A. Biochemistry Molecular Biology [A. Biochemistry/Molecular Biology] A-1 Ablation of Copper transporter 1 in adipose tissue impairs adaptive thermogenesis Young-Seung Lee¹, Thuy Nguyen Thi Phuong¹, Tamara Korolnek², Byung-Eun Kim², Tae-Il Jeon¹* ¹Department of Animal Science and Bioindustry, Chonnam National University, Gwangju 61186, Korea, ²Department of Animal and Avian science, University of Maryland, College Park MD 20742, USA Adipose tissues dissipate an energy as heat through uncoupling protein-1 (UCP1), a BAT-specific protein. In response to cold stress, adaptive thermogenesis is
induced through activation of the electron transport chain (ETC) including cytochrome c oxidase (CcO), a copper-dependent enzyme. Recent studies have reported copper is essential for maintaining cellular metabolism. However, the exact role of copper in adaptive thermogenesis of adipose tissue is still unclear. Here, we show that copper transporter 1 (Ctr1), which transports Cu across the plasma membrane with high affinity, is upregulated in BAT and iWAT by cold exposure with an increase in UCP1. Furthermore, Ctr1 adipose tissue specific knockout (Ctr1adq/adq) mice exhibited that decreased
adaptive thermogenesis and it related with impaired lipolysis. However, the Cu delivery compound elesclomol (ES) rescues these events in Ctr1adq/adq. Taken together, these findings indicate that Ctr1 plays an essential role in thermogenic adipocytes via Cu-dependent mechanisms. Ablation of Copper transporter 1 in adipose tissue impairs adaptive thermogenesis Young-seung Lee , Thuy Nguyen Thi Phuong , Tamara korolnek , Byung-Eun Kim Tae-Il Jeon , 1 1* 1 2 2, 1 Department of Animal Science and Bioindustry, Chonnam National University, Gwangju 61186, Korea 2 Department of Animal and Avian
Science, University of Maryland, College Park, MD 20742, USA Abstract Results Adipose tissues dissipate an energy as heat through uncoupling protein-1 (UCP1), a BAT-specific protein. In response to cold stress, adaptive thermogenesis is induced Ctr1 is upregulated in BAT and iWAT by CTR1 ablation in BAT impairs the through activation of the electron transport chain (ETC) acute cold exposure and adrenergic lipolysis during cold-induced including cytochrome c oxidase (CcO), a copper-dependent enzyme. Recent studies have reported copper is essential stimulation thermogenesis for maintaining
cellular metabolism. However, the exact role of copper in adaptive thermogenesis of adipose tissue is still unclear. Here, we show that copper transporter 1 (Ctr1), which transports Cu across the plasma membrane with high affinity, is upregulated in BAT and iWAT by cold exposure with an increase in UCP1. Furthermore, Ctr1 adipose tissue specific knockout (Ctr1 adq/adq ) mice exhibited that decreased adaptive thermogenesis and it related with impaired lipolysis. However, the Cu delivery compound elesclomol (ES) rescues these events in Ctr1 adq/adq . Taken together, these findings indicate that
Ctr1 plays an essential role in thermogenic adipocytes via Cu-dependent mechanisms. Introduction Adipose tissues, especially brown and beige adipose tissues, dissipate chemical energy as heat through thermogenesis. Non-shivering thermogenesis is induced by cold stress in BAT and WAT, which be converted to BAT- Figure 1. Mice were exposed to room temperature (RT) or like adipocyte via beigeing in response to cold or β3- 4℃ cold exposure for 12 hours. BAT and iWAT were adrenergic signaling. β3-adrenergic signaling activates adenylyl cyclase, which catalyzes the conversion of ATP to collected.
Arrowheads indicate the glycosylated full-length cyclic AMP (cAMP). cAMP activates protein kinase A (PKA) (g) and truncated (t) forms of Ctr1. Ctr1 expression in BAT and active PKA phosphorylates its target proteins such as was increased by cold exposure along with increased in the hormone-sensitive lipase (HSL). p-HSL converts thermogenic markers UCP1 and Pgc-1α (top panel). CL Figure 3. Cold exposure caused lipolysis in Ctr1 flox/flox BAT, triacylglycerols into free fatty acids. Uncoupling protein-1, a induced Ctr1 expression in iWAT (bottom panel). whereas this was prevented in Ctr1 adq/adq
BAT. In addition, master regulator in non-shivering thermogenesis, is CL administration induced pHSL expression in Ctr1 flox/flox , activated by β-oxidation of free fatty acids and produces but it suppressed in Ctr1 adq/adq . heat and four membrane-bound complexes in electron transport chain (ETC) are responsible for β-oxidation. Ctr1 in adipose tissue is essential for cold Copper is the enzymatic cofactor required for various adaption in mice biochemical processes. For example, copper-dependent enzyme cytochrome c oxidase plays an essential role in The Cu delivery compound elesclomol ETC to
generate ATP or heat. Recent data have shown that (ES) rescues cold-induced hypothermia copper modulates cAMP-dependent-lipolysis by altering the in Ctr1 adq/adq mice activity of cAMP degrading phosphodiesterase PDE3B. However, the exact role of copper in adaptive thermogenesis of adipose tissue is not fully understood. We hypothesized that genetic copper transporter 1 ablation in adipose tissue impairs adaptive thermogenesis during cold exposure. Our finding indicate that adipose tissue ctr1 deletion impairs cold-induced thermogenesis. Conclusions Ctr1 is the major copper influx transporter
to mediate the cellular copper level. Ctr1 is induced in BAT and iWAT by cold stress and β3-adrenergic signaling. Furthermore, ctr1 deficiency on adipose tissue inhibits cold-induced thermogenesis by impairing lipolysis. In Figure 2. The expression of Ctr1 was significantly decreased by Ctr1 adipose-specific knockout. Ctr1 adq/adq contrast, the copper delivery compound elesclomol mice exhibited that decreased rectal body temperature 4 restores cold-induced hypothermia in ctr1 adipose hours post cold exposure. Furthermore, core body tissue knockout mice. Taken together, these findings
temperature was statically decreased in Ctr1 adq/adq mice Figure 4. Mice were exposed to 4℃ for the indicated time suggest that ctr1 plays an essential role in controlling after cold exposure or CL administration at the indicate after vehicle (Veh) or ES injections. ES restored non- non-shivering thermogenesis in adipose tissues. time. shivering thermogenesis in Ctr1 adq/adq mice. [A. Biochemistry/Molecular Biology] A-2 Acyl chain length of sphingolipid regulates cytokine secretion via endoplasmic reticulum stress Su-Jung Kim¹, Min hee Kim¹, Hee-Yeon Kim², Hee-Soo Yoon², Woo-Jae Park¹*,
Joo-Won Park²* ¹Department of biochemistry, College of Medicine, Gachon University, Incheon 21999 , Korea, ²Department of biochemistry, College of Medicine, Ewha Womans University, Seoul 07084, Korea Distinct acyl chain length of ceramide is detemined by ceramide synthases (CerS). The current study investigated whether ceramide can have different effects on inflammatory cytokine production and inflammasome formation depending on its acyl chai length LPS treatment increased CerS6, but reduced CerS2 levels with concomitant increase of C16-ceramide. While CerS6 overexpression augmented
LPS-induced signaling cascades and inflammasome formation, CerS2 overexpression inhibited it. Since ER stress pathways are known to be involved in inflammatory cytokine production and inflammasome complex formation, we then examined phosphorylation of PERK and eIF2A. Phosphorylation of PERK and eIF2A were induced by LPS treatment, which was further intensified by CerS6 overexpression. However, CerS2 overexpression reduced LPS-induced ER stress response. In addition, bortezomib, which increases CerS2 and reduces CerS6 protein levels, diminished LPS-induced inflammatory cytokine production and
inflammasome complex formation. Taken together, long chain ceramides produced by CerS6 and veryl long chain ceramides generated by CerS2 plays an opposite role in LPS-induced inflammation and inflammsome complex formation by modulating ER stress, MAP kinase and NF-kB signalling pathways. Acyl chain length of sphingolipid regulates cytokine secretion via endoplasmic reticulum stress Su-Jung Kim , Min hee Kim , Hee-Yeon Kim , Hee-Soo Yoon , Woo-Jae Park *, and Joo-Won Park * 2 2, 1, 1 1 2 1 Department of Biochemistry, School of Medicine, Gachon University, Incheon 21999, Republic of Korea 2
Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul 07084, Republic of Korea Abstract CerS6 overexpression synergistically increased lipopolysaccharide (LPS)-induced inflammasome Ceramide has been emerged as an important regulator in various cellular processes such as apoptosis, differentiation, and inflammation. In the present study, we investigated formation and inflammatory cytokines release whether ceramide can have different effects on inflammatory cytokine production and inflammasome formation depending on its acyl chain length. LPS treatment increased CerS6,
but reduced CerS2 levels with concomitant increase of C16-ceramide. While CerS6 overexpression augmented LPS-induced signaling cascades and inflammasome formation, CerS2 overexpression inhibited it. Since ER stress pathways are known to be involved in inflammatory cytokine production and inflammasome complex formation, we then examined phosphorylation of PERK and eIF2A. Phosphorylation of PERK and eIF2A were induced by LPS treatment, which was further intensified by CerS6 overexpression. However, CerS2 overexpression reduced LPS-induced ER stress response. In addition, bortezomib, which
increases CerS2 and reduces CerS6 protein levels, diminished LPS-induced inflammatory cytokine production and inflammasome complex formation. Taken together, long chain ceramides produced by CerS6 and veryl long chain ceramides generated by CerS2 plays an opposite role in LPS-induced inflammation and inflammsome complex formation by modulating ER stress, MAP kinase and NF-kB signalling pathways. LPS treatment increased CerS6, but reduced CerS2 levels with concomitant increase of C16-ceramide After treatment with LPS (50 ng/ml) in CerS6 overexpressed RAW264.7 cells, (A) Representative western
blots of NF-κB (p-p65, p-IκB), iNOS, MAP kinase (p-p38, p-JNK, p-ERK), inflammasome (NRLP3, ASC, cleaved caspase-1, IL- 1)(upper panels) and C16-ceramide levels (lower panels) were examined, and (B) Levels of TNF-, IL-1, IL-6 and nitrite were measured in RAW264.7 cell culture medium using ELISA kits and griess reagent (n=3). CerS2 overexpression reduced LPS-induced inflammasome formation and inflammatory cytokines release After treating two concentrations (50 ng/ml, 1 µg/ml) of LPS for 48 hr, TNF- (A), IL-1 (B), IL-6 (C), nitrite (D), ceramide acyl chain length (E), mRNA levels of
ceramide synthases (F), representative western blots of CerS2, CerS6 (G) were examined (n=3). The values are expressed as means ± standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001. Three independent experiments were performed. CerS6 level, but not CerS5 level, affects lipopolysaccharide (LPS)-induced inflammasome formation and inflammatory cytokines release After treatment with LPS (50 ng/ml) in CerS2 overexpressed RAW264.7 cells, (A) Representative western blots of NF-κB (p-p65, p-IκB), iNOS, MAP kinase (p-p38, p- JNK, p-ERK), inflammasome (NRLP3, ASC, cleaved caspase-1,
IL-1)(upper panels) and C16-ceramide levels (lower panels) were examined, and (B) Levels of TNF-, IL- 1, IL-6 and nitrite were measured in RAW264.7 cell culture medium using ELISA kits and griess reagent (n=3). CerS2 and CerS6 oppositely regulate LPS- induced ER stress Representative western blots of the indicated proteins in (A) CerS6 After overexpression with CerS5 or CerS6 plasmids, representative western blots of NF- overexpressed or (B) κB (p-p65, p-IκB), iNOS, MAP kinase (p-p38, p-JNK, p-ERK), inflammasome (NRLP3, CerS2 overexpressed ASC, cleaved caspase-1, IL-1) were examined and
(B) levels of TNF-, IL-1, IL-6 and RAW264.7 cells that nitrite were measure
