Evidence for tuning adipocytes ICER levels for obesity care.

Abnormal adipokine production, along with defective uptake and metabolism of glucose within adipocytes, contributes to insulin resistance and altered glucose homeostasis. Recent research has highlighted one of the mechanisms that accounts for impaired production of adiponectin (ADIPOQ) and adipocyte glucose uptake in obesity. In adipocytes of human obese subjects and mice fed with a high fat diet, the level of the inducible cAMP early repressor (ICER) is diminished. Reduction of ICER elevates the cAMP response element binding protein (CREB) activity, which in turn increases the repressor activating transcription factor 3. In fine, the cascade triggers reduction in the ADIPOQ and GLUT4 levels, which ultimately hampers insulin-mediated glucose uptake. The c-Jun N-terminal kinase (JNK) interacting-protein 1, also called islet brain 1 (IB1), is a target of CREB/ICER that promotes JNK-mediated insulin resistance in adipocytes. A rise in IB1 and c-Jun levels accompanies the drop of ICER in white adipose tissues of obese mice when compared with mice fed with a chow diet. Other than the expression of ADIPOQ and glucose transport, decline in ICER expression might impact insulin signaling. Impairment of ICER is a critical issue that will need major consideration in future therapeutic purposes.

Systemic insulin resistance is one of the hallmarks of obesity that is associated with life-threatening complications., In obesity, enlarged adipocytes have numerous abnormalities that contribute to overall insulin resistance such as defective insulin-mediated glucose uptake. The latter is caused by diminished production and translocation of the glucose transporter GLUT4 and impaired insulin signaling. As a consequence, adipocytes cannot properly store glucose and increase the release of free fatty acids (FFAs) into the blood. Continuous overload of FFAs promotes an impairment in insulin signaling and glucose uptake in muscle cells. Adipocytes produce several adipokines including the insulin sensitizer adipocyte-specific product adiponectin (ADIPOQ). ADIPOQ is thought to decrease circulating FFAs by increasing their oxidation in skeletal muscle and liver. Increased ADIPOQ levels reduce the triglyceride contents, leading thereby to improvement of insulin sensitivity. Numerous studies report reduced adipose production and plasma circulating levels of the ADIPOQ in obese mice and human obese individuals. A mechanism that accounts for this reduction of ADIPOQ and glucose uptake has been shown. Adipocytes from obese subjects have a sustained increase in activity of the transcriptional activators cAMP response element (CRE) binding protein (CREB). The rise in CREB activity elevates the activating transcription factor 3 (ATF3) levels, which in turn represses the expression of ADIPOQ and the glucose transporter GLUT4.

CREB activity is tightly regulated by the level of the inducible cAMP early repressor (ICER), a natural antagonist that contains neither activating nor repressing domains. ICER acts as a passive repressor that competes with CREB for binding to target genes promoters. In the normal situation, ICER activity is transiently induced by the same stimuli that induce CREB, but repression occurs only when ICER reaches certain levels. This interplay between CREB and ICER constitutes part of an adaptive mechanism to answer environmental cues. While CREB rapidly induces the expression of target genes in response to stimuli, the repressor ICER restores their initial expression levels and thereby permits transient induction., Stimuli that evoke CREB and ICER activities include hypoglycemia, fasting, vagal and β-adrenergic response for example, and this typically occurs in response to an elevation of cAMP levels. Thus, as a passive repressor, ICER activity is directly correlated with its abundance., It is therefore predictable that inadequate levels of ICER impact to CREB activity, thereby leading to cells dysfunction and ultimately certain pathologies., For example, upregulation of ICER is thought to contribute to the development of type 2 diabetes (T2D).- The disease manifests when islets β cells from endocrine pancreas fail to release sufficient insulin to compensate for insulin resistance in target tissues. Loss of β-cells function includes a decrease in insulin secretion and amounts, which is caused by micro-environmental diabetogenic factors including chronic excess of FFAs, prolonged hyperglycemia and possibly oxidized LDL.- Persistent induction of ICER couples these factors to β-cell failure.- The prevalence of T2D is increasing dramatically as a result of the obesity epidemic. Identification of FFAs as diabetogenic factors, points to a crosstalk between adipocytes and β cells in the pathogenesis of diabetes. For this reason, in the opposite of β cells, we postulate that the loss of ICER elicits an increase in CREB mediated-adipocytes dysfunction in obesity. Quantification indeed revealed a collapse in the levels of ICER in adipocytes of mice obese fed with a high fat diet (HFD) and human obese individuals. Both constitutive and induced levels of ICER are affected in obesity. The decrease in the production of ICER is found in adipocytes of obese mice that were either underfed or fasting conditions and correlated with an augmentation in CREB activity. In vitro experiments realized in mouse differentiated adipocytes showed that ICER deficiency increases Atf3, which in turn inhibit Adipoq and Glut4 expression and insulin mediated-glucose uptake (Fig. 1).

Figure 1. Schematic representation for the impaired expression of Adipoq and GLUT4 ruled by the unbalance of ICER and CREB in obesity. In normal adipocytes, there is enough production of ICER that permits the formation of homo- (ICER/ICER) and/or heterodimers (ICER/CREB) with CREB. These interactions often occur when ICER reaches a certain level upon stimulation and aim to prevent CREB activity. Thus decreased CREB activity reduces the expression of target genes with a cAMP response element (CRE) such as activating transcription factor 3 (ATF3). In turn ATF3 cannot repress the expression of adiponectin and GLUT4. In obesity, the level of ICER is constitutively reduced, fostering CREB homodimers and heterodimers with other activators. Increased CREB activity promotes the expression of ATF3, and therefore, inhibition of adiponectin and GLUT4.

Insulin signaling fosters glucose entry in adipocytes. In obesity insulin signaling is defective and contributes to impaired glucose metabolism. A mechanism that triggers inhibition of this signaling involves the c-Jun N-terminal kinase (JNK) pathway. JNK achieves upstream inhibition of insulin signaling by phosphorylating the Ser-307 residue of the insulin receptor substrate 1 (IRS-1), thus suppressing signal transduction of insulin., In obesity, JNK activity is increased and its inhibition can prevent obesity-induced insulin resistance. The JNK interacting protein 1, also termed islet brain 1 (IB1) because of its high abundance in pancreatic islets cells and brain, is a scaffold protein that tethers kinases of the JNK pathway. At present the exact mechanism though which IB1 regulates the JNK pathway is not understood. IB1 can either inhibit or activate JNK in response to stimuli and the function depends of the cells type. In adipocytes, IB1 promotes activation of JNK. Homozygous disruption of the gene coding for IB1 in mice prevents activation of JNK in adipose tissue. As the consequence of the inhibition of JNK activity, the knockout mice have smaller adipose tissue, gain less weight and have higher systemic insulin sensitivity under a HFD compared with wild-type mice. Interestingly the promoter of IB1 contains a CRE that is conserved between mammalians and the level of the scaffold protein is regulated by CREB and ICER in β-cells. This observation leads to the hypothesis that the drop of ICER in obesity accounts for JNK activity and reduced insulin sensitivity in adipocytes in a mechanism that involve IB1. Phosphorylation of the transcription factor c-Jun by JNK is accompanied by an elevation of the c-Jun mRNA. The gene coding for c-Jun contains a site that is regulated by activated c-Jun itself in a positive feedback loop. For this reason we quantified the expression of c-Jun by quantitative real-time PCR in parallel to ICER and IB1 to mirror JNK activity in adipose tissues of obese mice fed with HFD. We found that the diminution of ICER correlated with an augmentation of c-Jun mRNA and IB1 protein levels in adipose tissues (Fig. 2A and B), supporting the idea that induction of IB1 might trigger JNK activity and insulin resistance in obesity. Adipose tissue expansion, which could be the consequence of both adipocytes hyperplasia and hypertrophy, accompanies adipocytes dysfunction. Some genes involved in cell proliferation and apoptosis are also targets of CREB/ICER., It is therefore possible that the rise in CREB activity promotes expansion of adipose cell mass in humans even though such role has been ruled out in mice.

Figure 2. Examination of IB1, Icer and c-Jun levels in epididymal white adipose tissues (WAT) of obese mice. (A) Measurement of IB1 protein contents by western blotting experiments. Total proteins were prepared from WAT of C57Bl6-Rj male mice that either fed for 16 weeks with a high fat diet (obese, H) or chow diet (control, C). All procedures on mice were performed according to the Swiss legislation for animal experimentation. Total proteins (50 μg) were loaded into a SDS-polyacrylamide gel as described., The figure shows the results of a representative experiment out of four. The corresponding quantitation is depicted below the blot. The values correspond to the ratio in band intensities of IB1 over β tubulin. Data are the mean ± SEM of four independent experiments. **p < 0.01. (B) Quantification of Icer and c-Jun mRNA levels. Total RNAs were prepared from WAT of obese mice (filled bars) and lean control mice (white bars) and were then subjected to quantitative real-time PCR. The primers sequence for Icer and c-Jun and conditions for PCR are those published elsewhere., The levels of the two mRNAs were normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene (Rplp0) gene and those of the lean mice (control) cells were set to 100%. Data are the mean ± SEM of five independent experiments. **p < 0.01, ***p < 0.001.

Constitutive reduction in the levels of ICER in obesity elicits a sustained rise in CREB activity, which in turn stimulates the expression of genes that contain a CRE. However not only the CREB transcriptional factors but also activators of the CRE modulators (CREM) regulate the expression of these CRE containing genes in response to stimuli., In addition, like CREB, CREM acts by either forming homo- or heterodimers with basic leucine zipper transcription factors and can be antagonized by ICER., Approximately 4,000 genes could be potentially regulated by CREB, CREM and ATF in mammals. In obesity, with the exception of ADIPOQ, expression of many adipokines is overproduced in adipocytes.,, The production of these adipose products could be affected by the reduced abundance of ICER in mechanisms that evolve CREB and CREM activators. If so, restoration in the adipose levels of ICER should be a major consideration for future therapeutic strategies to combat insulin resistance and its metabolic complications such as type 2 diabetes.