Adipocyte arrestin domain-containing 3 protein (Arrdc3) regulates uncoupling protein 1 (Ucp1) expression in white adipose independently of canonical changes in β-adrenergic receptor signaling

Adaptive thermogenesis and cold-induced activation of uncoupling protein 1 (Ucp1) in brown adipose tissue in rodents is well-described and attributed to sympathetic activation of β-adrenergic signaling. The arrestin domain containing protein Arrdc3 is a regulator of obesity in mice and also appears linked to obesity in humans. We generated a mouse with conditional deletion of Arrdc3, and here we present evidence that genetic ablation of Arrdc3 specifically in adipocytes results in increased Ucp1 expression in subcutaneous and parametrial adipose tissue. Although this increase in expression did not correspond with significant changes in body weight or energy expenditure, adipocyte-specific Arrdc3-null mice had improved glucose tolerance. It was previously hypothesized that Arrdc3 ablation leads to increased β-adrenergic receptor sensitivity; however, in vitro experiments show that Arrdc3-null adipocytes responded to β-adrenergic receptor agonist with decreased Ucp1 levels. Additionally, canonical β-adrenergic receptor signaling was not different in Arrdc3-null adipocytes. These data reveal a role for Arrdc3 in the regulation of Ucp1 expression in adipocytes. However, this adipocyte effect is insufficient to generate the obesity-resistant phenotype of mice with ubiquitous deletion of Arrdc3, indicating a likely role for Arrdc3 in cells other than adipocytes

Correction: Adipocyte arrestin domain-containing 3 protein (Arrdc3) regulates uncoupling protein 1 (Ucp1) expression in white adipose independently of canonical changes in β-adrenergic receptor signaling.

[This corrects the article DOI: 10.1371/journal.pone.0173823.]

Thioesterase superfamily member 1 suppresses cold thermogenesis by limiting the oxidation of lipid droplet-derived fatty acids in brown adipose tissue

Objective: Non-shivering thermogenesis in brown adipose tissue (BAT) plays a central role in energy homeostasis. Thioesterase superfamily member 1 (Them1), a BAT-enriched long chain fatty acyl-CoA thioesterase, is upregulated by cold and downregulated by warm ambient temperatures. Them1−/− mice exhibit increased energy expenditure and resistance to diet-induced obesity and diabetes, but the mechanistic contribution of Them1 to the regulation of cold thermogenesis remains unknown. Methods: Them1−/− and Them1+/+ mice were subjected to continuous metabolic monitoring to quantify the effects of ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C) on energy expenditure, core body temperature, physical activity and food intake. The effects of Them1 expression on O2 consumption rates, thermogenic gene expression and lipolytic protein activation were determined ex vivo in BAT and in primary brown adipocytes. Results: Them1 suppressed thermogenesis in mice even in the setting of ongoing cold exposure. Without affecting thermogenic gene transcription, Them1 reduced O2 consumption rates in both isolated BAT and primary brown adipocytes. This was attributable to decreased mitochondrial oxidation of endogenous but not exogenous fatty acids. Conclusions: These results show that Them1 may act as a break on uncontrolled heat production and limit the extent of energy expenditure. Pharmacologic inhibition of Them1 could provide a targeted strategy for the management of metabolic disorders via activation of brown fat

Thioesterase superfamily member 1 suppresses cold thermogenesis by limiting the oxidation of lipid droplet-derived fatty acids in brown adipose tissue

Objective: Non-shivering thermogenesis in brown adipose tissue (BAT) plays a central role in energy homeostasis. Thioesterase superfamily member 1 (Them1), a BAT-enriched long chain fatty acyl-CoA thioesterase, is upregulated by cold and downregulated by warm ambient temperatures. Them1−/− mice exhibit increased energy expenditure and resistance to diet-induced obesity and diabetes, but the mechanistic contribution of Them1 to the regulation of cold thermogenesis remains unknown. Methods: Them1−/− and Them1+/+ mice were subjected to continuous metabolic monitoring to quantify the effects of ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C) on energy expenditure, core body temperature, physical activity and food intake. The effects of Them1 expression on O2 consumption rates, thermogenic gene expression and lipolytic protein activation were determined ex vivo in BAT and in primary brown adipocytes. Results: Them1 suppressed thermogenesis in mice even in the setting of ongoing cold exposure. Without affecting thermogenic gene transcription, Them1 reduced O2 consumption rates in both isolated BAT and primary brown adipocytes. This was attributable to decreased mitochondrial oxidation of endogenous but not exogenous fatty acids. Conclusions: These results show that Them1 may act as a break on uncontrolled heat production and limit the extent of energy expenditure. Pharmacologic inhibition of Them1 could provide a targeted strategy for the management of metabolic disorders via activation of brown fat. Keywords: Energy expenditure, Fatty acyl-CoA, Acyl-CoA thioesterase, Mitochondria, Obesit

Characterization of adipocyte-specific <i>Arrdc3</i>-null mice.

<p>(A) To confirm adipocyte-specific deletion, Arrdc3 expression was measured in various tissues of Cre–(control) and Cre+ (<i>Arrdc3</i>-null) mice by quantitative PCR. Brown (BAT), parametrial (VAT) and subcutaneous adipose tissue (SAT) had significantly decreased <i>Arrdc3</i> expression while there was no significant difference in liver or kidney (n = 3–4). (B) Adipocyte-specific <i>Arrdc3</i>-null mice and littermate controls were weighed for 16 weeks and no differences in body weight were found (n = 4–10). (C) Specific adipose depots of female mice were weighed and normalized to total body weight. Subcutaneous (SAT) and parametrial (VAT) adipose tissue from adipocyte-specific <i>Arrdc3</i>-null mice weighed significantly less than controls (n = 5). (D) Representative macroscopic (formaldehyde fixed tissue) and microscopic appearance of subcutaneous (SAT), parametrial (VAT) and brown (BAT) adipose tissue from adipocyte-specific <i>Arrdc3</i>-null and control mice. Paraffin tissue sections were stained with hematoxylin and eosin and images were taken at 40x.</p

<i>Arrdc3</i> deletion increases PPAR target gene expression in adipocytes.

<p>Quantitative PCR array analysis of gene expression of PPAR target genes, PPAR cofactors and PPARs and associated transcription factors in control versus <i>Arrdc3</i> SVF-derived adipocytes <i>in vitro</i> (n = 3 mice per group). Only significantly different genes are displayed. *p≤ 0.05.</p

β-adrenergic signaling in <i>Arrdc3</i>-null adipocytes <i>in vitro</i>.

<p>(A) Quantification of total Oil Red O staining of control and adipocyte-specific <i>Arrdc3</i>-null stromal vascular fractions after adipogenic treatment (n = 4 mice/group). Representative Oil Red O staining of stromal vascular fraction-derived adipocytes from control and adipocyte-specific <i>Arrdc3</i>-null mice. (B) Quantitative PCR analysis of Ucp1 upregulation upon 4 hours of isoproterenol treatment of control and <i>Arrdc3</i>-null cells (n = 4 mice/group). (C) Western analysis of Ucp1 protein expression upon 24 hours of isoproterenol treatment in control and <i>Arrdc3</i>-null cells. (D) cAMP concentration in control and <i>Arrdc3</i>-null cells upon 5 minutes of isoproterenol treatment (n = 3 mice/group). (E) Glycerol concentrations of control and <i>Arrdc3</i>-null cell media after 3 hours of isoproterenol treatment (n = 4 mice/group). (F) Western analysis of CREB and HSL phosphorylation of control and <i>Arrdc3</i>-null cells upon 5 minutes of isoproterenol treatment (n = 3 mice/group).</p

Cdkal1, a type 2 diabetes susceptibility gene, regulates mitochondrial function in adipose tissue

Objectives: Understanding how loci identified by genome wide association studies (GWAS) contribute to pathogenesis requires new mechanistic insights. Variants within CDKAL1 are strongly linked to an increased risk of developing type 2 diabetes and obesity. Investigations in mouse models have focused on the function of Cdkal1 as a tRNALys modifier and downstream effects of Cdkal1 loss on pro-insulin translational fidelity in pancreatic β−cells. However, Cdkal1 is broadly expressed in other metabolically relevant tissues, including adipose tissue. In addition, the Cdkal1 homolog Cdk5rap1 regulates mitochondrial protein translation and mitochondrial function in skeletal muscle. We tested whether adipocyte-specific Cdkal1 deletion alters systemic glucose homeostasis or adipose mitochondrial function independently of its effects on pro-insulin translation and insulin secretion. Methods: We measured mRNA levels of type 2 diabetes GWAS genes, including Cdkal1, in adipose tissue from lean and obese mice. We then established a mouse model with adipocyte-specific Cdkal1 deletion. We examined the effects of adipose Cdkal1 deletion using indirect calorimetry on mice during a cold temperature challenge, as well as by measuring cellular and mitochondrial respiration in vitro. We also examined brown adipose tissue (BAT) mitochondrial morphology by electron microscopy. Utilizing co-immunoprecipitation followed by mass spectrometry, we performed interaction mapping to identify new CDKAL1 binding partners. Furthermore, we tested whether Cdkal1 loss in adipose tissue affects total protein levels or accurate Lys incorporation by tRNALys using quantitative mass spectrometry. Results: We found that Cdkal1 mRNA levels are reduced in adipose tissue of obese mice. Using adipose-specific Cdkal1 KO mice (A-KO), we demonstrated that mitochondrial function is impaired in primary differentiated brown adipocytes and in isolated mitochondria from A-KO brown adipose tissue. A-KO mice displayed decreased energy expenditure during 4 °C cold challenge. Furthermore, mitochondrial morphology was highly abnormal in A-KO BAT. Surprisingly, we found that lysine codon representation was unchanged in Cdkal1 A-KO adipose tissue. We identified novel protein interactors of CDKAL1, including SLC25A4/ANT1, an inner mitochondrial membrane ADP/ATP translocator. ANT proteins can account for the UCP1-independent basal proton leak in BAT mitochondria. Cdkal1 A-KO mice had increased ANT1 protein levels in their white adipose tissue. Conclusions: Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity