Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy

Abstract

Overexpression of the nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c/ADD1) in cultured 3T3-L1 preadipocytes was shown previously to promote adipocyte differentiation. Here, we produced transgenic mice that overexpress nSREBP-1c in adipose tissue under the control of the adipocyte-specific aP2 enhancer/promoter. A syndrome with the following features was observed: (1) Disordered differentiation of adipose tissue. White fat failed to differentiate fully, and the size of white fat depots was markedly decreased. Brown fat was hypertrophic and contained fat-laden cells resembling immature white fat. Levels of mRNA encoding adipocyte differentiation markers (C/EBPalpha, PPARgamma, adipsin, leptin, UCP1) were reduced, but levels of Pref-1 and TNFalpha were increased. (2) Marked insulin resistance with 60-fold elevation in plasma insulin. (3) Diabetes mellitus with elevated blood glucose (>300 mg/dl) that failed to decline when insulin was injected. (4) Fatty liver from birth and elevated plasma triglyceride levels later in life. These mice exhibit many of the features of congenital generalized lipodystrophy (CGL), an autosomal recessive disorder in humans.

Figure 1

Photograph of 7-day-old wild-type male mouse (left) and a transgenic aP2–SREBP-1c436 (line A) male littermate mouse (right). (A) Dorsal view of the mice, illustrating the enlargement of the interscapular brown fat pads in the transgenic mouse. (B) Exposed dorsal view of the interscapular brown fat pads. (C) Exposed ventral view of the mice, illustrating the enlarged fatty liver in the transgenic mouse.

Figure 2

Liver, interscapular brown fat, epididymal white fat/testis from a 3-month-old wild-type male mouse (left) and a transgenic aP2–SREBP-1c436 (line A) male littermate mouse (right). The wild-type and transgenic livers weighed 1.5 and 3.9 grams, respectively. The wild-type and transgenic interscapular fat weighed 58 and 110 mg, respectively. The wild-type and transgenic epididymal fat weighed 510 and 160 mg, respectively.

Figure 3

Histologic sections of liver (A,B), white fat (C,D), and brown fat (E,F) from an 8-day-old wild-type male mouse and a transgenic aP2–SREBP-1c436 male littermate mouse (hematoxylin and eosin stain; magnification, 112 ×). (A,B) Hepatocytes throughout the liver of the transgenic mouse are dramatically swollen. The cytoplasm stains palely eosinophilic and contains numerous microvesicular vacuoles. The alteration is uniform throughout the lobules. Scattered hematopoietic foci are present in both the wild-type and transgenic liver. (C) White fat pad of a wild-type mouse contains a heterogeneous population of cells consisting of both small immature adipocytes (left) and mature adipocytes, each of which contains a large unilocular vacuole (right). (D) White fat of the transgenic mouse contains numerous, small immature adipocytes with brightly eosinophilic cytoplasm and a distinct unilocular vacuole. (E,F) Adipocytes of the brown fat of the transgenic mouse are dramatically enlarged and stain palely. The cytoplasm is replaced by numerous small vacuoles that frequently coalesce to form large unilocular vacuoles, resembling those of immature white adipocytes.

Figure 4

Histologic sections of liver (A,B), white fat (C,D), and brown fat (E,F) from a 6-week-old wild-type mouse and a transgenic aP2–SREBP-1c436 littermate (hematoxylin and eosin stain; magnification, 112 ×). (A,B) Hepatocytes of the transgenic mouse are swollen and contain microvesicular vacuoles. (C,D) Adipocytes in white fat of the transgenic mouse show marked heterogeneity of size. Admixed with the mature adipocytes are smaller, immature adipocytes with bright eosinophilic cytoplasm, a round nucleus, and a distinct unilocular vacuole. This is in contrast to the WAT from a wild-type mouse, which consists of adipocytes of uniform size filled with a large, unilocular vacuole. (E,F) Interscapular brown fat of the transgenic mouse consists of markedly enlarged adipocytes containing large unilocular fat droplets. The interstitium shows signs of mild chronic inflammation.

Figure 5

Amounts of various mRNAs in liver, white fat (WF), and brown fat (BF) of wild-type mice (WT) and transgenic aP2-SREBP-1c436 (line A) mice (Tg) as measured by blot hybridization. The radioactivity in each band was quantified as described in Materials and Methods. The fold change in each mRNA of transgenic mice relative to that of wild-type mice was calculated after correction for loading differences by measuring the amount of 18S rRNA. (A) Total RNA was isolated from two of the seven wild-type and two of the seven transgenic mice (line A) described in Table 1. The RNA was pooled, and 10-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe. The probe for stearoyl CoA desaturase (SCD) was a mouse SCD1 cDNA fragment that can detect both SCD1 and SCD2 mRNAs (Shimomura et al. 1998). (B) Total RNA was isolated from the remaining five wild-type and five littermate transgenic mice (line A) described in Table 1. The RNA was pooled, and 10-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe.

Figure 5

Amounts of various mRNAs in liver, white fat (WF), and brown fat (BF) of wild-type mice (WT) and transgenic aP2-SREBP-1c436 (line A) mice (Tg) as measured by blot hybridization. The radioactivity in each band was quantified as described in Materials and Methods. The fold change in each mRNA of transgenic mice relative to that of wild-type mice was calculated after correction for loading differences by measuring the amount of 18S rRNA. (A) Total RNA was isolated from two of the seven wild-type and two of the seven transgenic mice (line A) described in Table 1. The RNA was pooled, and 10-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe. The probe for stearoyl CoA desaturase (SCD) was a mouse SCD1 cDNA fragment that can detect both SCD1 and SCD2 mRNAs (Shimomura et al. 1998). (B) Total RNA was isolated from the remaining five wild-type and five littermate transgenic mice (line A) described in Table 1. The RNA was pooled, and 10-μg aliquots were subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe.

Figure 6

Changes in amounts of mRNA for Pref-1 (A) and TNFα (B) in wild-type mice and transgenic aP2–SREBP-1c436 (line A) mice as measured by Rnase protection assay. Aliquots of total RNA (20 μg) from pooled samples (see below) isolated from the indicated genotype were hybridized in solution for 10 min at 68°C to the ³²P-labeled cRNA probes for Pref-1 and TNFα, all in the presence of a cRNA probe for β-actin as described in Materials and Methods. After RNase digestion, the protected fragments were separated by gel electrophoresis and exposed to film for 16 hr at −80°C. The radioactivity in the gels was quantified, normalized to the β-actin signal, and expressed as the fold change relative to the mRNA level of wild-type mice. (A) Total RNA was isolated from the white fat (WF) and brown fat (BF) of five wild-type (WT) mice and five littermate transgenic (Tg) mice (line A) described in Fig. 5B. (B) Total RNA was isolated from the gastrocnemius muscle and spleen of three of the seven wild-type and four of the seven littermate transgenic mice (line A) described in Table 1. The total RNA from white fat and brown fat was obtained from an aliquot of the same sample in A. Total RNA of liver was isolated from two wild-type and two littermate transgenic mice (line A) described in Fig. 5A.

Figure 7

Plasma concentrations of insulin (A,C) and glucose (B,D) in wild-type (open bars) and two lines of transgenic aP2–SREBP-1c436 (solid bars) mice. Blood was drawn in the early-to-middle phase of the light cycle in the nonfasted state. Each value represents the mean ±s.e.m. of seven mice (A,B) or four mice (C,D).

Figure 8

Insulin tolerance tests in wild-type (○) and transgenic aP2-SREBP-1c436 (•) mice. Ad-libitum fed animals were injected intraperitoneally with human crystalline insulin in the middle of the light cycle as described in Materials and Methods, and blood was taken at the indicated time for measurement of glucose content. Results are expressed as percent of the plasma glucose concentration at zero time and are plotted as mean ±s.e.m. of the indicated number of animals. The zero-time plasma glucose levels were as follows: Exp. 1, 164 ± 23 and 310 ± 38 mg/dl in wild-type and transgenic mice, respectively; Exp. 2, 163 ± 11 and 412 ± 72; Exp. 3, 183 ± 7 and 279 ± 91.

Figure 9

Uptake of [³H]2-deoxyglucose by isolated soleus muscle in wild-type and transgenic aP2–SREBP-1c436 mice. Soleus muscles from 12-week-old female mice (line A) were first incubated for 30 min with (solid bars) or without (open bars) the indicated concentration of insulin, followed by a 15-min incubation with [³H]2-deoxyglucose as described in Materials and Methods. Each value denotes the mean ± s.e.m. of the indicated number of animals. Plasma glucose and insulin were measured in the mice used in B. Plasma glucose levels (mean ± s.e.m.) were 170 ± 12 and 334 ± 32 mg/dl for wild-type and transgenic mice, respectively. Plasma insulin levels were 0.07 ± 0.02 and 3.2 ± 0.7 nm for wild-type and transgenic mice, respectively.