Life without white fat: a transgenic mouse

Abstract

We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model.

Figure 1

(A) Schematic of A-ZIP/F dominant-negative action. (Left) Cartoon of the B-ZIP homodimer, C/EBP. The amino-terminal part of the protein is the activation domain. Carboxy-terminal to the activation region is the basic DNA-binding domain, which in the absence of DNA is unstructured. This is followed by the α-helical leucine zipper dimerization domain. The dashed lines represent physical interactions between the leucines in the d positions that are critical for forming a coiled coil dimer structure. (Middle) C/EBP homodimer bound to DNA. This binding causes the basic region to form an α-helical extension of the leucine zipper, which enhances stability of the dimer by ∼100-fold. (Right) Heterodimerization between a C/EBPα monomer and a A-ZIP/F monomer. The carboxy-terminal F-zipper interacts specifically with the C/EBPα (or Jun) leucine zipper whereas the amino-terminal acidic extension forms a coiled coil with the basic domain of B-ZIP proteins. This heterodimeric coiled coil structure is more stable than C/EBPα bound to DNA by a further ∼100-fold. (B) A-ZIP/F inhibits the DNA binding of C/EBPα (left) and Fos+Jun (middle), but not CREB (right) in gel-shift assays. (Lanes 1) No protein; (lanes 2) 10 nmoles of the indicated B-ZIP domain, which forms a complex with the labeled oligonucleotide probe; (lanes 3,4,5) B-ZIP domains as well as 1-, 10-, or 100-fold molar equivalent of recombinant A-ZIP/F protein, respectively. Equimolar A-ZIP/F abolishes the DNA-binding of both C/EBPα and Fos+Jun, but has no effect on CREB DNA binding, even in 100-fold molar excess.

Figure 1

(A) Schematic of A-ZIP/F dominant-negative action. (Left) Cartoon of the B-ZIP homodimer, C/EBP. The amino-terminal part of the protein is the activation domain. Carboxy-terminal to the activation region is the basic DNA-binding domain, which in the absence of DNA is unstructured. This is followed by the α-helical leucine zipper dimerization domain. The dashed lines represent physical interactions between the leucines in the d positions that are critical for forming a coiled coil dimer structure. (Middle) C/EBP homodimer bound to DNA. This binding causes the basic region to form an α-helical extension of the leucine zipper, which enhances stability of the dimer by ∼100-fold. (Right) Heterodimerization between a C/EBPα monomer and a A-ZIP/F monomer. The carboxy-terminal F-zipper interacts specifically with the C/EBPα (or Jun) leucine zipper whereas the amino-terminal acidic extension forms a coiled coil with the basic domain of B-ZIP proteins. This heterodimeric coiled coil structure is more stable than C/EBPα bound to DNA by a further ∼100-fold. (B) A-ZIP/F inhibits the DNA binding of C/EBPα (left) and Fos+Jun (middle), but not CREB (right) in gel-shift assays. (Lanes 1) No protein; (lanes 2) 10 nmoles of the indicated B-ZIP domain, which forms a complex with the labeled oligonucleotide probe; (lanes 3,4,5) B-ZIP domains as well as 1-, 10-, or 100-fold molar equivalent of recombinant A-ZIP/F protein, respectively. Equimolar A-ZIP/F abolishes the DNA-binding of both C/EBPα and Fos+Jun, but has no effect on CREB DNA binding, even in 100-fold molar excess.

Figure 2

(A) Schematic of the transgene for fat-specific expression of the A-ZIP/F dominant-negative protein. (Open region) 7.6-kb aP2 promoter/enhancer; (closed region) 298-bp A-ZIP/F open reading frame with a Flag epitope; (striped region) ∼1.0-kb SV40 small t antigen splice and poly(A) sites. The unique restriction sites used to form junctions between these three different elements are shown, as well as the 5′ and 3′ sites used for isolating DNA for microinjection from the plasmid. (B) Lack of WAT in A-ZIP/F-1 mice. Dorsal view of male littermates, aged 26 weeks, after removal of the skin. (Left) Wild-type mouse; (right) A-ZIP/F-1 mouse. Note the absence of visible WAT and the residual interscapular adipose tissue in the A-ZIP/F-1 animal.

Figure 2

(A) Schematic of the transgene for fat-specific expression of the A-ZIP/F dominant-negative protein. (Open region) 7.6-kb aP2 promoter/enhancer; (closed region) 298-bp A-ZIP/F open reading frame with a Flag epitope; (striped region) ∼1.0-kb SV40 small t antigen splice and poly(A) sites. The unique restriction sites used to form junctions between these three different elements are shown, as well as the 5′ and 3′ sites used for isolating DNA for microinjection from the plasmid. (B) Lack of WAT in A-ZIP/F-1 mice. Dorsal view of male littermates, aged 26 weeks, after removal of the skin. (Left) Wild-type mouse; (right) A-ZIP/F-1 mouse. Note the absence of visible WAT and the residual interscapular adipose tissue in the A-ZIP/F-1 animal.

Figure 3

Expression analysis of the transgene. (A) Total RNA was isolated from wild-type BAT and the 11 indicated A-ZIP/F-1 tissues (interscapular fat is BAT), electrophoresed, blotted, and probed successively for A-ZIP/F and aP2. All lanes contain 10 μg of RNA, except for the interscapular fat, which contains 5 μg. Quantitation by PhosphorImager showed that A-ZIP/F expression in the heart lane is 3% of the level in BAT. Ethidium bromide staining confirmed RNA loading (bottom). (B) Anti-Flag antibody was used to probe expression of the Flag-tagged A-ZIP/F protein in BAT of A-ZIP/F-1 and littermate wild-type mice. (DN) Mobility of the A-ZIP/F molecule. A recombinant standard (20, 5, 2, 0.5, and 0.2 pmole) was run for quantitation. Molecular mass is indicated on the right.

Figure 3

Expression analysis of the transgene. (A) Total RNA was isolated from wild-type BAT and the 11 indicated A-ZIP/F-1 tissues (interscapular fat is BAT), electrophoresed, blotted, and probed successively for A-ZIP/F and aP2. All lanes contain 10 μg of RNA, except for the interscapular fat, which contains 5 μg. Quantitation by PhosphorImager showed that A-ZIP/F expression in the heart lane is 3% of the level in BAT. Ethidium bromide staining confirmed RNA loading (bottom). (B) Anti-Flag antibody was used to probe expression of the Flag-tagged A-ZIP/F protein in BAT of A-ZIP/F-1 and littermate wild-type mice. (DN) Mobility of the A-ZIP/F molecule. A recombinant standard (20, 5, 2, 0.5, and 0.2 pmole) was run for quantitation. Molecular mass is indicated on the right.

Figure 4

(A) Histology of adipose tissue. Wild-type, A-ZIP/F-1, and ob/ob interscapular BAT, and wild-type and A-ZIP/F-1 epididymal WAT are shown as indicated. Hematoxylin and eosin staining magnification 150× (top and middle); 60× (bottom). (B) Expression of C/EBP regulated genes in adipose tissue. Three separate Northern blots containing total RNA from A-ZIP/F-1 interscapular BAT and from wild-type interscapular BAT and epididymal WAT were prepared and hybridized with DNA probes for UCP1, leptin, C/EBPα, and PPARγ. The UCP1, leptin, and C/EBPα blots contain 10 μg RNA per lane and the PPARγ blot, 5 μg of RNA per lane. Ethidium bromide staining is shown as a loading control.

Figure 4

(A) Histology of adipose tissue. Wild-type, A-ZIP/F-1, and ob/ob interscapular BAT, and wild-type and A-ZIP/F-1 epididymal WAT are shown as indicated. Hematoxylin and eosin staining magnification 150× (top and middle); 60× (bottom). (B) Expression of C/EBP regulated genes in adipose tissue. Three separate Northern blots containing total RNA from A-ZIP/F-1 interscapular BAT and from wild-type interscapular BAT and epididymal WAT were prepared and hybridized with DNA probes for UCP1, leptin, C/EBPα, and PPARγ. The UCP1, leptin, and C/EBPα blots contain 10 μg RNA per lane and the PPARγ blot, 5 μg of RNA per lane. Ethidium bromide staining is shown as a loading control.

Figure 5

Absence of WAT in A-ZIP/F-1 mice at day 1. Transverse sections at the level of the neck were made from ∼18-hr wild-type (top) and A-ZIP/F-1 littermate (bottom) mice. (B) BAT; (W) WAT; (M) muscle; (S) skin. Hematoxylin and eosin staining magnification, 30×.

Figure 6

Appearance of skin, liver, and pancreas in mice at 22 weeks of age. Tissues from female wild-type (WT, left) and A-ZIP/F-1 (right) mice are shown at the same magnifications. (A) Skin, with WAT visible below the dermis in wild-type but not A-ZIP/F-1 mice. Grossly, livers of A-ZIP/F-1 mice are enlarged and lighter in color (B). Microscopically the A-ZIP/F-1 liver is filled with lipid droplets (C). Pancreatic islet β cell hypertrophy and hyperplasia were present in all A-ZIP/F-1 mice examined. A particularly striking example is in D (the lighter staining cells are the islets and the darker region is the exocrine pancreas). Hematoxylin and eosin staining magnification, 30× (A,C) and 16× (D).

Figure 7

Growth and survival of A-ZIP/F-1 mice. (Top) A-ZIP/F-1 (•) and littermate [wild-type (○)] controls were weighed, and the data were binned using 1-week (3–12 weeks) or 2-week (12–28 weeks) intervals. Data for the 1-, 7-, and 14-day-old mice include both sexes (gender differences did not appear until 4 weeks) and are presented in both panels. Data are mean ± s.e.m., n = 6–35. The A-ZIP/F-1 mice weighed less than wild-type mice (P < 0.05) from 1 to 7 weeks. Female A-ZIP/F-1 mice were significantly heavier for all points >11 weeks, whereas the difference in males was significant only at weeks 20 and 22. (Bottom) Mortality from weaning to 30 weeks in A-ZIP/F-1 mice. Survival analysis (Kaplan-Meier; Altman 1991) is based on 34 males and 51 females. None of the littermate controls died.

Figure 8

Adult A-ZIP/F-1 mice are diabetic. Blood was obtained from A-ZIP/F-1 mice and nontransgenic wild-type controls, aged 6–23 weeks in the nonfasting state (males, solid bars; females, open bars). Serum glucose, insulin, triglycerides, free fatty acids, and β-hydroxybutyrate were measured as detailed in Materials and Methods. The insulin panel uses a log scale. Data are mean ± s.e.m., with n = 16–20 per group (except for β-hydroxybutyrate, with n = 10 and wild-type insulin, with n = 6 and 7 per group). The glucose, insulin, triglyceride, and free fatty acids levels were elevated in A-ZIP/F-1 mice compared with their sex-matched controls at P<0.01. The insulin (P = 0.04) and free fatty acids (P = 0.005) levels differed between the male and female A-ZIP-1 mice.

Figure 9

Development of diabetes in A-ZIP/F-1 mice after birth. Glucose, insulin, and free fatty acids were measured in A-ZIP/F-1 (•) and littermate (○) control mice. Samples were serum from exsanguination for weeks 1–3 and tail vein plasma for weeks 4 and 5. Data are mean ± s.e.m., n = 5–6 per group.

Figure 10

Effect of fasting on A-ZIP/F-1 and wild-type mice. Male A-ZIP/F-1 and littermate wild-type controls, aged 23 weeks, were fasted for 24 hr (1:00 pm to 1:00 pm). Tail vein blood was obtained at the start (fed, solid bars) and conclusion (fasted, open bars) of the fast. Serum glucose, insulin, free fatty acids, and β- hydroxybutyrate were measured as detailed in Materials and Methods. Insulin was not measured in the wild-type mice and was undetectable in the fasted A-ZIP/F-1 mice (detection limit: 3 ng/ml). Data are mean ± s.e.m., n = 5 per group. (*) Difference at P ≤ 0.001, except for P = 0.02 for free fatty acids.