Genes and networks expressed in perioperative omental adipose tissue are correlated with weight loss from Roux-en-Y gastric bypass.

CONTEXT: Gastric bypass surgery is the most commonly performed bariatric surgical procedure in the United States. Variable weight loss following this relatively standardized intervention has been reported. To date, a method for reliable profiling of patients who will successfully sustain weight loss for the long term has not been established. In addition, the mechanisms of action in accomplishing major weight loss as well as the explanation for the variable weight loss have not been established. OBJECTIVE: To examine whether gene expression in perioperative omental adipose is associated with gastric bypass-induced weight loss. DESIGN: Cross-sectional study of gene expression in perisurgical omental adipose tissues taken/available at the time of operation and total excess weight loss (EWL). SUBJECTS: Fifteen overweight individuals who underwent Roux-en-Y gastric bypass (RYGB) surgery at the University of California Davis Medical Center (BMI: 40.6-72.8 kg/m(2)). MEASUREMENTS: Body weight before and following weight stabilization 18-42 months after surgery. Perioperative omental adipose RNA isolated from 15 subjects was hybridized to Affymetrix HG-U133A chips for 22,283 transcript expression measurements. RESULTS: Downstream analysis identified a set of genes whose expression was significantly correlated with RYGB-induced weight loss. The significant individual genes include acyl-coenzyme A oxidase 1 (ACOX1), phosphodiesterase 3A cGMP-inhibited (PDE3A) and protein kinase, AMP-activated, beta 1 non-catalytic subunit (PRKAB1). Specifically, ACOX1 plays a role in fatty acid metabolism. PDE3A is involved in purine metabolism and hormone-stimulated lipolysis. PRKAB1 is involved in adipocytokine signaling pathway. Gene network analysis revealed that pathways for glycerolipid metabolism, breast cancer and apoptosis were significantly correlated with long-term weight loss. CONCLUSION: This study demonstrates that RNA expression profiles from perioperative adipose tissue are associated with weight loss outcome following RYGB surgery. Our data suggest that EWL could be predicted from preoperative samples, which would allow for informed decisions about whether or not to proceed to surgery.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice. DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates. RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice. Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks. CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

Transcriptional control of the pref-1 gene in 3T3-L1 adipocyte differentiation. Sequence requirement for differentiation-dependent suppression.

Preadipocyte factor-1 (Pref-1) is a transmembrane epidermal growth factor-like domain-containing protein highly expressed in 3T3-L1 preadipocytes, but is undetectable in mature fat cells; this down-regulation is required for adipocyte differentiation. We show here that pref-1 transcription is markedly suppressed during adipose conversion and results in decreased Pref-1 RNA levels. Using 3T3-L1 cells stably transfected with Pref-1 5'-deletion constructs truncated at -6000, -2100, -1300, -692, -300, -235, -193, -183, -170, -93, and -45 base pairs, we determined that the -183 to -170 region is responsible for the suppression of the pref-1 gene during adipogenesis. This is distinct from the -93 to -45 sequence important for pref-1 promoter activity in preadipocytes. The placement of a 40-base pair -193 to -154 pref-1 sequence containing the putative SAD (suppression in adipocyte differentiation) element upstream of the SV40 promoter decreased promoter activity by 85% upon adipocyte differentiation, compared with 40% observed with the SV40 promoter alone. The SAD element is therefore sufficient for adipocyte differentiation-dependent down-regulation of a heterologous promoter. A DNA-protein complex was observed when the -193 to -174 sequence was used with 3T3-L1 nuclear extracts in gel mobility shift assays. Competition with oligonucleotides harboring base substitution mutations identified a core sequence of -183AAAGA-179 as crucial for DNA-protein complex formation. UV cross-linking predicts that an approximately 63-kDa protein specifically binds the SAD element.

Cloning and developmental regulation of a novel member of the insulin-like gene family in Caenorhabditis elegans.

Aging, metabolism and fat accumulation in Caenorhabditis elegans (C. elegans) are influenced by mutations in DAF-2, a putative insulin-like receptor. Ten putative insulin-like genes have been recently identified from the C. elegans genome database. However, it is unclear if these genes are orthologues of human insulin since they lack the C-peptide dibasic amino acid proteolysis sites. We have identified and measured mRNA expression during development of two novel members of the C. elegans insulin-like gene family. We also report the sequence characterization and gene structure for one of these, the insulin-like protein-1 (ILP1). We focused on ILP1 characterization because it has structural features consistent with its being a candidate insulin ligand for the DAF-2 insulin-like receptor. For example, ILP1 has a putative C-peptide flanked by dibasic amino acids, exhibits conserved cysteine residues that could provide disulfide bonds between the A and B chains, and has two introns. Northern blot analysis revealed that ILP1 mRNA is expressed at very high levels in embryos and is downregulated very early during postnatal development, suggesting that it may influence embryonic development, but not Dauer formation. We also identified a novel insulin-like growth factor-1-like protein (T28B8/IGF-I) that exhibits a very different developmental expression profile than ILP1. Our results are consistent with the hypothesis that members of the unusually large and complex C. elegans insulin-like protein family exhibit complex and perhaps redundant roles.

Arsenate suppression of human keratinocyte programming.

The human keratinocyte line SCC-9 has been used as a model for arsenate-induced perturbations of differentiation. Growth of these cells in 10 microM arsenate permitted the cultures to reach confluence, but prevented expression of 6 markers of suprabasal differentiation (involucrin, loricrin, filaggrin, spr 1, keratin 1 and keratin 10) as assayed by Northern blotting. By contrast, only slight alterations in mRNA levels were observed for one differentiation marker (keratinocyte transglutaminase) and for keratin 5, keratin 14, AP2 or glyceraldehyde phosphate dehydrogenase. The transition metal oxyanions vanadate and chromate had essentially the same suppressive effect on these markers as arsenate, while chronic treatment with tetradecanoylphorbol acetate was generally less effective in suppressing differentiation. To determine whether the previously observed arsenate-mediated alteration in AP1 and AP2 activities could account for the suppression of involucrin, a promoter analysis was conducted. Putative AP1 and AP2 response elements were identified in regions important for transcriptional activity of the 5'-flanking DNA. Mutations in two AP1 sites and one AP2 site were observed to decrease promoter activity significantly, and in combination, to reduce it to approximately 10% of that conferred by the native sequence. These results lend support to the working hypothesis that arsenate suppresses involucrin expression, and, more generally, keratinocyte programming, by altering the transcription factors AP1 and AP2.

Arsenate perturbation of human keratinocyte differentiation.

Treatment of cultured malignant human keratinocytes with sodium arsenate greatly suppressed expression of involucrin, a specific marker of keratinocyte differentiation. This action was primarily attributable to inhibition of involucrin transcription according to message run-on and stability measurements. Involucrin was suppressed in nontumorigenic keratinocytes as well, although the efficacy of suppression was less dramatic in cells derived from clinically normal epidermis. Several transition metal oxyanions (vanadate, molybdate, and tungstate) also substantially suppressed involucrin expression, but okadaic acid was ineffective. Immunoblotting detected marked increases in tyrosine phosphorylation of several proteins as a consequence of arsenate treatment of the cultures, while mobility shift analysis revealed a dramatic loss of DNA binding by the transcription factor AP2. These findings support a proposed role for altered levels of protein tyrosine phosphorylation in keratinocyte differentiation. They also suggest that arsenate perturbs the differentiation program in target cells by altering this phosphorylation level and transcription factor activity.