Insulin is one of several factors regulating SREBP-1c and hepatic triglyceride metabolism (). Our data, together with data in mice with liver-specific knockout of both IRS-1 and IRS-2 (Guo et al., 2009
), or Akt2 (Leavens et al., 2009
), show that feeding is able to activate mTORC1 and induce SREBP-1c mRNA via insulin-independent signaling pathways. The activation of ChREBP and lipogenic gene expression by fructose is also independent of hepatic insulin signaling, as is the regulation of non-esterified fatty acid flux from the adipocyte, an important driver of hepatic triglycerides. Hepatic insulin signaling is, however, required for the accumulation of nuclear SREBP-1c protein. Since SREBP-1c activates its own transcription, completing a feed-forward loop (Chen et al., 2004
; Dif et al., 2006
), insulin is also necessary for the full induction of SREBP-1c mRNA. These insulin-dependent signaling pathways are critical for the pathological induction of SREBP-1c and lipogenic gene expression observed in obesity.
Interaction between insulin-dependent and insulin-independent signaling pathways in the control of SREBP-1c and hepatic triglycerides
One important regulator of SREBP-1c is Insig2a. In vitro
, insulin increases degradation of the Insig2a
transcript (Yellaturu et al., 2009
) but it may also increase Insig2a
transcription, since activation of Liver X Receptor, a nuclear receptor that is induced by insulin, increases Insig2a
mRNA (Hegarty et al., 2005
). Our data add further complexity. First, Insig2a
expression in vivo
can be suppressed independently of hepatic insulin signaling, as Insig2a
mRNA levels are decreased in the livers of re-fed LIRKO mice (), Ad-Cre treated mice on the high-fat diet (Fig. S4F
), and mice treated with IR ASO (Fig. S6K
). Such in vivo regulation by feeding could be mediated by a factor other than insulin. One intriguing possibility is that in vivo Insig2a
is suppressed by leptin, as Insig2a
mRNA is increased by leptin deficiency (Kammoun et al., 2009
), even after knockdown of the insulin receptor by ASO treatment (Fig. S6K
). Second, insulin may regulate Insig2 post-transcriptionally, as Insig2 protein is increased in LIRKO livers despite lower levels of Insig2a
mRNA. Moreover, insulin is required for other aspects of SREBP-1c processing, nuclear import or stability, as knockdown of Insig2 does not fully restore nuclear SREBP-1c protein in LIRKO livers (Fig. S3B
The role of hepatic insulin signaling in the induction of SREBP-1c becomes more important in the setting of obesity/Type 2 diabetes, as modeled by the ob/ob
mouse. That is, though feeding induces mRNA levels of SREBP-1c and its targets via insulin-independent signaling pathways, the further induction which occurs in obesity is entirely insulin dependent. Neither leptin deficiency, nor any of the other metabolic or hormonal changes associated with the Type 2 diabetic state is capable of inducing lipogenic gene expression in the absence of hepatic insulin signaling. This is consistent with concept of “selective insulin resistance.” Although the specific signaling pathways that remain sensitive to insulin in obesity remain unclear, continued insulin signaling through phosphatidylinositol 3-kinase (Anai et al., 1999
) and its downstream targets mTORC1 (Um et al., 2004
; Khamzina et al., 2005
), as well as PKC-λ (Standaert et al., 2004
), have been reported.
Both mTORC1 dependent and independent signals are necessary for the full activation of SREBP-1c (Yecies et al., 2011
; Wan et al., 2011
). We therefore propose a model of obesity in which over-nutrition, independently of insulin, drives mTORC1, while insulin signaling through a distinct pathway, such as PKC-λ, permits the accumulation of nuclear SREBP-1c and activation of its feed-forward transcriptional loop. However, our data do not rule out the possibility that insulin, in the setting of hyperinsulinemia and obesity, becomes an important driver of mTORC1, even though it is not required for the induction of mTORC1 by feeding.
Fructose can bypass the requirement for insulin in the regulation of the lipogenic genes, as the fructose diet increases Acc
mRNA and protein in LIRKO livers to levels equal to or higher than those found in chow-fed controls. This is due, in part, to activation of ChREBP, as nuclear ChREBP levels and expression of the ChREBP target, pyruvate kinase, are increased in the livers of fructose fed LIRKO mice. The activation of ChREBP requires glucokinase, which in turn requires insulin for its transcription (Dentin et al., 2004
). However, fructose, which activates glucokinase post-transcriptionally by promoting its translocation from the nucleus to the cytosol, could drive ChREBP independently of insulin (Doiron et al., 1994
; Mayes, 1993
; Petersen et al., 2001
In human Type 2 diabetes, hyperinsulinemia, dietary fructose, and other lipogenic stimuli are present. Our data suggest that identifying and targeting the specific signaling pathways by which insulin stimulates lipogenesis, as well as decreasing dietary fructose, could be extremely effective in reducing SREBP-1c and ameliorating hepatic steatosis in Type 2 diabetes.
Animals, Diets and Treatments
Generation and genotyping of LIRKO (Cre+/−
, IR lox/lox
) mice and their littermate controls (Cre−/−
, IR lox/lox
) has been described previously (Michael et al., 2000
). LIRKO mice were maintained on a mixed genetic background. Unless otherwise indicated, the mice used in these experiments were male, eight to ten weeks of age, and sacrificed at 2 p.m. For fasting and re-feeding studies, mice were sacrificed under the following conditions: ad libitum fed; after a 24 hour fast; or after a 24 hour fast followed by re-feeding a high carbohydrate diet (TD. 88122, Harlan Teklad Diets). For adenoviral mediated knockdown of the insulin receptor, two month old mice homozygous for the floxed allele of the insulin receptor were injected via tail vein with 5 × 109
pfu of adenovirus encoding Cre or GFP (Viraquest), and sacrificed 21 days later. For the fructose feeding experiments, mice were fed ad libitum with a 60% fructose diet for one week (TD. 00202, Harlan Teklad Diets). For the antisense oligonucleotide mediated knockdown of the insulin receptor, C57Bl/6J mice and ob/ob
mice(Jackson Labs) were given 50 mg/kg IP of the chemically modified ASO each week for five weeks and sacrificed one day after the final dose. All animal experiments were performed with the approval of the Institutional Animal Care and Research Advisory Committee at Children’s Hospital Boston.
Gene Expression Analysis
Gene expression was measured using real-time PCR. Results were normalized to the house keeping gene, Tbp, and the value of the control group was set to 1.
Microsomal and nuclear protein extracts for measurements of SREBP-1 (Horton et al., 1998
), microsomal extracts for the measurements of the Insig proteins (Engelking et al., 2004
), and nuclear extracts for the measurements of ChREBP (Miao et al., 2009) were prepared as previously described, and subjected to western blotting.
Phenotypic and Histological Characterization
Blood glucose and ketone levels were measured using a glucometer and ketone meter. Plasma insulin (ALPCO) was measured using a commercial kit. Plasma measurements of non-esterified fatty acids (Wako Chemicals) and total triglycerides (ThermoScientific) were made using colorimetric assays. Hepatic triglycerides were measured as previously described (Biddinger et al., 2008
). Hematoxylin and eosin staining of the liver was performed by the Dana-Farber/Harvard Cancer Center Rodent Histopathology Core.
De novo Lipogenesis
Mice were fasted for 24 hours, re-fed a high carbohydrate diet for 24 hours, and then sacrificed. One hour prior to re-feeding, mice were injected IP with 24 μl/g body weight of deuterated normal saline, and their drinking water was replaced with 4% D2O. The fraction of newly synthesized palmitate was measured using gas chromatography-electron impact ionization mass spectrometry as previously described (Leavens et al., 2009
Primary hepatocytes were isolated from eight to ten week old female control and LIRKO mice as previously described (Biddinger et al., 2008
). After plating the hepatocytes, they were cultured overnight in DMEM (5mM glucose), without serum or insulin. They were then stimulated for ten minutes with DMEM (5 or 25mM glucose), with or without 10 nM insulin, or 10% fetal bovine serum.
Differences between groups were assessed by Student’s t-test using the Bonferroni correction for multiple testing. Bars and error bars correspond to the mean and SEM, respectively.