End-product feedback inhibition of cholesterol synthesis was first demonstrated in living animals by Schoenheimer 72 years ago. Current studies define Insig proteins as essential elements of this feedback system in mouse liver. In cultured cells, Insig proteins are required for sterol-mediated inhibition of the processing of sterol regulatory element-binding proteins (SREBPs) to their nuclear forms. We produced mice with germline disruption of the Insig2 gene and Cre-mediated disruption of the Insig1 gene in liver. On a chow diet, these double-knockout mice overaccumulated cholesterol and triglycerides in liver. Despite this accumulation, levels of nuclear SREBPs and mRNAs for SREBP target genes in lipogenic pathways were not reduced. Whereas cholesterol feeding reduced nuclear SREBPs and lipogenic mRNAs in wild-type mice, this feedback response was severely blunted in the double-knockout mice, and synthesis of cholesterol and fatty acids was not repressed. The amount of HMG-CoA reductase protein was elevated out of proportion to the mRNA in the double-knockout mice, apparently owing to the failure of cholesterol to accelerate degradation of the enzyme. These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in liver as they do in cultured cells. IntroductionIn 1933, Rudolph Schoenheimer, a physician then working in Germany, performed a metabolic balance study in mice and observed that the animals synthesized large amounts of cholesterol when fed a low-cholesterol diet and that synthesis stopped when the mice were fed cholesterol (1). This experiment provided the first evidence for end-product feedback regulation of a biosynthetic pathway, a discovery that predated by 2 decades the demonstration of end-product feedback inhibition in bacteria and prefigured much of modern molecular biology and metabolic science. With the advent of radioisotopes, Gordon Gould showed in the early 1950s that the cholesterol-feedback system operated in the liver of living animals (2, 3).In subsequent years many details of cholesterol feedback were elucidated, including the identification of HMG-CoA reductase as a highly regulated rate-controlling reaction (reviewed in ref. 4). However, the fundamental molecular mechanism by which liver cells sense cholesterol and use that information to regulate cholesterol production remained obscure until recent studies were conducted in cultured mammalian cells.The cultured cell studies showed that transcription of HMG-CoA reductase and other genes in the cholesterol biosynthetic pathway is dependent upon sterol regulatory element-binding proteins (SREBPs), a family of sterol-regulated, membrane-bound transcription factors (5). The SREBPs begin life as integral proteins of

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 aP 2 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 aP 2, peroxisome proliferator-activated receptor ␥2, 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.

To determine whether the antilipogenic actions of insulin-induced gene 1 (insig-1) demonstrated in cultured preadipocytes also occur in vivo, we infected Zucker diabetic fatty (ZDF) (fa͞fa) rats, with recombinant adenovirus containing insig-1 or -2 cDNA. An increase of both proteins appeared in their livers. In control ZDF (fa͞fa) rats infected with adenovirus containing the ␤-galactosidase (␤-gal) cDNA, triacylglycerols in the liver and plasma rose steeply whereas the insig-infected rats exhibited substantial attenuation of the increase in hepatic steatosis and hyperlipidemia. Insig overexpression was associated with a striking reduction in the elevated level of nuclear sterol regulatory element-binding protein (SREBP)-1c, the activated form of the transcription factor. The mRNA of SREBP-1c lipogenic target enzymes also fell. The mRNA of endogenous insig-1, but not -2a and -2b, was higher in the fatty livers of untreated obese ZDF (fa͞fa) rats compared with controls, but the elevation was not sufficient to block the Ϸ3-fold increase in SREBP-1c expression and activity. In normal animals, adenovirusinduced overexpression of the insigs reduced the increase in SREBP-1c mRNA and its target enzymes caused by refeeding. The findings demonstrated that both insigs have antilipogenic action when transgenically overexpressed in livers with increased SREBP1c-mediated lipogenesis. However, the increase in endogenous insig-1 expression associated with augmented lipogenesis may limit it, but is insufficient to prevent it. Insulin-induced gene 1 (insig-1) was originally cloned by Peng et al.(1) in regenerating liver and was subsequently shown to be dramatically elevated in the fat tissue of rats at the onset of diet-induced obesity (DIO) (2). Its function was unknown until Yang et al. (3) demonstrated that it binds sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), thereby preventing it from leaving the endoplasmic reticulum to escort SREBPs to the Golgi. SREBP proteins are inactive until converted by proteolytic processing in the Golgi into active transcription factors (4). In other words, insig-1 effectively blocks the activation of SREBPs.Although this seminal discovery was directed at the liver's cholesterogenic transcription factor, SREBP-2, it obviously was relevant to other members of the SREBP family. It was subsequently shown that transfection of insig-1 into preadipocytes completely blocks glucose-derived lipogenesis in 3T3-L1 adipocytes (5). This finding led to the proposal that the rise in insig-1 in the expanding adipocytes of diet-induced obesity ''brakes'' the lipogenesis so as to avoid an overaccumulation of fat that would exceed their storage capacity and damage them.This antilipogenic effect of insig-1 had been demonstrated in vitro without any corroborating evidence that the same antilipogenic effect also occurred in vivo in adult animals. To obtain such evidence, we chose to determine whether overexpression of insig-1 [and͞or insig-2, a second SCAP-binding protein discov...

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

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