Gunnar Mellgren

FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

BACKGROUND Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).

Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats

Conjugation of bile acids (BAs) to the amino acids taurine or glycine increases their solubility and promotes liver BA secretion. Supplementing diets with taurine or glycine modulates BA metabolism and enhances fecal BA excretion in rats. However, it is still unclear whether dietary proteins varying in taurine and glycine contents alter BA metabolism, and thereby modulate the recently discovered systemic effects of BAs. Here we show that rats fed a diet containing saithe fish protein hydrolysate (saithe FPH), rich in taurine and glycine, for 26 days had markedly elevated fasting plasma BA levels relative to rats fed soy protein or casein. Concomitantly, the saithe FPH fed rats had reduced liver lipids and fasting plasma TAG levels. Furthermore, visceral adipose tissue mass was reduced and expression of genes involved in fatty acid oxidation and energy expenditure was induced in perirenal/retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism.

Switch from stress response to homeobox transcription factors in adipose tissue after profound fat loss.

Background In obesity, impaired adipose tissue function may promote secondary disease through ectopic lipid accumulation and excess release of adipokines, resulting in systemic low-grade inflammation, insulin resistance and organ dysfunction. However, several of the genes regulating adipose tissue function in obesity are yet to be identified. Methodology/Principal Findings In order to identify novel candidate genes that may regulate adipose tissue function, we analyzed global gene expression in abdominal subcutaneous adipose tissue before and one year after bariatric surgery (biliopancreatic diversion with duodenal switch, BPD/DS) (n = 16). Adipose tissue from lean healthy individuals was also analyzed (n = 13). Two different microarray platforms (AB 1700 and Illumina) were used to measure the differential gene expression, and the results were further validated by qPCR. Surgery reduced BMI from 53.3 to 33.1 kg/m2. The majority of differentially expressed genes were down-regulated after profound fat loss, including transcription factors involved in stress response, inflammation, and immune cell function (e.g., FOS, JUN, ETS, C/EBPB, C/EBPD). Interestingly, a distinct set of genes was up-regulated after fat loss, including homeobox transcription factors (IRX3, IRX5, HOXA5, HOXA9, HOXB5, HOXC6, EMX2, PRRX1) and extracellular matrix structural proteins (COL1A1, COL1A2, COL3A1, COL5A1, COL6A3). Conclusions/Significance The data demonstrate a marked switch of transcription factors in adipose tissue after profound fat loss, providing new molecular insight into a dichotomy between stress response and metabolically favorable tissue development. Our findings implicate homeobox transcription factors as important regulators of adipose tissue function.

ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes

The cell surface markers ASC-1, PAT2, and P2RX5 can be used to mark and identify brown, beige, and white adipocytes in both rodents and humans. Fat Cells Gain New Identities There’s “good fat” and there’s “bad fat.” Good fat is considered to be brown adipose tissue (BAT), which burns calories. Bad fat can be white adipose tissue (WAT), which stores lipids as energy and, in excess, contributes to obesity. When brown fat cells, or adipocytes, develop within white fat, they are called “beige.” Sorting out these different adipocyte subtypes within the human body has been challenging but will be important in uncovering the underlying mechanisms for obesity and its comorbidities, such as type 2 diabetes. To this end, Ussar and colleagues have now identified three new surface markers of white, beige, and brown fat cells. These markers—ASC-1, PAT2, and P2RX5—were first selected in silico, then confirmed in mouse WAT and BAT, and lastly verified in human adipose tissue biopsies. ASC-1, PAT2, and P2RX5 are located in the plasma membrane of adipocytes, thus making them prime targets for imaging fat locations within the body and for directing therapeutics toward particular fat depots. White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte–specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.

Efficacy of tamoxifen based on cytochrome P450 2D6, CYP2C19 and SULT1A1 genotype in the Italian Tamoxifen Prevention Trial

The role of pharmacogenomics and tamoxifen was investigated by analyzing several polymorphisms of cytochrome P450 and SULT1A1 gene in a nested case control study from the Italian Tamoxifen Prevention Trial. This study included 182 Caucasian subjects, 47 breast cancer (BC) cases and 135 matched controls. We used the AmpliChip CYP450 Test to screen 33 alleles of CYP2D6 and 3 of CYP2C19. One more variant for CYP2C19*17 and two single-nucleotide polymorphisms for the gene SULT1A1 were also performed. By using the AmpliChip CYP450 Test, out of 182 subjects, we identified 8 poor metabolizer (PM), 17 intermediate metabolizer (IM), 151 extensive metabolizer (EM) and 3 ultrarapid metabolizer (UM). PM women allocated to the tamoxifen arm showed a higher risk of developing BC compared to the remaining phenotypes (P=0.035). In an exploratory analysis, among 58 women with a CYP2D6*2A allele, 9 BCs were diagnosed in the placebo arm and only 1 in the tamoxifen arm (P=0.0001). CYP2C19 and SULT1A1 polymorphisms did not show any correlation with tamoxifen efficacy. Tamoxifen showed reduced efficacy in CYP2D6 PMs in the chemoprevention setting. Conversely, the CYP2D6*2A allele may be associated with increased efficacy of tamoxifen. These findings support the relevance of pharmaco-genomics in tailoring tamoxifen treatment.

Associations between tamoxifen, estrogens, and FSH serum levels during steady state tamoxifen treatment of postmenopausal women with breast cancer

BackgroundThe cytochrome P450 (CYP) enzymes 2C19, 2D6, and 3A5 are responsible for converting the selective estrogen receptor modulator (SERM), tamoxifen to its active metabolites 4-hydroxy-tamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen). Inter-individual variations of the activity of these enzymes due to polymorphisms may be predictors of outcome of breast cancer patients during tamoxifen treatment. Since tamoxifen and estrogens are both partly metabolized by these enzymes we hypothesize that a correlation between serum tamoxifen and estrogen levels exists, which in turn may interact with tamoxifen on treatment outcome. Here we examined relationships between the serum levels of tamoxifen, estrogens, follicle-stimulating hormone (FSH), and also determined the genotypes of CYP2C19, 2D6, 3A5, and SULT1A1 in 90 postmenopausal breast cancer patients.MethodsTamoxifen and its metabolites were measured by liquid chromatography-tandem mass spectrometry. Estrogen and FSH levels were determined using a sensitive radio- and chemiluminescent immunoassay, respectively.ResultsWe observed significant correlations between the serum concentrations of tamoxifen, N-dedimethyltamoxifen, and tamoxifen-N-oxide and estrogens (p < 0.05). The genotype predicted CYP2C19 activity influenced the levels of both tamoxifen metabolites and E1.ConclusionsWe have shown an association between tamoxifen and its metabolites and estrogen serum levels. An impact of CYP2C19 predicted activity on tamoxifen, as well as estrogen kinetics may partly explain the observed association between tamoxifen and its metabolites and estrogen serum levels. Since the role of estrogen levels during tamoxifen therapy is still a matter of debate further prospective studies to examine the effect of tamoxifen and estrogen kinetics on treatment outcome are warranted.

Nutritional Regulation of Bile Acid Metabolism Is Associated with Improved Pathological Characteristics of the Metabolic Syndrome

Bile acids (BAs) are powerful regulators of metabolism, and mice treated orally with cholic acid are protected from diet-induced obesity, hepatic lipid accumulation, and increased plasma triacylglycerol (TAG) and glucose levels. Here, we show that plasma BA concentration in rats was elevated by exchanging the dietary protein source from casein to salmon protein hydrolysate (SPH). Importantly, the SPH-treated rats were resistant to diet-induced obesity. SPH-treated rats had reduced fed state plasma glucose and TAG levels and lower TAG in liver. The elevated plasma BA concentration was associated with induction of genes involved in energy metabolism and uncoupling, Dio2, Pgc-1α, and Ucp1, in interscapular brown adipose tissue. Interestingly, the same transcriptional pattern was found in white adipose tissue depots of both abdominal and subcutaneous origin. Accordingly, rats fed SPH-based diet exhibited increased whole body energy expenditure and heat dissipation. In skeletal muscle, expressions of the peroxisome proliferator-activated receptor β/δ target genes (Cpt-1b, Angptl4, Adrp, and Ucp3) were induced. Pharmacological removal of BAs by inclusion of 0.5 weight % cholestyramine to the high fat SPH diet attenuated the reduction in abdominal obesity, the reduction in liver TAG, and the decrease in nonfasted plasma TAG and glucose levels. Induction of Ucp3 gene expression in muscle by SPH treatment was completely abolished by cholestyramine inclusion. Taken together, our data provide evidence that bile acid metabolism can be modulated by diet and that such modulation may prevent/ameliorate the characteristic features of the metabolic syndrome.

Activation of cAMP-Dependent Protein Kinase Increases the Protein Level of Steroidogenic Factor-1

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is an essential regulator of endocrine organogenesis, sexual differentiation, and steroidogenisis. SF-1 is a transcriptional regulator of cAMP responsive genes, but the exact mechanisms by which cAMP-dependent PKA modulates SF-1 dependent transcription leading to increased steroidogenic output have not been determined. In this report the effects of PKA activation on SF-1 in living cells have been examined by the use of full-length SF-1 cDNA fused to the cDNA encoding green fluorescent protein (GFP). The GFP-SF-1 fusion protein localized to the nucleus of both steroidogenic Y1 cells and nonsteroidogenic COS-1 cells, and the functional properties of wild-type SF-1 were conserved. When the catalytic subunit of PKA was coexpressed with GFP-SF-1, we observed that the fluorescence emission was markedly elevated. These findings were confirmed by Western blot analysis, showing that stimulation of PKA increased SF-1 protein levels. The PKA- induced expressi...

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Associations of Plasma Kynurenines With Risk of Acute Myocardial Infarction in Patients With Stable Angina Pectoris

Objective—Enhanced tryptophan degradation, induced by the proinflammatory cytokine interferon-&ggr;, has been related to cardiovascular disease progression and insulin resistance. We assessed downstream tryptophan metabolites of the kynurenine pathway as predictors of acute myocardial infarction in patients with suspected stable angina pectoris. Furthermore, we evaluated potential effect modifications according to diagnoses of pre-diabetes mellitus or diabetes mellitus. Approach and Results—Blood samples were obtained from 4122 patients (median age, 62 years; 72% men) who underwent elective coronary angiography. During median follow-up of 56 months, 8.3% had acute myocardial infarction. Comparing the highest quartile to the lowest, for the total cohort, multivariable adjusted hazard ratios (95% confidence intervals) were 1.68 (1.21–2.34), 1.81 (1.33–2.48), 1.68 (1.21–2.32), and 1.48 (1.10–1.99) for kynurenic acid, hydroxykynurenine, anthranilic acid, and hydroxyanthranilic acid, respectively. The kynurenines correlated with phenotypes of the metabolic syndrome, and risk associations were generally stronger in subgroups classified with pre-diabetes mellitus or diabetes mellitus at inclusion (Pint⩽0.05). Evaluated in the total population, hydroxykynurenine and anthranilic acid provided statistically significant net reclassification improvements (0.21 [0.08–0.35] and 0.21 [0.07–0.35], respectively). Conclusions—In patients with suspected stable angina pectoris, elevated levels of plasma kynurenines predicted increased risk of acute myocardial infarction, and risk estimates were generally stronger in subgroups with evidence of impaired glucose homeostasis. Future studies should aim to clarify roles of the kynurenine pathway in atherosclerosis and glucose metabolism.