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Animal Models

Studies on the antiobesity effect of zinc-α2-glycoprotein in the ob/ob mouse

Abstract

Objective:

To investigate the mechanism of the lipid depletion by zinc-α2-glycoprotein (ZAG).

Design:

Studies were conducted in the ob/ob mouse, or on isolated adipocytes from these animals or their lean counterparts.

Results:

Treatment of these animals for 15 days with ZAG (100 μg, intravenously, daily) resulted in a reduction of body weight of 6.55 g compared with phosphate-buffered saline-treated controls, without a change in food or water intake, but with a 0.4 °C rise in rectal temperature. ZAG-treated mice had a 30% reduction in carcass fat mass and a twofold increase in weight of brown adipose tissue. Epididymal adipocytes from ZAG-treated mice showed an increased expression of ZAG and hormone-sensitive lipase (HSL), and this was maintained for a further 3 days in the absence of ZAG. There was an increased lipolytic response to isoproterenol, which was retained for 3 days in vitro in the absence of ZAG. Expression of HSL was also increased in subcutaneous and visceral adipose tissue, as was also adipose triglyceride lipase (ATGL). There was a rapid loss of labelled lipid from epididymal adipose tissue of ZAG-treated mice, but not from the other depots, reflecting the difference in sensitivity to lipolytic stimuli. The increased expression of HSL and ATGL may involve the extracellular signal-regulated kinase (ERK) pathway, as the active (phospho) form was upregulated in all adipose depots after ZAG administration, whereas in vitro studies showed induction of HSL and ATGL by ZAG to be attenuated by PD98059, an inhibitor of the ERK pathway.

Conclusion:

These results suggest that ZAG not only induces direct lipolysis, but also sensitizes adipose tissue to other lipolytic stimuli.

Introduction

Obesity, and its associated health effects, is a major problem for the Western World, and is thought to arise through both genetic and environmental influences.1 Obesity has been linked with insulin resistance and type 2 diabetes through release from adipocytes of non-esterified fatty acids and proinflammatory cytokines.2 The current approach to management involves lifestyle alteration combined with pharmacological intervention, although the options for treatment are limited.

We have investigated the potential use of zinc-α2-glycoprotein (ZAG) for the treatment of obesity using ex-breeder male NMRI mice.3 ZAG was initially identified as the lipid-mobilizing factor associated with loss of adipose tissue in cancer cachexia,4 and was shown to be tumour derived.5 However, later studies showed that ZAG was also produced by a range of normal tissues including both white (WAT) and brown (BAT) adipose tissues,6 with major increases in expression in mice bearing a cachexia-inducing tumour, which induced loss of fat mass. In contrast with cachexia expression of ZAG in WAT is low in obese human subjects7 and correlated negatively with body weight, body mass index, fat mass, waist and hip circumference, as well as plasma insulin levels.8, 9 The expression level of ZAG may be responsible for some of the effects of obesity, as ZAG ‘knockout’ animals gain more weight, especially on a high fat diet, while adipocytes from these animals showed a decreased lipolytic response to various agents including catecholamines, β3-adrenoreceptor (β3-AR) agonists and agents which increase cyclic AMP.10 In contrast, overexpression of ZAG in mice was associated with a reduced body weight and percentage of epididymal fat when they were fed a high-fat diet.8 A reduced catecholamine-induced lipolysis and fat oxidation is seen in obese individuals,11 and in first-degree relatives of obese subjects,12 and may have a role in the development and maintenance of the increased fat stores. A reduced hormone-sensitive lipase (HSL) expression is the best-characterized defect contributing to this resistance to catecholamines.13 ZAG has been suggested as a possible candidate gene for obesity using the KK/Ta mouse as an animal model of spontaneous type 2 diabetes.14

ZAG has been shown to induce loss of adipose tissue through a lipolytic effect on WAT, combined with an increased expression of uncoupling protein-1 in BAT, which would result in an increase in energy expenditure.3, 15 The lipolytic effect arises from activation of adenylyl cyclase to produce cyclic AMP in a guanosine triphosphate-dependent process,4 which is attenuated by the specific β3-AR antagonist SR59230A,16 suggesting that it is mediated through the β3-AR. Using the ob/ob mouse as an experimental model, this study evaluates the effect of ZAG on lipolytic enzymes and the sensitivity of adipocytes to lipolytic stimuli.

Materials and methods

Materials

Freestyle media and RPMI 1640 were purchased from Invitrogen (Paisley, UK) and fetal calf serum was from Biosera (Sussex, UK). Rabbit polyclonal antibodies to phospho (Thr-202) and total extracellular signal-regulated kinase-1 (ERK1), and total HSL were purchased from Abcam (Cambridge, UK). Rabbit antisera for adipose triglyceride lipase (ATGL) was from New England Biolabs (Herts, UK). Mouse monoclonal antibody to human ZAG was from Santa Cruz (Santa Cruz, CA, USA). Phosphosafe Extraction Reagent was from Calbiochem (via Merck Chemicals, Nottingham, UK). Peroxidase-conjugated goat anti-rabbit antibody, Hybond A nitrocellulose membranes and enhanced chemiluminescence development kits were from GE Healthcare (Amersham, Bucks, UK). Rabbit anti-mouse antibody was purchased from Dako (Cambridge, UK). Polyclonal rabbit antibody to mouse β-actin, PD98059, BRL 37344, endotoxin standard, endotoxin-free water and the triglyceride assay kit were purchased from Sigma Aldrich (Poole, Dorset, UK). A WAKO colorimetric assay kit for non-esterified fatty acids was purchased from Alpha Laboratories (Hampshire, UK), and a mouse insulin ELISA kit was purchased from DRG (Marburg, Germany). Endotoxin was measured with a LAL Pyrogent single test kit from Lonza (Bucks, UK). Glucose measurements were carried out using a Boots (Nottingham, UK) plasma glucose kit.

Animals

Obese hyperglycaemic (ob/ob) mice (73–77 g; Table 1) were bred in our own colony. The origin and characteristics of these animals have been described in detail previously.17 Expression of the ob gene on this background produces a more severe form of diabetes than C57BL/6J ob/ob mice. Mice were housed in an air-conditioned room at 22±2 °C and fed ad libitum, a rat and mouse breeding diet (Special Diet Services, Witham, UK), and tap water. Male mice (20–21 weeks old) were grouped into three per cage and administered ZAG (35, 50 or 100 μg daily) by intravenous administration (n=6) or phosphate-buffered saline (PBS) (n=6). Both body weight and food and water intake were monitored daily, as was body temperature, with use of a rectal thermometer (RS Components, Northants, UK), and urinary glucose output was also measured.

Table 1 Body composition of ob/ob mice treated with ZAG (100 μg) or PBS for 15 days

Determination of body composition

Body composition was determined gravimetrically as previously described.3 Briefly, after termination, animals were heated to 80–90 °C until constant weight was achieved, and the water content was determined from the difference between the wet and dry weight. Lipids were extracted from the dried carcass by extraction with chloroform/methanol (1:1), ethanol/acetone (1:1) and diethyl ether; the solvents were combined and evaporated. The weight of the residue gave the total carcass fat, and the carcass non-fat mass was calculated as the difference between the initial weight of the carcass and the weight of water and fat.

Production and purification of recombinant human ZAG

Human HEK 293F cells were transfected with the mammalian cell expression vector pcDNA 3.1, containing human ZAG, and selected for growth in neomycin (50 μg ml−1) in Freestyle medium under an atmosphere of 5% CO2 in air at 37 °C. Protein levels in the culture medium increased progressively with time reaching plateau levels within about 2 weeks of seeding. Cells were removed by centrifugation at 700 g for 15 min, and the medium (200 ml) was concentrated into a volume of 1 ml of sterile PBS using an Amicon Ultra-15 centrifugal filter with a cut-off of 10 kDa (Millipore, Watford, Middlesex, UK). The concentrate (containing about 2 mg protein) was then added to diethylaminoethyl cellulose (2 g) suspended in 20 ml 10 mM Tris, pH 8.8, and stirred at 4 °C for 2 h. ZAG bound to the diethylaminoethyl cellulose cellulose, which was sedimented by centrifugation (1500 g for 15 min), and was eluted by stirring for 30 min at 4 °C with 20 ml 10 mM Tris, pH 8.8, containing 0.3 M NaCl. After sedimentation the supernatant fluid, containing ZAG, was concentrated to a volume of 1 ml in sterile PBS using the Amicon centrifugal filter. The ZAG was free of endotoxin as determined by a LAL Pyrogent single test kit (Lonza). The purity of the recombinant ZAG was >95% as previously reported.15

Preparation of human and murine adipocytes

Adipose tissue was minced into small fragments and digested in Krebs-Ringer bicarbonate containing 1 g l−1 collagenase and 4% bovine serum albumin under an atmosphere of 95% oxygen/5% CO2 at 37 °C, as described.18 After 30 min, the adipocytes were filtered through nylon mesh (pore size 250 μm), centrifuged at 500 g for 2 min, and washed three times with PBS before suspension in RMPI 1640 medium containing 10% fetal calf serum and maintained under an atmosphere of 5% CO2 in air at 37 °C. The culture medium was replaced daily. Adipocyte number was determined with a haemocytometer.

Lipolytic assay

For lipolytic assays, 105 to 2 × 105 adipocytes were incubated with the lipolytic agent for 2 h in 1 ml Krebs-Ringer bicarbonate buffer, pH 7.2, and the extent of lipolysis was determined by measuring glycerol released, expressed as μmol glycerol per 105 adipocytes per 2 h.19 Control samples containing adipocytes alone were analysed to determine the spontaneous glycerol release.

Lipolysis in vivo by direct injection of tracer into fat pads

The detailed methodology has been previously published.20, 21Briefly, (U-14C) plamitic acid (50 μCi sp.act 828mCimmol−1) was evaporated under a stream of nitrogen to remove the toluene and converted to a soap by addition of 500 μl 30% (w/v) KOH. After drying, the soap was dissolved in 200 μl of 0.9% (w/v) NaCl, with heating, and portions (15 μl) were injected into the epididymal, subcutaneous and visceral (perirenal) fat of ob/ob mice, which had been administered either PBS or ZAG (50 μg) daily by intravenous injection for 4 days. The mice were anaesthetized using a mixture of halothane, oxygen and nitrous oxide during this procedure. After injection, the wound was clipped together, and at specific time points the animals were killed, and the fat pad removed, together with selected organs. The amount of labelled lipid was determined by the method of Stansbie et al.22 Pads were transferred to scintillation vials with 30% KOH, heated to 70 °C for 15 min, after which 3 ml 95% ethanol was added, and the heating continued for 2 h. The vials were cooled and the saponified material was acidified with 3 ml 9 M sulphuric acid. Acid soluble lipids were extracted three times by shaking with petroleum ether (boiling point 40–60), centrifugation at 3000 g for 15 min, and the supernatants from the three fractions were pooled and evaporated under nitrogen. Lipids were redissolved in 10 ml Optiphase scintillation fluid (Fisons, Leics, UK) and the radioactivity determined using a Packard Tri Carb 3100TR liquid scintillation analyser (PerkinElmer, Cambridge, Cambs, UK).

Western blot analysis

Freshly excised WAT was washed in PBS and lysed in Phosphosafe Extraction Reagent for 5 min at room temperature, followed by sonication at 4 °C. Samples of cytosolic protein formed by centrifugation at 18 000 g for 5 min at 4 °C were resolved on 12% SDS-polyacrylamide gel electrophoresis at 180 V for approximately 1 h, and transferred to 0.45 μm nitrocellulose membranes, which were blocked with 5% Marvel in Tris-buffered saline, pH 7.5, at 4 °C overnight. Both primary and secondary antibodies were used at a dilution of 1:1000. Incubation was for 1 h at room temperature and development was by enhanced chemiluminescence. Blots were scanned by a densitometer to quantify differences.

Statistical analysis

Results are shown as mean±s.e.m. for at least three replicate experiments. Differences in means between groups was determined by one-way analysis of variance, followed by Tukey–Kramer multiple comparison test. P-values <0.05 were considered significant.

Results

Preliminary experiments were carried out in vitro to determine the lipolytic effect of ZAG and isoproterenol in adipocytes of ob/ob mice from various sites. ZAG induced lipolysis in a dose-related manner in epididymal adipocytes from both lean and obese (ob/ob) mice, but with a significantly reduced effect in adipocytes from ob/ob mice at low concentrations (Figure 1a). Epididymal adipocytes from ob/ob mice also showed a lower lipolytic response to isoproterenol (Figure 1a). The lipolytic response to both isoproterenol and ZAG was significantly less in adipocytes from both subcutaneous and visceral deposits (Figure 1b), although there was no significant difference in response of adipocytes from obese and non-obese animals. Freshly isolated human subcutaneous adipocytes also showed lipolysis in the presence of ZAG (Figure 1c) and isoproterenol, and the lipolytic response was comparable with murine adipocytes (Figure 1b). The lipolytic response of human adipocytes to ZAG was comparable with that for isoproterenol (Figure 1c). To determine the mechanism for the increased lipolysis by ZAG, adipocytes from non-obese animals were incubated with ZAG for 3 h and the expression of HSL was determined by Western blotting (Figure 1d). Both ZAG and isoproterenol increased the protein expression of HSL, and this was completely attenuated by the selective and cell permeable inhibitor of mitogen-activated protein kinase kinase PD98059 (10 μM),23 suggesting a role for the ERK pathway in its induction. Similar results were obtained for protein expression of ATGL, which was increased twofold by both ZAG and isoproterenol, and this was completely attenuated by PD98059 (Figure 1e), again suggesting a role for ERK. Indeed, lipolysis induced in isolated adipocytes by ZAG and to a lesser extent isoproterenol was attenuated by PD98059, although this did not return to basal values (Figure 1f). These results suggest that the ERK pathway is involved in the increased protein expression of HSL and ATGL in WAT.24, 25

Figure 1
figure1

Lipolytic activity of ZAG in adipocytes isolated from lean and ob/ob mice, human adipocytes and effect of ZAG on expression of HSL. (a) Induction of lipolysis in murine epididymal adipocytes from lean (▪) and ob/ob () mice by isoproterenol or ZAG at the concentrations indicated over a 2 h period. Differences from lean animals are shown as *P<0.05; **P<0.01 or ***P<0.001. (b) Lipolysis in adipocytes from subcutaneous (sc) and visceral (vis) deposits from lean and ob/ob mice in the presence of isoproterenol (10 μM; □) or ZAG (0.58 μM; ) compared with basal levels (▪). Differences from epididymal adipocytes in (a) are shown as **P<0.01. (c) Lipolysis in human sc adipocytes in response to isoproterenol or ZAG over a 2 h time period. Differences from control are shown as **P<0.01, or ***P<0.001. (d) Western blot showing expression of HSL in mouse epididymal adipocytes after incubation for 3 h with isoproterenol (10 μM) or ZAG (0.58 μM), alone, or in the presence of PD 98059 (10 μM). Actin was used as a loading control. (e) Western blot showing expression of ATGL in epididymal adipocytes in the presence of ZAG and isoproterenol, as detailed in (d). The densitometric analysis is an average of three separate Western blots and is expressed as a percentage of control in the absence of stimulation. (f) Lipolytic effect of isoproterenol (10 μM) and ZAG (0.58 μM) in isolated epididymal adipocytes in the absence and presence of PD98059 (10 μM). Differences from control are shown as ***P<0.001, whereas differences in the presence of PD98059 are shown as P<0.01 or ††P<0.001.

In vivo studies were carried out in ob/ob mice (73–78 g) over a 15-day period. Administration of ZAG (100 μg, intravenously, daily) resulted in a 6.55 g reduction in body weight compared with PBS-treated controls (Figure 2a and Table 1). There was no change in food or water intake, but rectal temperature was elevated by approximately 0.4 °C within 3 days of treatment, and remained at this level during the course of the study even on days when ZAG was not administered (Figure 2b). The body composition of the animals is shown in Table 1. Treatment with ZAG was associated with a 30% reduction in total body fat compared with PBS-treated controls, although there was a 53% increase in non-fat mass (predominantly lean body mass). The magnitude of these changes in absolute amounts, were similar, with a 7.8 g loss of fat and a 6.8 g increase in non-fat mass, which explains why the body weight of these animals was relatively constant during the time course of this study (Figure 2a). As previously reported,15 the increase in lean body mass was manifest as an increase in weight of the gastrocnemius muscles, although there was no effect on the weight of the soleus muscles. There was almost a twofold increase in the weight of BAT (PBS 0.42±0.12 g, ZAG 0.79±0.06 g; P<0.001), which was probably responsible for the decrease in plasma levels of non-esterified fatty acids and triglycerides (Table 2), and the increase in body temperature (Figure 2b). As previously reported,15 the plasma levels of glucose and insulin were decreased, as were those of non-esterified fatty acids and triglycerides, whereas glycerol levels increased, indicating an increased lipolysis (Table 2). The magnitude of these changes were similar to those reported after 5 days administration of ZAG.15 Urinary excretion of glucose was also decreased (Figure 2c), but the major decrease occurred in the first 5 days after ZAG administration, following which levels remained constant.

Figure 2
figure2

Effect of ZAG on body weight, temperature and urinary glucose excretion in ob/ob mice. (a) Effect of ZAG (100 μg; intravenous, ▪) or PBS (♦) on body weight of ob/ob mice over 15 days. The days when ZAG was not administered are indicated by ↑. (b) Rectal temperature of ob/ob mice administered PBS (♦) or ZAG (▪) as shown in (a). (c) Urinary glucose excretion of ob/ob mice administered PBS (♦) or ZAG (▪) over a 15-day period. Differences from PBS controls are shown as *P<0.001.

Table 2 Plasma metabolite and insulin levels in ob/ob mice after administration of ZAG for 15 days

Administration of ZAG led to an increase in the expression of intracellular ZAG, where levels increased about twofold in epididymal, subcutaneous and visceral adipose deposits after 5 days of treatment, compared with PBS controls (Figure 3a). The increased expression of ZAG was maintained in tissue culture for a further 3 days in the absence of ZAG (Figure 3b). This suggests that it may not be necessary to administer ZAG on a daily basis for it to exert its biological effects. As shown in Figures 2a and b, there was no significant change in the body weight loss or rectal temperature on those days when ZAG was not administered, suggesting that the effects were mediated by intracellular ZAG.

Figure 3
figure3

Expression of HSL and lipolytic response of adipocytes from ob/ob mice administered ZAG. (a) Western blot showing expression of ZAG in epididymal (ep), subcutaneous (sc) and visceral (vis) adipose tissue of ob/ob mice after 5 days continuous ZAG administration (35 μg; intravenously per day). Actin served as a loading control. (b) Adipocytes (ep) were removed from mice at the end of 5 days treatment (day 0) and maintained in RPMI 1640 medium containing 10% fetal calf serum in the absence of ZAG for a further 4 days. ZAG expression was determined by Western blotting. (c) Western blot showing expression of phospho HSL in ep adipocytes directly after removal from mice (day 0) and after a further 4 days in tissue culture in the absence of ZAG. (d) Lipolytic response in vitro of ep adipocytes from ob/ob mice treated with either PBS (▪) or ZAG () with no additions, or after the addition of isoprenaline (10 μM) to adipocytes isolated from PBS- (□) or ZAG ()-treated mice, as described above. Differences from the respective controls are shown as *P<0.001, whereas the difference in response to isoprenaline between ZAG- and PBS-treated mice are shown as **P<0.01 or ***P<0.001.

This conclusion is substantiated by changes in the protein expression of HSL. As expected from in vitro studies, ZAG administration to ob/ob mice also caused an increased protein expression of HSL in WAT, and this also remained elevated in tissue culture in the absence of ZAG for 3 days (Figure 3c). In addition, adipocytes from ZAG-treated mice showed an increased lipolytic response to isoproterenol, and this was also retained for 3 days when the adipocytes were maintained in tissue culture in the absence of ZAG (Figure 3d). These results again suggest that the biological effects of ZAG are due to its raised intracellular expression.

Protein expression of HSL (Figure 4a) and ATGL (Figure 4b) was significantly upregulated by ZAG in epididymal (ep), subcutaneous (sc) and visceral (vis) adipose tissues. This correlated with expression of the active (phospho) form of ERK, which showed selective upregulation only in epididymal adipose tissue (Figure 4c). This, together with the data in Figure 1d, suggests that ERK may be responsible for the increase in phospho HSL triggered by ZAG. To determine the rate of lipolysis in the different adipose depots, [U-14C] palmitic acid complexed with albumin was directly injected into fat deposits at the three sites. This has been shown20 to reflect the behaviour of the fat pad triacylglycerol fatty acid. The rate of loss of [U-14C] palmitic acid at the three sites is shown in Figure 5. There was little loss from control animals at any site during the 60 min of investigation, showing that there was minimal loss due to leakage. Although there was no significant change in [U-14C] palmitic acid in subcutaneous (Figure 5b) or visceral (peri-renal) adipose tissue (Figure 5c) in ZAG-treated animals during the course of the experiment, there was a rapid and significant loss in epididymal adipose tissue, extending to about 35% during 60 min. These results confirm that the rate of lipolysis after ZAG administration is highest in epididymal adipose tissue. The major portion of the liberated palmitic acid was transported to the liver, and levels were significantly higher in ZAG-treated animals (Figure 5d). Levels of palmitate in BAT were also higher in ZAG-treated animals, but the difference was not as large, probably because of an increased utilization rate.15

Figure 4
figure4

Protein expression of HSL, ATGL and phospho ERK in WAT of ob/ob mice treated with ZAG. Western blots showing expression of HSL (a), ATGL (b) and phospho ERK (c) in epididymal (ep), subcutaneous (sc) and visceral (vis) adipocytes removed from ob/ob mice treated with either PBS or ZAG (35 μg; intravenous per day) for 5 days. The densitometric analysis represents an average of three separate blots. Differences from PBS-treated animals are shown as *P<0.001.

Figure 5
figure5

Rate of loss of radioactivity from [U-14C] palmitate-labelled (a) epididymal, (b) subcutaneous, (c) mesenteric (visceral) fat pads of ob/ob mice after treatment with either ZAG (50 μg; intravenous (i.v.) per day) (▪) or PBS (♦) for a 5-day period. (d) [U-14C] Palmitate accumulation in various organs 1 h after direct injection into epididymal fat pads of ob/ob mice after receiving either ZAG (50 μg; i.v. per day) () or PBS (▪). Differences from PBS controls are shown as *P<0.05 or **P<0.001.

The increased release of [U-14C] palmitic acid from epididymal fat deposits would also correlate with the increased lipolytic response to ZAG (Figure 1a), compared with subcutaneous and visceral adipocytes (Figure 1b). This effect is also seen with the β3-AR agonist, BRL 37344 (Figure 6a), which caused an increased stimulation of lipolysis in epididymal adipocytes from ZAG-treated animals, whereas in subcutaneous and visceral adipocytes, pretreatment with ZAG had no effect on the lipolytic response. These results suggest that ZAG may act synergistically with β3-AR agonists to mobilize lipids. The sensitization of adipocytes to BRL 37344 was seen even in short-term culture after 2 h incubation with ZAG, but not with isoproterenol (Figure 6b).

Figure 6
figure6

Effect of ZAG treatment on response to β3-AR agonists. (a) Glycerol release from epididymal adipocytes treated with PBS (▪) or ZAG (□), subcutaneous adipocytes treated with PBS () or ZAG () and visceral adipocytes isolated from ob/ob mice treated with PBS () or ZAG () in response to BRL 37344. Differences from PBS-treated mice are shown as *P<0.001. (b) Lipid-mobilizing effect of BRL 37344 in epididymal adipocytes pretreated with either isoprenaline (1 μM) or ZAG (0.58 μM) for 2 h before measurement of lipolytic activity in the absence (▪), or presence (□) of BRL 37344 at the concentrations shown. Differences from control are shown as *P<0.001, whereas differences in the presence of BRL 37344 are shown as **P<0.001.

Discussion

This study shows the potent anti-obesity effect of ZAG in ob/ob mice, reducing the body mass by 6.55 g in 15 days, with a 30% reduction in weight of carcass fat, but without a decrease in food or water intake. In comparison sibutramine reduced the body weight gain of ob/ob mice by about 8 g over a 6-week period,26 but there was no measurement of body fat. Sibutramine was also less effective than ZAG in the glucose tolerance test with 17% lower mean area under curve,26 compared with 53% for ZAG.15 As described in the previous 5-day study, ZAG also produced an increase in lean body mass in ob/ob mice, due to an increase in protein synthesis and decrease in degradation.15 The mechanism by which this occurs will be the subject of a further study.

In vitro studies on isolated adipocytes show ZAG to be as effective as isoproterenol in inducing lipolysis with a maximum effectiveness at 0.58 μM (25 μg ml−1). This concentration of ZAG is close to the serum level (26 μg ml−1) reported using an immunoassay,27 but is much higher than the serum level reported using liquid chromatography mass spectrometry (3.65 μg ml−1).28 The reason for the disparity between the two methods of measuring ZAG has not been explained, or commented on. Both ZAG and isoproterenol showed a lower effectiveness in inducing lipolysis in adipocytes from subcutaneous and visceral depots than in epididymal depots. The decreased lipolytic effect of catecholamines and β-AR agonists towards subcutaneous and visceral adipocytes has been previously reported,29 and may be due to differences in number of β3-AR.30 However, epididymal adipocytes show a more marked reduction in lipolysis after isoproterenol pretreatment than those from subcutaneous fat.29 In addition, subcutaneous adipocytes possess a lower steady-state level of mRNA for HSL, consistent with the reduced lipolysis rate.30 Differences in expression of HSL could explain the lower lipolytic response of epididymal adipocytes from ob/ob mice to both isoproterenol and ZAG. Treatment of epididymal adipocytes with ZAG and isoproterenol increased the expression of HSL. Previous studies have shown that catecholamines translocate HSL to its substrate on the surfaces of lipid droplets in fat cells.31 A recent study8 has reported an increased expression of HSL mRNA in epididymal adipose tissue in mice overexpressing ZAG.

Activation of ERK is required for the induction of protein expression of HSL and ATGL by ZAG, as it was attenuated by the specific inhibitor PD98059. Activation of the ERK pathway has also been shown to increase lipolysis by phosphorylating HSL at Ser600,32 but there have been no reports to show that it is involved in the expression of HSL or ATGL. Mice lacking mitogen-activated protein kinase phosphatase-1 have increased activities of ERK and p38 mitogen-activated protein kinase in WAT, and are resistant to diet-induced obesity, due to enhanced energy expenditure.33 Expression of HSL in adipose tissue is high in cachexia,34 in which ZAG levels are also high,6 and low in obesity,13 in which ZAG levels are low.7 The increased expression of HSL in cachexia sensitizes the adipocytes to lipolytic stimuli.34 Thus, cachectic mice bearing the MAC16 tumour showed an increased rate of release of [U-14C] palmitate from epididymal fat pads than either non-tumour-bearing mice or mice bearing the MAC13 tumour, which does not induce cachexia.21 Using the same technique, this study shows an enhanced rate of loss of radioactivity from [U-14C] palmitate-labelled epididymal fat pads from ob/ob mice treated with ZAG, whereas there was no difference in the rate of release from PBS controls in either subcutaneous or visceral adipose tissue over the 60 min period of investigation. This could reflect the slower rate of lipolysis in these adipose tissue depots. The released [U-14C] palmitate was directed mainly to the liver, and to a lesser extent to BAT.

HSL was initially considered to be the rate-limiting enzyme for lipolysis, but recent data35 suggest that ATGL may be rate limiting. As with HSL,13 levels of ATGL in subcutaneous adipose tissue of obese subjects have been shown to be reduced, despite an increase in mRNA expression,36 although other studies37, 38 report a decrease in both HSL and ATGL mRNA and protein. There is a significant correlation between mRNA expression of ATGL and HSL in both visceral and subcutaneous adipose tissues suggesting a common regulatory mechanism for their expression.37, 38 This may be related to the activation of the ERK pathway, as suggested from in vitro studies, as ERK was activated in all adipose tissue depots after ZAG administration to ob/ob mice. Both insulin resistance and hyperinsulinemia in obese subjects have been shown to be negatively correlated with ATGL and HSL protein expression independent of fat mass.38 Thus, the ability of ZAG to increase the expression of both HSL and ATGL would correlate with its ability to attenuate insulin resistance,15 although other mechanisms such as an increase in uptake and oxidation of glucose and fatty acids are probably more important.

Treatment of ob/ob mice with ZAG also increased protein expression of ZAG in epididymal, subcutaneous and visceral adipose tissue. Although expression of ZAG is low in obesity,7, 8, 9 its expression in WAT has been shown to be increased 10-fold in mice with cachexia.6 This has been shown to be due to an increase in serum cortisol,39 although tumour necrosis factor-α has been shown to result in a fourfold decrease in ZAG expression in human Simpson–Golabi–Behmel syndrome adipocytes,40 providing a potential mechanism to explain the low levels of ZAG found in adipose tissue of obese subjects.41 Induction of ZAG in 3T3-L1 adipocytes by administration of exogenous ZAG has been shown to be attenuated by the selective β3-AR antagonist SR59230A, suggesting that it is mediated through a β3-AR.39

ZAG may be necessary for optimal β3-AR action, as ZAG knockout mice showed a lower response to the specific β3-AR agonist CL316243.10 As ZAG levels are low in obesity,7, 8, 9 the expression of β3-AR may also be suboptimal. Thus, β3-AR agonists may require ZAG for optimal activity, as evidenced by the increased lipolytic effect of both isoprenaline and BRL 37344 in adipocytes from ob/ob mice treated with ZAG. In addition, many of the effects of ZAG in diabetes in this model15 are possibly related to its β3-AR agonist activity.16 β3-AR agonists induce lipolysis in WAT, both through the classical cyclic AMP and protein kinase A pathway, and through the ERK pathway, which accounts for between 15 and 25% of total lipolysis.32

Adipocytes from obese mice also express twofold lower levels of Gαs, a stimulatory subunit of the guanosine triphosphate-binding protein, which stimulates adenylyl cyclase.42 ZAG has been shown to increase the expression of Gαs and decrease the expression of the inhibitory G-protein, Gαi, in 3T3 adipocytes,43 suggesting a mechanism by which ZAG could increase lipolytic responsiveness, in addition to the induction of HSL and ATGL.

These results show that ZAG caused a major reduction in adipose mass in ob/ob mice. The effect was exerted through its lipolytic activity, together with the induction in adipocytes of protein expression of HSL and ATGL, which would cause sensitization to other lipolytic stimuli. In addition, the released lipids would be converted to heat through an increased mass of BAT. These results suggest that ZAG may overcome some of the metabolic alterations associated with the obese state. Further studies will investigate the role of β-adrenergic receptors in the action of ZAG.

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Acknowledgements

This work was supported by Halsa Pharmaceuticals, TX, USA, which also provided salary for one of us (ST Russell).

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Correspondence to M J Tisdale.

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Russell, S., Tisdale, M. Studies on the antiobesity effect of zinc-α2-glycoprotein in the ob/ob mouse. Int J Obes 35, 345–354 (2011). https://doi.org/10.1038/ijo.2010.150

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Keywords

  • lipolysis
  • hormone-sensitive lipase
  • adipose triglyceride lipase
  • zinc-α2-glycoprotein
  • extracellular signal-regulated kinase

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