development of insulin resistance ⁸⁷. Interestingly, TN was a novel scecretory protein identified in the adipocyte culture medium in the current study.

B A

Fig 3-1 Tetranectin (TN) was a secreted cytokine from adipocytes. Culture medium from with primary human adipocytes (A) and without adipocytes (B) which was isolated from breast tissue and was analyzed by two-dimensional polyacrylamide gel-electrophoresis of proteins in culture medium of human primary adipocytes.

Proteins were visualized by Zinc Staining and characterized by MALDI-TOF analysis.

The red circle indicates TN with pI = 5.5 and Mr. = 22 kDa, respectively.

3.4.2 Expression of TN Correlates with Adipocyte Differentiation, Lipid Accumulation and BMI

To test the hypothesis that TN plays a role in adipogenesis, we analyzed TN expression during the differentiation of human preadipocytes and found that it was increased during the 18 days of differentiation period (Fig. 3-2A) with 70~ 80% of the cells containing lipid droplets (Fig.3-2B). This led us to exam its expression in the adipose tissues of people with different BMI and found that it also positively correlates with BMI (Fig. 3-2C). Taken together, our data suggest that TN potentially serves as an adipocytokine and plays roles in regulating lipid homeostasis.

Fig.3-2 A-C Correlation of TN mRNA Expression adipogenesis. Light microscopy picture of human adipocytes, differentiated from preadipocyte, has 70-80% of lipid accumulation rate (A). Quantification of TN mRNA expression in adipocytes during the differentiation periods of day 0, 6, 12 and 18 by real-time PCR (B). Quantification of TN mRNA expression in adipose tissue of donors with

3.4.3 TN is primarily involved in lipid metabolism in human adipocytes To explore the function of TN in human adipocytes, we over-expressed TN in human adipocytes and the resultant protein profile was characterized by proteomic analysis, which showed that 40 proteins are up-regulated and 80 down-regulated at least 1.5 folds by TN, as compared to the control. They were then annotated by molecular function and pathway analysis using the Ingenuity Pathway Analysis database (IPA). It was revealed that 49% of proteins which were affected by TN overexpression is related to lipid metabolism. There are 19% belongs to the second-ranking functional network of connective tissue, skeletal and muscular system development and function and 17% is involved in the functional network of dermatological diseases and conditions, infectious disease and developmental disorder.

13% belongs to the hematological system development and function, immune cell trafficking and inflammatory response (Fig. 3-3A). Output of the highest-scoring functional network of lipid metabolism from IPA was displayed graphically as nodes (proteins) and edges (the biological relationship between the nodes). In Figs.3-3B is the second-scoring functional network of lipid metabolism from IPA. Those two lipid metabolism functional networks gave us a hint about the function of TN in adipocytes. As a result, proteins regulated by TN overexpression in human adipocytes are involved primarily in lipid metabolism.

Detailed information of lipid metabolism-associated proteins regulated by TN is summarized in Table 3-1. Interestingly, TN dramatically decreases the protein levels of ATP-binding cassette, sub-family D, member 3 (ABCD3) and HSL. It also increases the perilipin levels, which coats intracellular lipid droplets in adipocytes ^6,19. TN increased the expression of solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1) which provides instructions for producing a protein called the

glucose transporter protein type 1 (GLUT1). Prostacyclin synthase (PTGIS) protein was inhibited by TN overexpression in adipocytes, then the inhibition of prostaglandin I2 (PGI2) production may be vanquished to lead to various cardiovascular disorder ⁸⁸. We also found that TN may modulate MMPs family and its inhibitor, TIMPs in adipose tissue which initiate us to interested in knowing the relationship between TN and summarize above observations, TN promotes the proteins which involves in energy storage and suppresses energy expending in human adipocytes.

Lipid

Relationship

Network shapes B

Fig 3-3. Functional analysis of Tetranectin in human adipocytes by proteomic analysis. Function network analysis of identified proteins which were influenced by TN overespressed in primary human adipocytes. Classification was performed using the Ingenuity Pathway Analysis Knowledge Base (A). High-level functions for each network are reported, as are canonical pathways that were significant in the whole data set. The proteins of top 1-scoring lipid metabolism were represented by nodes, with their shape representing the functional class of the gene product, and edges indicate the biological relationship between the nodes (see legend). According to their score, nodes are color coded (red, overexpression; green, underexpression) (B). The

C

second canonical pathways of lipid metabolism were represented by nodes and edges (C).

Table 3-1. Examples of function analysis from large scale analysis of the total cell lysate of TN-overexpressed human adipocytes.

Biological process Protein name Fold Accession number

Lipid metabolism

Prostacyclin synthase (PTGIS) -999 IPI00003411 Nicotinamide phosphoribosyltransferase

Solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)

1.68 IPI00220194

CD9

Prostacyclin synthase (PTGIS) -999 IPI00003411 Nicotinamide phosphoribosyltransferase

(NAMPT)

1.74 IPI00018873

Lipolysis Hormone-sensitive lipase (HSL) -999 IPI00419542 ATP-binding cassette sub-family D member 3

Glutathione S-transferase P (GSTP1) 3.53 IPI00219757 Isoform 1 of Beta-galactosidase (GLB1)

3.4.4 Ectopic expression of TN induced lipogenesis, but reduced lipolysis in PHA and which was decreased by TN knockdown

We examined the effect of TN on human adipocytes with TN overexpression, which results in an averaged 13.5-fold increase in the TN mRNA (Fig. 3-4A) and increased mRNA expression of genes associated with lipogenesis, including LPL, FAS, PPARγ, FAS,SREBP1c and fatty acid binding protein 4 (FABP4) (Fig. 3-4B).

In contrast, the expression of genes associated with lipid degradation are down-regulated by TN overexpression. This is also seen in lipolytic genes, including TNFα, IL-6, SAA1 and PPARα only had 50% mRNA expression and HSL had 30% (Fig. 3-4D).

PHA was infected by TN knockdown virus has opposite result from above observations from TN overexpression in PHA. Decrease TN expression in PHA bring in the reduction of lipid accumulation and promotion of lipolysis through the change of mRNA level of lipogenesis and lipolysis related genes (Fig. 3-4 E and 3-4 F).

Furthermore, overexpression of TN renders cells with considerably stronger oil red O staining for lipids than control cells, confirming an enhanced lipogenesis and less lipid accumulate in adipocytes in TN knockdown group compare with control group separately (Fig. 3-4F).

49

F

Fig 3-4 Effects of TN on lipid metabolism in primary human adipocytes. Isolated human preadipocytes were differentiated into adipocytes in vitro. At day 3 of differentioation, treated TN overexpressed virus to differentiating primary human adipocytes for 3 folds. TN was overexpressed in human adipocytes compare to control group which was infected by control virus. Light microscopy (top left panel), fluorescent microscopy (top right panel) and quantification of TN expression (bottom panel) by real-time PCR (A). Quantification of the effects of TN-overexpression and -knockdown on mRNA expression of genes associated to lipogenesis. TN indicate TN-overexpression group and KO indicate TN-knockdown group in PHA (B) (C).

Quantification of the effects of TN-overexpression and -knockdown on mRNA expression of genes associated to lipolysis in human in PHA (D) and (E). Oil-red O staining on the lipid droplets in PHA which is infected by TN-overexpression and -knockdown virus (F). The mRNA concentration was quantified by real-time PCR analysis and related to the β-actin mRNA concentration in the same sample.

Data are expressed as mean ± SEM, n=6 independent experiments. Different

superscripts indicate a statistical significance (P<0.05).

3.4.5 Potential role of TN in lipid metabolism, but not for extracellular matrix remodeling, by regulating MMPS and its tissue inhibitor 1 (TIMP1) in adipose tissues

MMP3 and TIMP1 are expressed in white adipose tissue and MMP/TIMP balance is shifted toward increased matrix degradation in obesity. TIMP1 is strongly induced in adipose tissue⁸⁹. TN also increases TIMP1 mRNA and decrease MMP3 expression in PHA to promote lipid accumulation. Expression of the metalloproteinase, MMP3 and the metallopeptidase inhibitor, TIMP1 were down- and up-regulated for 0.5 fold and 21 fold by overexpression of TN in PHA.

Overexpression of TN in human adipocytes decreased the tPA mRNA concentration (Figure 3-5) which may be promote fibrosis in adipose tissue. Moreover, downregulation of TN expression in PHA had opposite results to overexpression (Figure 3-5). TN-overexpressed and knockdown lentivirus by real-time PCR. Data are expressed as mean ± SEM, n=6 independent experiments. Different superscripts indicate a statistical significance (P<0.05).

53

3.4.6 TN levels are higher in the adipose tissues of obese mice than the lean ones

From above in vitro results (Fig 3-4, 3-5), we suspected that TN may express higher in obese mice and promote obesity. Therefore, we fed high fat diet (HFD) and chow diet to mice for 16 weeks to generate obese and control group then detect protein level of TN in adipose tissue. The physiologic result indicated that TN expression is higher in HFD treated mice in the top and middle panel (Fig 3-6). After 16 weeks HFD diet treatment to mice and then shifted diet into DHA for 2 weeks, the TN expression was dramatically decreased in mice adipose tissue (button panel of fig 3-6). Therefore, we observed that TN has high expression in obese mice and its expression was reversed by DHA treatment.

Fig 3-6 TN expresses higher in obese mice than lean and decrease expression by DHA treatment. Immunoflouroscent images of TN level in adipose tissue of high fat diet, chow diet and DHA diet treated mice. First panel is the adipose tissue from the mice fed with chow diet for 18 weeks. Middle panel is from the mice fed with high fat diet for 18 weeks. The third panel is from the mice fed with high fat diet for 16 weeks and 2 weeks for DHA. Data are expressed as mean ± SEM, n=5 independent experiments. Different superscripts indicate a statistical significance (P<0.05).

3.4.7 Polyunsaturated Fatty Acids (PUFA) inhibit the expression of TN and tissue type plasminogen (tPA)

Since PUFA have been reported to exert a beneficial effect on obesity-associated diseases and we previously showed that DHA reduced fat deposition in vivo ¹⁹, and TN seems to enhance adipogenesis and lipogenesis, we thought TN may be the downstream target of PUFA and tested this hypothesis in vitro. As shown in Fig. 7A, the expression of TN in human adipocyte culture is reduced at least 50% by the treatments of 100 μM of n-3 PUFA, including arachidonic acid (AA), eicosapentaenoic acid (EPA), linoleic acid (LA) and DHA (Fig 3-6A). The TN and tPA mRNA expression were decreased followed by inceasing DHA concentration (Fig 3-6 B and C ).

Fig. 3-7 The mRNA concentration of TN and tPA in human adipocytes were significantly inhibited by PUFA. Quantification of mRNA level of TN in PHA with 100μM docosahexanoic acid (DHA), arachidonic acid (AA), eicosapentaenoic acid (EPA), linoleic acid (LA) (A). Quantification of mRNA level of TN and tissue type plasminogen activator (tPA) in PHA with 50 and 100μM DHA (B) (C).decreased the mRNA expression of TN about 0.3 to 0.4 fold of control (group) in human adipocytes. Treatment of different concentration of DHA, 50μM and 100μM DHA in

A

human adipocytes for 24hr, dominantly inhibited the mRNA expression of TN and tissue type plasminogen (tPA). Data are expressed as mean ± SEM, n=6 independent experiments. Different superscripts indicate a statistical significance (P<0.05). *, P<0.05; #, P<0.01.

3.5 Discussion

In this study, we have demonstrated the expression patten of TN in the adipose tissue and its expression has positive pattern with obesity which was according to BMI. We then provide provided further evidences of changes of lipogenesis and lipolysis-related gene expression and genes in human adipocytes by TN overexpression. In the study, we found that the mRNA expression of TN is positively correlated with the adipogenesis and lipid accumulation in human adipocytes. Overexpression and knockdown of TN in human primary adipocytes demonstrated that TN promotes adipogenic and lipogenic gene expression, including PPARγ, LPL, FAS and FABP4, but suppresses those related to lipolysis, including PPARα, HSL, IL-6, TNF α and SAA. Analyze by quantitative proteomic analysis and quantitative real-time PCR further confirmed a altered profiles of proteins primarily responsible for lipid metabolism. From our observation, we propose that TN might play an integral role in lipogenesis.

Interestingly, the protein levels of ABCD3 and HSL were dramatically suppressed in TN overexpressing human adipocytes. ABCD3 is involved in the metabolic transport of long- and very long-chain FAs into peroxisomes for oxidation,

90. HSL converts cholesteryl esters to free cholesterol for steroid hormone production,⁹¹. It also increases the perilipin levels, which coats intracellular lipid droplets. We recently showed when perilipin expression was decreased, intracellular lipid droplets were susceptible to hydrolysis to the access of HSL and thereby to increased lipolysis in pig and human adipocytes ^6,19. TN also promoted the expression

of SLC2A1, which provides sequential process to produce GLUT1 protein. GLUT1 protein is suited on part of the membranes of cells, where they transport glucose (a simple sugar) from the blood into the cells for use as fuel or storage in adipocytes ⁹².

It has long been known that TN stimulates the tPA to clever cleavage of plasminogen by tPA and then promote fibrinolysis ⁹³. TN and tPA specifically bound together as observedligand blot analysis and ELISA ³⁶. Insulin, dexamethasone and IBMX induced 3T3L1 differentiation and caused lower activity of tPA, which is the major type of plasminogen activator in adipocytes ⁹⁴. Mice lacking tPA gene expression exhibited higer than that of WT mice ⁹⁵. tPA in adipose tissue thus is regulated to diminishe the development and lipogenesis or adipogenesis.

In our results, TN overexpression inhibited the mRNA expression of tPA, which thus may cause hypertrophy at adipocytes. In another aspect, the plasminogen acitivators (urokinase and tissue types) are suppressed by a physiological inhibitor, plasminogen activator inhibitor (PAI)-1 which is expressed in adipose tissue. Since plasma PAI-1 are increased in obesity and reduced with weight loss people, PAI-1 may have contribution to obesity through indirect effects on insulin signaling to influence adipocyte differentiation and cause weight gain and induction of obesity in mice ^96,97. Lipogenic gene expressions were promoted by TN overexpression in human adipocytes cause accumulation, and may promote expression of PAI-1. According our data TN decrease the gene expression of tPA. Our observation not only matche previous results but also demonstrated that TN inhibits tPA expression and promote fat accumulation in adipocytes and TN may play a role of induction of obesity development.

There is multitude functional properties attributed to the MMP system. MMP can

maintain the balance between the deposition and degradation of extra cellular matrix (ECM) ^98,99 and act in concert to lead to the development of adipose mass.

Adipogenesis, angiogenesis and tissue remodeling of the ECM are associated with an extensive reorganization of the adipose tissue with obesity development. The matrix is increased by MMPs which facilitate adipose tissue remodeling and /or adipocytes hypertrophy ⁸⁹. When mouse 3T3-L1 preadipocytes are induced to differentiate into adipocytes, they change from an extended fibroblast-like morphology to become rounded ones, with extracellular matrix remodeling charges the change of the morphology, a process known to be mediated in part by MMP ¹⁰⁰. MMPs also participat in cellular remodeling during adipogenesis ¹⁰¹. MMP3 is one of MMPs expressed in adipocytes. MMP levels diminishes in preadipocytes cells isolate from obese subjects compare to lean ones. MMP3 expression levels are negatively correlated to adiposity in human subcutaneous adipose tissue ¹⁰². MMP activity is further regulated through interaction with tissue inhibitors of MMPs (TIMPs) ¹⁰³. In the transgenic means, mice lack MMP3 had increased adipocytes hypertrophy when fed a high-fat diet ¹⁰⁴. MMP3^-/- mice or overexpress the tissue inhibitor of metalloproteinases, TIMP-1 had accelerated adipocyte differentiation during mammary gland involution ¹⁰⁵. In relation to adipose tissue development, TIMP1 has been studied the most. A recent report suggests that, beyond direct adipose tissue ECM remodeling, TIMP1 may have an effect on adipose tissue development by acting in the hypothalamus to regulate food intake ¹⁰⁶. We may demonstrate that diminished expression of MMP3 and/or facilitated TIMP1 would promote lipid accumulation in adipocytes. However, one of cytokines which is secreted by adipocytes, tumor necrosis factor α (TNFα) which is also a lipolysis related protein treated to adipoyctes to induced substantial increases in MMP3 mRNA expression and

MMP3 protein release from adipocytes ¹⁰⁷. And the activated form of MMP3 promoted glycerol release as well as TNFα protein secretion from 3T3L1 adipocytes

108. MMP3 and TNFα have similar pattern in lipid metabolism and may involve in lipolysis in adipocytes. Mature adipocytes appear to spend much energy on the modulation of ECM. The balance of constructive and destructive enzymes together, such as MMP3 with one of its inhibitors, TIMP1 mediates the ECM remodeling to affect the dynamics and physiology of the adipose tissue. We speculate that TN suppresses the expression of MMP3 and simultaneously promotes the expression of TIMP1 to further lipid accumulation in adipoyctes and may help develop the obesity in vivo.

PUFA is being identified as one of anti-obesity nutrient. In patients with metabolic syndrome, n-3 PUFA contained in the diet is represented to improve their health and prevent cardiovascular diseases by decreasing triacylglyceral and HDL-cholesterol levels in plasma. PUFA prevent the expression of obesity and advance glucose sensitivity in rodents ¹⁰⁹. Mitochondrial biogenesis and oxidative metabolism related genes, such as PPARγ co-activator 1a and nuclear respiratory factor-1 were increased respectively with 3-fold expression in abdominal fat. The stearoyl-CoA desaturase gene, Scd-1 was down-regulated by n-3 PUFA in white fat to induct lipid oxidation as in other tissues ^110,111. Thus n-3 PUFA which include DHA induce metabolic switch to lipid oxidation. In our observation, n-3 and n-6 PUFA inhibits the gene expression of TN which may indicate that PUFA modulate lipid metabolism through TN to inhibit lipid accumulation. Therefore, the regulation pathway between PUFA and TN is an important issue to regulate the expression of TN to suppress lipogenesis in fat.

chapter4 General Discussion

Obesity is defined as chronic inflammation in adipose tissue. Obesity and dyslipidemia induced by high fat diet (HFD) are intimately linked to chronic low-grade inflammation, which are characterized by elevated levels of circulating adipocytokines. Numerous results showed that adipocytokines regulate the physiology of adipose tissue. For example: RBP4, adiponectin, TNFα, IL-6 etc. can influence adipose tissue hypertrophy through different ways. RBP4 has a detrimental effect on lipolysis ^112,113, but adiponectin protects acipocytes against lipid accumulation through MAPK pathway¹¹³. Moreover, TNFαand IL-6 are identified as pro-inflammation cytokines in obese individula significant increase of IL-1β and TNFα, in adipose tissue in obese group compare to control¹¹⁴. We demonstrated SAA and TN as novel adipocytokines in human and studied their functions in adipose tissue.

Evidences suggested that PUFAs regulate some adipocytokines to affect lipid metabolism. We found that SAA is upregulated by DHA in porcine hepatic cells and SAA also induced IL-6, TNFα, glycerol release and decreased prilipin which indicating increased lipolysis³³. The same results were observed in the study. DHA promotes the expression of SAA1 to induce lipolysis by downregulating peilipin expression in human adipocytes⁶. In porcine adipocytes, we proved that SAA induces lipolysis by downregulating

Evidences suggested that PUFAs regulate some adipocytokines to affect lipid metabolism. We found that SAA is upregulated by DHA in porcine hepatic cells and SAA also induced IL-6, TNFα, glycerol release and decreased prilipin which indicating increased lipolysis³³. The same results were observed in the study. DHA promotes the expression of SAA1 to induce lipolysis by downregulating peilipin expression in human adipocytes⁶. In porcine adipocytes, we proved that SAA induces lipolysis by downregulating