Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.

Unconventional Secretion of Adipocyte Fatty Acid Binding Protein 4 Is Mediated By Autophagic Proteins in a Sirtuin-1-Dependent Manner.

Fatty acid binding protein 4 (FABP4) is a leaderless lipid carrier protein primarily expressed by adipocytes and macrophages that not only functions intracellularly but is also secreted. The secretion is mediated via unconventional mechanism(s), and in a variety of species, metabolic dysfunction is correlated with elevated circulating FABP4 levels. In diabetic animals, neutralizing antibodies targeting serum FABP4 increase insulin sensitivity and attenuate hepatic glucose output, suggesting the functional importance of circulating FABP4. Using animal and cell-based models, we show that FABP4 is secreted from white, but not brown, adipose tissue in response to lipolytic stimulation in a sirtuin-1 (SIRT1)-dependent manner via a mechanism that requires some, but not all, autophagic components. Silencing of early autophagic genes such as Ulk1/2, Fip200, or Beclin-1 or chemical inhibition of ULK1/2 or VPS34 attenuated secretion, while Atg5 knockdown potentiated FABP4 release. Genetic knockout of Sirt1 diminished secretion, and serum FABP4 levels were undetectable in Sirt1 knockout mice. In addition, blocking SIRT1 by EX527 attenuated secretion while activating SIRT1 by resveratrol-potentiated secretion. These studies suggest that FABP4 secretion from adipocytes is regulated by SIRT1 and requires early autophagic components.

Serum FABP4 concentrations decrease after Roux-en-Y gastric bypass but not after intensive medical management.

BACKGROUND: Serum concentrations of fatty acid binding protein 4, an adipose tissue fatty acid chaperone, have been correlated with insulin resistance and cardiovascular risk factors. The objective of this study were to assess relationships among Roux-en-Y gastric bypass, intensive lifestyle modification and medical management protocol, fatty acid binding protein 4, and metabolic parameters in obese patients with severe type 2 diabetes mellitus; and to evaluate the relative contribution of abdominal subcutaneous adipose and visceral adipose to the secretion of fatty acid binding protein 4. METHODS: Participants were randomly assigned to intensive lifestyle modification and medical management protocol (n = 29) or to intensive lifestyle modification and medical management protocol augmented with Roux-en-Y gastric bypass (n = 34). Relationships among fatty acid binding protein 4 and demographic characteristics, metabolic parameters, and 12-month changes in these values were examined. Visceral and subcutaneous adipose tissue explants from obese nondiabetic patients (n = 5) were obtained and treated with forskolin to evaluate relative secretion of fatty acid binding protein 4 in the different adipose tissue depots. RESULTS: The intensive lifestyle modification and medical management protocol and Roux-en-Y gastric bypass cohorts had similar fasting serum fatty acid binding protein 4 concentrations at baseline. At 1 year, mean serum fatty acid binding protein 4 decreased by 42% in Roux-en-Y gastric bypass participants (P = .002) but did not change significantly in the intensive lifestyle modification and medical management protocol cohort. Percentage of weight change was not a significant predictor of 12-month fatty acid binding protein 4 within treatment arm or in multivariate models adjusted for treatment arm. In adipose tissue explants, fatty acid binding protein 4 was secreted similarly between visceral and subcutaneous adipose tissue. CONCLUSION: After Roux-en-Y gastric bypass, fatty acid binding protein 4 is reduced 12 months after surgery but not after intensive lifestyle modification and medical management protocol in patients with type 2 diabetes mellitus. Fatty acid binding protein 4 was secreted similarly between subcutaneous and visceral adipose tissue explants.

The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage.

Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.

Mcm10 self-association is mediated by an N-terminal coiled-coil domain.

Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.