Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref-1)

Abstract

Preadipocyte factor-1 (Pref-1) is a transmembrane protein highly expressed in preadipocytes. Pref-1 expression is, however, completely abolished in adipocytes. The extracellular domain of Pref-1 undergoes two proteolytic cleavage events that generate 50 and 25 kDa soluble products. To understand the function of Pref-1, we generated transgenic mice that express the full ectodomain corresponding to the large cleavage product of Pref-1 fused to human immunoglobulin-gamma constant region. Mice expressing the Pref-1/hFc transgene in adipose tissue, driven by the adipocyte fatty acid-binding protein (aP2, also known as aFABP) promoter, showed a substantial decrease in total fat pad weight. Moreover, adipose tissue from transgenic mice showed reduced expression of adipocyte markers and adipocyte-secreted factors, including leptin and adiponectin, whereas the preadipocyte marker Pref-1 was increased. Pref-1 transgenic mice with a substantial, but not complete, loss of adipose tissue exhibited hypertriglyceridemia, impaired glucose tolerance, and decreased insulin sensitivity. Mice expressing the Pref-1/hFc transgene exclusively in liver under the control of the albumin promoter also showed a decrease in adipose mass and adipocyte marker expression, suggesting an endocrine mode of action of Pref-1. These findings demonstrate the inhibition of adipogenesis by Pref-1 in vivo and the resulting impairment of adipocyte function that leads to the development of metabolic abnormalities.

Figure 1

aP2-Pref-1/hFc transgene expression. (a) Analysis of transgene expression by Northern blot analysis in 3AS line of aP2-Pref-1/hFc mice. Total mRNA was extracted from the tissues of 10-week-old transgenic mice (TG) and wild-type littermates and probed with radiolabeled Pref-1 cDNA probe. S, spleen; Th, thymus; Li, liver; Lu, lung; K, kidney; SM, skeletal muscle; BAT, brown adipose tissue; Rep, reproductive fat pad; Ing, inguinal fat pad; Ren, renal fat pad. (b) Western blot analysis for Pref-1/hFc fusion protein in serum of three lines (1AS, 2AS, and 3AS) of aP2-Pref-1/hFc transgenic mice. The serum proteins were separated by SDS-PAGE and probed with human Fc antibody. The antibody detected the 75-kDa of Pref-1/hFc fusion protein in three founder lines of transgenic but not in wild-type mice.

Figure 2

Growth curve and organ weight of 3AS aP2-Pref-1/hFc transgenic mice. (a) Growth curves for male and female wild-type (open circles) and transgenic (filled circles) (TG) mice fed a chow diet. Body weight of mice measured at 5-day intervals is shown; each point represents mean ± SEM from 6 to 13 mice. Body weight of Pref-1/hFc transgenic mice was significantly lower (P < 0.01) than those of wild-type mice at all ages. (b) Organ weights of 10-week-old mice are presented as percentage of body weight (n = 8–10 per group). Statistically significant differences between the groups are indicated as *P < 0.05 and **P < 0.01.

Figure 3

Reduced fat pad weight in 3AS aP2-Pref-1/hFc transgenic mice. Fat depot weights from 10-week-old mice are presented (n = 6 per group). BAT, brown adipose tissue; Renal, renal fat pad; Ing, inguinal fat pad; Para, parametrial fat pad; Epi, epididymal fat pad. Statistically significant differences between the groups are indicated as *P < 0.05 and **P < 0.01.

Figure 4

Histological analysis of adipose tissue and cell size distribution. (a) Paraffin-embedded sections of renal white adipose tissue from 10-week-old male mice were stained with hematoxylin and eosin. Scale bar, 50 μm. (b) Distribution of the adipocyte volume in wild-type and aP2-Pref-1/hFc transgenic mice. The volume of at least 300 cells per sample (mean of four mice per group) was determined with the NIH image software.

Figure 5

Gene expression in adipose tissue from aP2-Pref-1/hFc transgenic mice. (a) Northern blot analysis of adipocyte marker expression in white adipose tissue of wild-type and 3AS aP2-Pref-1/hFc transgenic mice. Total RNA from three different fat pads (Ing, inguinal; Rep, reproductive; Ren, renal) was probed with cDNA probes for different adipocyte markers. (b) Endogenous Pref-1 expression analyzed by RT-PCR. A primer set was designed to amplify only the endogenous Pref-1 message (see Methods). β-actin was used as an internal control. (c) Northern blot analysis for adipocyte markers in interscapular brown adipose tissue.

Figure 6

Reduced fat pad weight and decreased expression of adipocyte markers in Alb-Pref-1/hFc transgenic mice expressing Pref-1/hFc specifically in liver. (a) Circulating Pref-1/hFc was detected by Western blot analysis in three different lines of Alb-Pref-1/hFc using Pref-1 antibody. Serum Pref-1/hFc levels in Alb-Pref-1/hFc mice were compared with those in 3AS aP2-Pref-1/hFc mice (upper panel). The liver-specific expression of the transgene in Alb-Pref-1/hFc mice was assessed by Northern blot analysis (lower panel). H, heart; Li, liver; S, spleen; Lu, lung; Th, thymus; K, kidney; BAT, brown adipose tissue; WAT, white adipose tissue. (b) Weight of fat pads was reduced in 21Alb-Pref-1/hFc mice (black bars) compared with wild-type littermates (white bars). Results are mean ± SEM of fat pads from nine to ten mice, and statistical significance is indicated as *P < 0.05, **P < 0.01. Ren, renal fat pad; Ing, inguinal fat pad; Rep, reproductive fat pad. (c) Northern blot analysis of adipocyte marker expression in inguinal (Ing) and reproductive (Rep) depots of white adipose tissue from 21Alb-Pref-1/hFc mice. TG, transgenic.

Figure 7

Reduction in fat pad mass was correlated with the expression levels of Pref-1/hFc in various transgenic lines. The high expresser line 3AS aP2-Pref-1/hFc showed a 45% reduction in total fad pad weight while lines 2AS aP2-Pref-1/hFc, 21Alb and 4Alb Alb-Pref-1/hFc, which express approximately 50% of that observed in 3AS, exhibited a 27, 21, and 25% reduction in total fat pad weight, respectively.

Figure 8

Glucose intolerance and insulin resistance in aP2-Pref-1/hFc transgenic mice that express high levels of Pref-1/hFc fusion protein. (a) Glucose-tolerance test. Overnight-fasted mice were given an intraperitoneal injection of glucose (2 mg/g body weight). Blood samples were collected from the tail at indicated time points and analyzed for glucose concentration. Results are means ± SEM from six animals in each group. (b) Insulin tolerance test. Insulin (0.5 U/kg body weight) was intraperitoneally injected after fasting for 5 hours. Data are presented as mean percentage of 0 min glucose value ± SEM from six mice in each group. Statistically significant differences between the groups are indicated as *P < 0.05 and **P < 0.01.

Figure 9

Growth retardation and bone malformation in aP2-Pref-1/hFc transgenic mice. (a) Pref-1 expression in aP2-Pref-1/hFc embryos. Total protein extracts from embryo day 12 (E12) embryo from wild-type and 3AS line were subjected to Western blot analysis for Pref-1. Pref-1/hFc fusion protein was detected as a 75-kDa band only in transgenic embryo, while multiple forms of endogenous Pref-1 were detected at around 50-kDa in both wild-type and transgenic mice. (b) The appearances of wild-type and aP2-Pref-1/hFc transgenic littermates were compared at E18, showing the growth retardation of transgenic embryos. (c) The transgenic mice had kinky tails, the severity of which is correlated with levels of transgene expression. (d) Bone and cartilage staining. Embryos (E17) were stained with alizarin red for bone and alcian blue for cartilages. The Pref-1/hFc transgenic embryos had a smaller thoracic cavity with short ribs. Vertebrae were fused and disorganized, resulting in scoliosis.