Insig-1 "brakes" lipogenesis in adipocytes and inhibits differentiation of preadipocytes

Abstract

We have examined gene expression in the fat tissue of normal mice at the onset of diet-induced obesity. Insulin-induced gene 1 (insig-1) mRNA rose progressively with a high-fat diet and declined on a restricted diet. Because insig-1 binds sterol regulatory element-binding protein cleavage-activating protein in the endoplasmic reticulum, thereby blocking proteolytic processing required for sterol regulatory element-binding protein activation, we tested its influence on lipogenesis. In differentiating 3T3-L1 cells, insig-1 and -2 rose in parallel with aP2 mRNA during differentiation. The mRNA of the lipogenic transcription factor, carbohydrate response element-binding protein, was undetectable in undifferentiated 3T3-L1 preadipocytes but rose dramatically during differentiation in 25 mM, but not in 5 mM, glucose. Transfection of mouse or human insig-1 into 3T3-L1 preadipocytes completely prevented oil red O staining and blocked upregulation of aP2, peroxisome proliferator-activated receptor gamma2, and carbohydrate response element-binding protein, while reducing down-regulation of preadipocyte factor 1. The results suggest that insig-1 expression restricts lipogenesis in mature adipocytes and blocks differentiation in preadipocytes.

Fig. 1.

Changes in insig-1 expression during expansion and contraction of adipose tissue. (A) Weight of epididymal fat pad in mice on a 60% (•—•) or 4% (□—□) fat diet. n = 3 at 1 and 2 weeks; n = 4 at 4 and 5 weeks. (B) Plasma leptin levels in these mice. (C) Insig-1 mRNA/18S mRNA ratio of epididymal fat pad of mice on a 60% (•—•) or 4% (□—□) fat diet. (n = 3). (D) Insig-1 mRNA/18S mRNA ratio in the epididymal fat pad of rats on an unrestricted 4% fat diet (□) or after 4 days of 50% restriction (▪) (n = 4). *, P < 0.05; **, P < 0.01.

Fig. 2.

Northern blots of 3T3-L1 cells for insig-1 and -2 mRNA and aP₂ during 9 days of differentiation into mature adipocytes. Bottom shows equal amount of total RNA was loaded.

Fig. 3.

Insig-1 (▪) and -2 (□) mRNA/18S ratio and oil red O staining of 3T3-L1 cells. (A Lower Left) Undifferentiated 3T3-L1 cells. (Lower Center) 3T3-L1 cells cultured in standard medium (25 mM glucose, Ins/Dex/IBMX). (Lower Right) Cells cultured in the foregoing medium plus 1 mM of a 2:1 oleate/palmitate mixture. (B Lower Left) Undifferentiated 3T3-L1 cells. (Lower Center) Cells cultured with Ins/Dex/IBMX but only 5 mM glucose. (Lower Right) Cells were cultured in the foregoing medium plus 1 mM fatty acid mixture. Numbers below panels indicate the percent area of oil red O stain. *, Significant (P < 0.01) lowering of oil red O staining compared with A. FA, fatty acids.

Fig. 4.

Expression in 3T3-L1 cells of genes involved in lipid metabolism before and after differentiation in Ins/Dex/IBMX-containing medium with 5 mM glucose (A) or 25 mM glucose (B). ▪, undifferentiated 3T3-L1 cells; □, 3T3-L1 cells cultured for 9 days in Ins/Dex/IBMX without added fatty acids;, cells cultured in Ins/Dex/IBMX with added oleate/palmitate (2:1). Data are mean ± SEM of the relative amount of mRNA of interest calculated with 18S as standard. *, P < 0.05; **, P < 0.01 (n = 3). FAS, fatty acid synthase. ACC, acetyl CoA carboxylase.

Fig. 5.

Effects of insig-1 transfection into 3T3-L1 cells on their differentiation. (A) Immunoblotting for V5-tagged insig-1 of 3T3-L1 cells transfected with vector (Left) or with insig-1 (Right). (B) Oil red O staining of cells transfected with empty vector (Left) or with insig-1 (Right) before and 9 days after culture Ins/Dex/IBMX and 25 mM glucose (Left).

Fig. 6.

Expression of relevant genes in 3T3-L1 cells transfected with insig-1. □, mouse insig-1 (m) or human insig-1 (h) transfection; ▪, transfection with empty vector (EV). All cells were cultured in 25 mM glucose with Ins/Dex/IBMX. Data are mean ± SEM of the relative amount of mRNA of interest calculated with 36B4 as standard. *, P < 0.05; **, P < 0.01 (n = 3). Pref-1, preadipocyte factor 1; ACC, acetyl CoA carboxylase.