Insulin depletion leads to adipose-specific cell death in obese but not lean mice

Abstract

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory "overexpression" of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

Figure 1

Functional neutralization of insulin by antibody. (A) Cultured 3T3-L1 adipocytes were assayed for their rate of 2-deoxyglucose uptake in the absence or presence of 5 nM porcine insulin (Lilly), alone, or in combination with guinea pig anti-porcine insulin (Sigma). Values represent mean ± SEM. (B) After an initial blood sample, C57BL/6J mice (n = 3) were injected i.p. with 2 g/kg glucose along with 50 μl anti-insulin antibody (•) or a nonimmune control serum (○). Serial blood samples were drawn from the tail vein and serum glucose levels were determined by using a hexokinase glucose assay kit (Sigma) Values represent mean ± SEM.

Figure 2

Effect of insulin depletion on tissue total RNA in obese and lean. (A) Male C57BL/6J ob/ob or lean mice were given two i.v. injections of 100 μl of anti-insulin antibody (–6) or nonimmune control serum (–3) at 0 and 3 hr. At 6 hr, mice were killed and epididymal white adipose tissue excised and snap frozen. (B) ob/ob mice were given a single i.p. injection of 100 μl of anti-insulin antibody alone (1) or in combination with 20 (2) or 60 μg (3) of porcine insulin. After 4 hr, adipose tissue was excised and snap frozen. (C) C57BLS/J db/db mice were given a single i.p. injection of 100 μl of antibody or a saline control. Eight hr postinjection, adipose (A), liver (L), brain (B), spleen (S), and skeletal muscle (M) tissues were isolated and snap frozen. In all cases, RNA was extracted from the isolated tissues by the guanidinium thiocyanate method, separated by formaldehyde/agarose gel electrophoresis, capillary blotted, and stained with methylene blue (18). The 28S ribosomal RNA band is shown.

Figure 3

Histological analysis of adipose tissue from control and anti-insulin antibody injected obese mice. Epidiymal adipose tissue was excised from control (A) or antibody-treated (100 μl i.p.) (B–D) male C57BLS/J db/db mice eight hr postinjection. After routine overnight fixation in neutral buffered formalin and paraffin embedding, 4-μm sections were prepared and stained with hematoxylin and eosin for light microscopy.

Figure 4

Effect of complement depletion on adipocyte cell death in obese mice in response to anti-insulin antibody. C57BL/6 ob/ob mice were injected i.p. with either saline (–3) or 20 units/kg body weight cobra venom anti-complementarity protein (1, 4, 5) 16 hr before injection with either saline (1) or anti-insulin antibody (–5). After 5 hr, epidydimal fat was excised and RNA was purified, electrophoresed, blotted, and stained with methylene blue.

Figure 5

DNA laddering analysis of adipose tissue DNA from insulin-depleted obese mice. (A) Genomic DNA was isolated from adipose tissue of control (3, 4) or anti-insulin antibody treated (1, 2) C57BL6 ob/ob mice described in Fig. 2A. (B). Male C57BL/6J ob/ob mice were injected with 200 mg/kg streptozotocin (2) or saline vehicle (1) as an i.v. bolus in 100 μl, adipose tissue was excised after 24 hr, and genomic DNA was prepared. DNA ladder was visualized by Southern blot analysis with a total mouse DNA probe.