One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

A qPCR technology for direct quantification of methylation in untreated DNA.

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants.

Reverse transcription quantitative PCR (RT-qPCR) assays are gold standard in diagnosing SARS-CoV-2 infection and play a major role in viral subtyping for rapid detection and monitoring of important mutations, containing the spread of new virus variants. We wanted to compare RT-qPCR melting curve analysis assays to Sanger Sequencing for detection of variants within the SARS-CoV-2 spike glycoprotein and examined their sensitivity and specificity. Samples positive for SARS-CoV-2 (n = 663 + 82) were subtyped using both Sanger sequencing and five RT-qPCR melting curve analysis assays specific for the mutations N501Y, P681H, E484K, K417N/T, and N439K. The results of the two methods were compared. The training cohort and the clinical validation cohort showed equally, or significantly better sensitivity of the assays compared to the Sanger sequencing. The agreement of the Sanger sequencing and the assays ranged from 92.6 to 100% for the training cohort and 99.4-100% for the clinical validation. The sensitivity, specificity, and turn-around time of the RT-qPCR melting curve analysis assays are well-suited for clinical monitoring of VOCs, making the assays an important tool in contact tracing and risk stratification. Furthermore, the assays were able to indicate the presence of new mutations in the complementary sequence to the mutation-specific probes.

Adipose MDM2 regulates systemic insulin sensitivity.

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies.

BACKGROUND: A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. RESULTS: Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven-log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. CONCLUSION: For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

Effect of a long-term high-protein diet on survival, obesity development, and gut microbiota in mice.

Female C57BL/6J mice were fed a regular low-fat diet or high-fat diets combined with either high or low protein-to-sucrose ratios during their entire lifespan to examine the long-term effects on obesity development, gut microbiota, and survival. Intake of a high-fat diet with a low protein/sucrose ratio precipitated obesity and reduced survival relative to mice fed a low-fat diet. By contrast, intake of a high-fat diet with a high protein/sucrose ratio attenuated lifelong weight gain and adipose tissue expansion, and survival was not significantly altered relative to low-fat-fed mice. Our findings support the notion that reduced survival in response to high-fat/high-sucrose feeding is linked to obesity development. Digital gene expression analyses, further validated by qPCR, demonstrated that the protein/sucrose ratio modulated global gene expression over time in liver and adipose tissue, affecting pathways related to metabolism and inflammation. Analysis of fecal bacterial DNA using the Mouse Intestinal Tract Chip revealed significant changes in the composition of the gut microbiota in relation to host age and dietary fat content, but not the protein/sucrose ratio. Accordingly, dietary fat rather than the protein/sucrose ratio or adiposity is a major driver shaping the gut microbiota, whereas the effect of a high-fat diet on survival is dependent on the protein/sucrose ratio.

p53 regulates expression of uncoupling protein 1 through binding and repression of PPARγ coactivator-1α.

The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP1-dependent uncoupled respiration and increased oxidation of glucose and fatty acids in brown or brown-like adipocytes, termed brite or beige, in relation to energy balance and homeostasis has been highlighted recently. UCP1-dependent uncoupled respiration in classic interscapular brown adipose tissue is central to cold-induced thermogenesis, whereas brite/beige adipocytes are of special importance in relation to diet-induced thermogenesis, where the importance of UCP1 is only clearly manifested in mice kept at thermoneutrality. We challenged wild-type and TRP53-deficient mice by high-fat feeding under thermoneutral conditions. Interestingly, mice lacking TRP53 gained less weight compared with their wild-type counterparts. This was related to an increased expression of Ucp1 and other PPARGC1a and PPARGC1b target genes but not Ppargc1a or Ppargc1b in inguinal white adipose tissue of mice lacking TRP53. We show that TRP53, independently of its ability to bind DNA, inhibits the activity of PPARGC1a and PPARGC1b. Collectively, our data show that TRP53 has the ability to regulate the thermogenic capacity of adipocytes through modulation of PPARGC1 activity.

Macronutrient composition determines accumulation of persistent organic pollutants from dietary exposure in adipose tissue of mice.

Accumulation of persistent organic pollutants (POPs) has been linked to adipose tissue expansion. As different nutrients modulate adipose tissue development, we investigated the influence of dietary composition on POP accumulation, obesity development and related disorders. Lifespan was determined in mice fed fish-oil-based high fat diets during a long-term feeding trial and accumulation of POPs was measured after 3, 6 and 18months of feeding. Further, we performed dose-response experiments using four abundant POPs found in marine sources, PCB-153, PCB-138, PCB-118 and pp'-DDE as single congeners or as mixtures in combination with different diets: one low fat diet and two high fat diets with different protein:sucrose ratios. We measured accumulation of POPs in adipose tissue and liver and determined obesity development, glucose tolerance, insulin sensitivity and hepatic expression of genes involved in metabolism of xenobiotics. Compared with mice fed diets with a low protein:sucrose ratio, mice fed diets with a high protein:sucrose ratio had significantly lower total burden of POPs in adipose tissue, were protected from obesity development and exhibited enhanced hepatic expression of genes involved in metabolism and elimination of xenobiotics. Exposure to POPs, either as single compounds or mixtures, had no effect on obesity development, glucose tolerance or insulin sensitivity. In conclusion, this study demonstrates that the dietary composition of macronutrients profoundly modulates POP accumulation in adipose tissues adding an additional parameter to be included in future studies. Our results indicate that alterations in macronutrient composition might be an additional route for reducing total body burden of POPs.

Tissue Inhibitor Of Matrix Metalloproteinase-1 Is Required for High-Fat Diet-Induced Glucose Intolerance and Hepatic Steatosis in Mice.

BACKGROUND: Plasma levels of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are elevated in obesity and obesity-related disorders, such as steatosis, but the metabolic role of TIMP-1 is unclear. Here we investigated how the presence or absence of TIMP-1 affected the development of diet-induced glucose intolerance and hepatic steatosis using the Timp1 null mice. METHODS: Timp1 knockout (TKO) and wild type (TWT) mice were fed chow, high-fat diet (HFD) or intermediate fat and sucrose diet (IFSD). We determined body weight, body composition, lipid content of the liver, energy intake, energy expenditure, oral glucose tolerance, as well as insulin tolerance. In addition, the histology of liver and adipose tissues was examined and expression of selected genes involved in lipid metabolism and inflammation in liver and adipose tissues was determined by RT-qPCR. RESULTS: TKO mice gained less weight and had lower energy efficiency than TWT mice when fed HFD, but not when fed chow or IFSD. Importantly, TKO mice were protected from development of HFD- as well as IFSD-induced glucose intolerance, hepatic steatosis, and altered expression of genes involved in hepatic lipid metabolism and inflammation. CONCLUSION: Collectively, our results indicate that TIMP-1 contributes to the development of diet-induced hepatic steatosis and glucose intolerance and may be a potential therapeutic target.

PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation.

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation.

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes.

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0-8 or day 8-10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0-8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.

Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes.

Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.

Identification of odorant-receptor interactions by global mapping of the human odorome.

The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors (hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1 and OR5P3. As it remains largely unknown how human perception of odorants influence or prevent diseases, we also developed an odorant-protein matrix to explore global relationships between chemicals, biological targets and disease susceptibilities. We successfully experimentally demonstrated interactions between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma (PPARγ). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.

Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

PPARgamma-PGC-1alpha activity is determinant of alcohol related breast cancer.

Alcohol is a risk factor for postmenopausal breast cancer. One of several proposed mechanisms is that alcohol-related breast cancer is caused by increased sex hormone levels. PPARγ inhibits aromatase transcription in breast adipocytes. We reproduced previously found allele-specific effects of the wildtype Pro-allele of PPARG Pro(12)Ala in alcohol related breast cancer. In transiently transfected cells, transcriptional activation by PPARγ and the PPARγ-PGC-1α complex was inhibited by ethanol. PPARγ 12Ala-mediated transcription activation was not enhanced by PGC-1α, resulting in allele-specific transcription activation by the PPARγ 12Pro-PGC-1α complex. Our results suggest that PPARγ and PGC-1α activity is an important determinant of alcohol related breast cancer.

Sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.

BACKGROUND: Polyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced obesity and inflammation in adipose tissue. Here we aimed to investigate if the amount of sucrose in the background diet influences the ability of n-3 PUFAs to protect against diet-induced obesity, adipose tissue inflammation and glucose intolerance. METHODOLOGY/PRINCIPAL FINDINGS: We fed C57BL/6J mice a protein- (casein) or sucrose-based high fat diet supplemented with fish oil or corn oil for 9 weeks. Irrespective of the fatty acid source, mice fed diets rich in sucrose became obese whereas mice fed high protein diets remained lean. Inclusion of sucrose in the diet also counteracted the well-known anti-inflammatory effect of fish oil in adipose tissue, but did not impair the ability of fish oil to prevent accumulation of fat in the liver. Calculation of HOMA-IR indicated that mice fed high levels of proteins remained insulin sensitive, whereas insulin sensitivity was reduced in the obese mice fed sucrose irrespectively of the fat source. We show that a high fat diet decreased glucose tolerance in the mice independently of both obesity and dietary levels of n-3 PUFAs and sucrose. Of note, increasing the protein∶sucrose ratio in high fat diets decreased energy efficiency irrespective of fat source. This was accompanied by increased expression of Ppargc1a (peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha) and increased gluconeogenesis in the fed state. CONCLUSIONS/SIGNIFICANCE: The background diet influence the ability of n-3 PUFAs to protect against development of obesity, glucose intolerance and adipose tissue inflammation. High levels of dietary sucrose counteract the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.

Structure-Activity Study of Dihydrocinnamic Acids and Discovery of the Potent FFA1 (GPR40) Agonist TUG-469.

The free fatty acid 1 receptor (FFA1 or GPR40), which is highly expressed on pancreatic ß-cells and amplifies glucose-stimulated insulin secretion, has emerged as an attractive target for the treatment of type 2 diabetes. Several FFA1 agonists containing the para-substituted dihydrocinnamic acid moiety are known. We here present a structure-activity relationship study of this compound family suggesting that the central methyleneoxy linker is preferable for the smaller compounds, whereas the central methyleneamine linker gives higher potency to the larger compounds. The study resulted in the discovery of the potent and selective full FFA1 agonist TUG-469 (29).

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes.

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.

cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids.

The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-adipogenic action of n-6 PUFAs was dependent on a cAMP-dependent protein kinase-mediated induction of cyclooxygenase expression and activity. We show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice. The high protein diet increased the glucagon/insulin ratio, leading to elevated cAMP-dependent signaling and induction of cyclooxygenase-mediated prostaglandin synthesis. Mice fed the high protein diet had a markedly lower feed efficiency than mice fed the high carbohydrate diet. Yet, oxygen consumption and apparent heat production were similar. Mice on a high protein diet had increased hepatic expression of PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha) and genes involved in energy-demanding processes like urea synthesis and gluconeogenesis. We conclude that cAMP signaling is pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity.