UCP1-independent Thermogenesis in White Adipose Tissue of Cold-acclimated Ucp1-/- Mice

Abstract

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 °C, to adapt to long term exposure at 4 °C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1α, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation. Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 °C, to adapt to long term exposure at 4 °C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1α, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation. Specific regulatable thermogenic mechanisms in mammals have two important functions as follows: one is for the control of body temperature, and the other is for the maintenance of body weight. The former can be served through several well defined mechanisms, depending on physiological circumstances, that include shivering, fever, restriction of blood flow to the periphery, and nonshivering thermogenesis in brown adipocytes (1Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4550) Google Scholar, 2Wang Y. Kimura K. Inokuma K. Saito M. Kontani Y. Kobayashi Y. Mori N. Yamashita H. Pfluegers Arch. 2006; 452: 363-369Crossref PubMed Scopus (20) Google Scholar). However, understanding the regulation of body weight by thermogenic mechanisms is problematic. It is probable that in adult humans the primary mechanism for burning off excess calories is through physical activity (3Hill J.O. Wyatt H.R. Reed G.W. Peters J.C. Science. 2003; 299: 853-855Crossref PubMed Scopus (1681) Google Scholar); however, in severely obese individuals where physical activity is not possible finding alternative thermogenic mechanisms that can be activated to burn off excess calories is an important goal. Mice with a targeted inactivation of the Ucp1 gene, which encodes the brown adipocyte-specific mitochondrial uncoupling protein, have phenotypes relating to both body temperature and body weight regulation (4Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1088) Google Scholar, 5Liu X. Rossmeisl M. McClaine J. Riachi M. Harper M.E. Kozak L.P. J. Clin. Investig. 2003; 111: 399-407Crossref PubMed Scopus (236) Google Scholar). Because these mice cannot be protected from acute cold exposure by shivering thermogenesis and require a period of slow adaptation to the cold for survival, they can be to identify thermogenic mechanisms X. Harper Kozak L.P. J. PubMed Scopus Google Scholar, A. Jacobsson A. B. Nedergaard J. J. PubMed Scopus Google Scholar). We have that mice for Ucp1 are to adapt to mice for both Ucp1 and the cannot in the they are with J. Y. Kozak L.P. 2006; PubMed Scopus Google Scholar). The in muscle sarcoendoplasmic expression by a thermogenic mechanism for in the the with mice that muscle the to through sarcoendoplasmic serum sarcoendoplasmic long term cold AMP sarcoendoplasmic serum sarcoendoplasmic long term cold AMP mechanisms, an induction of in muscle at the Nedergaard and A. Jacobsson A. B. Nedergaard J. J. PubMed Scopus Google the induction of associated with muscle as a of shivering in mice with brown a in However, shivering, which is to be an term thermogenic is to to body temperature in Ucp1-/- mice to 4 X. Harper Kozak L.P. J. PubMed Scopus Google in mice deficient for Nature. 1997; 387: PubMed Scopus Google C. K. Kozak L.P. J. Clin. Investig. PubMed Scopus Google H. S. Science. PubMed Scopus Google Scholar). is that shivering is a thermogenic mechanism activated in to body temperature, mechanisms are to enable mice to adapt to the shivering is the we its capacity can be can be by and and other mechanisms, and we the molecular for that Ucp1-/- mice on to to the a thermogenic mechanisms that be in the maintenance of body temperature, we have and molecular phenotypes of UCP1-deficient mice on two and a gradual adaptation to the the physiological the of UCP1-deficient mice to the cold is to capacity to oxygen consumption as indicated by The of the molecular analysis was on in these cannot be associated with shivering The showed an of white fat with the of brown adipocytes but Ucp1 The tissue was by fat increased AMP kinase induced levels of total and phosphorylated a of sarcoendoplasmic reticulum as well as induced levels of other that The that tissue in cold-adapted UCP1-deficient mice the for enhanced and mice on a as (4Enerback S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1088) Google Scholar, X. Harper Kozak L.P. J. PubMed Scopus Google Scholar). and Ucp1-/- mice to type mice from The the two of Ucp1-/- and type mice with and of to °C, one the from to the cold by the temperature 2 4 °C, whereas the other at °C. Because was to a of Ucp1-/- mice at one and also of the that of Ucp1-/- mice to an was a of mice of a was and to of Ucp1-/- The total of mice was cold-adapted and Ucp1-/- mice and cold-adapted and Ucp1+/+ on two of Ucp1-/- and type the Ucp1-/- mice on and with a type to 4 by the temperature 2 was body and of the in physiological by and tissue and at °C. was and at °C. the by the and in with of for the and of of temperature was with a and body was analyzed by was by with to 2 to the in to the oxygen and and in an temperature The to be and a period in activity was by of that in and and as a a to in a and for a the to its body the was for the and DNA was by the and of from by was with The and of by and the the and capacity for was total with and with for of gene was for of other gene the gene expression to the of Specific of and for gene expression analysis are on Western tissue from tissue and skeletal muscle in and for the of phosphorylated in 2 of and in 2 and and K. A. PubMed Scopus Google Scholar). was by the the on and to with an and Specific and the and as to the of oxygen was to the oxygen consumption of inguinal was to the in the was from the for consumption was for The was for and the of the oxygen consumption was the to the DNA and in the from the tissue C. K. PubMed Scopus Google Scholar). oxidation was by of and of inguinal fat with G.W. J. Physiol. 2003; PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). of inguinal fat was and and The tissue was and the was in a with a to for H. Y. J. A. H. 1997; PubMed Scopus Google Scholar). was at for and the fat was was and for of of a inguinal fat with a 2 2 serum with serum and of at °C, of to the the was with of The and in a as M. PubMed Scopus Google Scholar). The was and for by The of the was at °C, and at for and of the was and by was as of and of DNA by a of J. 2003; PubMed Scopus Google Scholar). dissected inguinal and in in and a in a and a of and was at for at 4 °C. The was and was to a of The was at for at 4 °C. The was and a in a and The was at for at 4 °C. The was and was at for at 4 °C. The reticulum a in and and and The was a the of the reticulum was as J. Physiol. 2004; Scopus Google Scholar). sarcoendoplasmic to a and and of the 2 The was in a of an The was to for and was by the of the was was to a of the of of was to the the sarcoendoplasmic reticulum a no on the of of was to for of on reticulum as by J. 2003; PubMed Scopus Google with as the in for to of and of was from and glucose levels by the blood glucose was by sensitive and was with and levels with and in the glycogen was by from the as A. J. J. Clin. Investig. 2003; 111: PubMed Scopus Google Scholar). the are as analysis of with the of between Analysis of with was two and in Ucp1-/- type and mice and Ucp1-/- mice on these to the in mice and at they for acute to the cold adaptation to the cold with that of the type mice, of to the cold an acute and for of mice, they long term cold adaptation by the temperature 2 is no in the of these type to the Ucp1-/- showed in both the acute and long term to to acute cold mice sensitive mice of the cold whereas mice to type mice with the of one to acute cold exposure to the cold by gradual of the temperature 4 and mice to the of the mice to adapt occurred the Ucp1-/- mice, the temperature cold in Ucp1-/- and control mice is on cold in a We have that at and mice an fat levels of oxygen but the Ucp1-/- mice a of fat oxidation X. Rossmeisl M. McClaine J. Riachi M. Harper M.E. Kozak L.P. J. Clin. Investig. 2003; 111: 399-407Crossref PubMed Scopus (236) Google Scholar). We energy and the and mice adaptation to the cold a fat We from the to for these the was in the and was we not to an in a on the analysis of cold adaptation. consumption increased with the and at a in the two the temperature was °C, at which temperature oxygen consumption in the Ucp1-/- mice the temperature 4 °C, oxygen consumption was in Ucp1-/- mice in Ucp1+/+ in the which between and be between the of body fat and at the adaptation a of body weight in which is associated with fat that is with increased energy in Ucp1-/- mice to body temperature in consumption both showed a in a gradual in physical activity was in both the that the of Ucp1-/- mice to the cold upon to oxygen is with the of and on oxygen consumption in mice to in the cold the temperature J. Y. Kozak L.P. 2006; PubMed Scopus Google of energy balance cold adaptation. weight fat and physical activity in Ucp1-/- and Ucp1+/+ mice in a of the gradual from to 4 °C. are as the for mice of We the in cold Ucp1-/- mice on showed to levels of oxygen of oxygen and activity a of robust metabolism and physical activity in the mice in was the the mice 2 the consumption was in the mice and for the energy with of body balance phenotypes of Ucp1-/- mice on and body at consumption the period of of exposure to and change in body weight associated with gradual to 4 °C. of the mice is in the for mice of in and occurred in metabolite levels in serum and cold adaptation of Ucp1+/+ and Ucp1-/- mice from to 4 The between in in the and but in the Ucp1-/- levels in serum with increased levels increased oxidation in both but in the Ucp1-/- in liver glycogen levels in cold-adapted Ucp1-/- mice that glycogen is an important of for thermogenesis and in Ucp1-/- mice in Ucp1+/+ The in serum also a for these in in Ucp1-/- the of in the glycogen of skeletal muscle that muscle metabolism is not a for enhanced metabolism to thermogenesis in of cold on serum and tissue of and type mice and are by analysis of with at glucose glycogen muscle muscle glycogen in a of in have on the robust induction of brown adipocytes in inguinal fat of Ucp1-/- mice X. Rossmeisl M. McClaine J. Riachi M. Harper M.E. Kozak L.P. J. Clin. Investig. 2003; 111: 399-407Crossref PubMed Scopus (236) Google Scholar). The of of the inguinal fat was these adipocytes are of UCP1 of by that However, the in between inguinal fat from type and Ucp1-/- mice to as a of cold be To analyzed and to have the brown was and in a with a oxygen to oxygen consumption with these not show in oxygen consumption with glucose as to a in oxygen consumption in from Ucp1-/- mice to the cold from Ucp1-/- mice at Ucp1+/+ mice showed a in oxygen with an in oxygen consumption that is for oxidation in of inguinal fat in from cold-adapted Ucp1-/- mice with Ucp1-/- mice at type mice analysis of inguinal fat tissue showed enhanced AMP kinase activity as by increased of the in cold-adapted Ucp1-/- mice and that the enhanced oxidation is as a of enhanced and in and mice can adapt to the cold they are J. Y. Kozak L.P. 2006; PubMed Scopus Google Scholar). of brown adipocytes was not in of these however, the induction of levels in muscle in to and a mechanism based upon To the thermogenic mechanisms that be induced in cold-adapted mice, the levels of several associated with energy metabolism in from mice at and to an temperature of 4 The finding for gene expression in skeletal muscle was the of in the muscle and The was in the levels of increased oxidation in Ucp1-/- with Ucp1+/+ and oxidation in mice at 4 at °C. levels also in the muscle of Ucp1-/- mice at 4 expression and mitochondrial DNA in the of Ucp1-/- and Ucp1+/+ mice to with and are at and in gene expression at and 4 in Ucp1+/+ and Ucp1-/- mice, change in gene expression between and Ucp1-/- mice at 4 in a expression and mitochondrial DNA in the of Ucp1-/- and Ucp1+/+ mice to with and are at and in gene expression at and 4 in Ucp1+/+ and Ucp1-/- mice, in gene expression between and Ucp1-/- mice at 4 in a expression and mitochondrial DNA in the inguinal tissue of Ucp1-/- and Ucp1+/+ mice to with and are at and in gene expression at and 4 in Ucp1+/+ and Ucp1-/- mice, in gene expression between and Ucp1-/- mice at 4 in a in gene expression was in the inguinal fat as in The in type 2 is a that white adipocytes to brown adipocytes, and the in gene expression to mitochondrial for mitochondrial glycerol 3-phosphate dehydrogenase, PGC1α, CoxII, and mitochondrial DNA these in type mice cold the of induction was robust in Ucp1-/- The other induction was in the of in Ucp1-/- We have in in skeletal but not in the of analyzed by To in thermogenic mechanisms associated with cycling be in Ucp1-/- mice cold for and and for total and phosphorylated phospholamban in and white and no be in skeletal muscle for of these by with to in of these in fat a expression in brown fat was as in and in the white fat analyzed in the levels of total phospholamban occurred in of cold-adapted Ucp1-/- phosphorylated we a change in the and of the a in the of of the phospholamban and increased of the phospholamban on is of as the of expression of phosphorylated in inguinal fat of cold-adapted Ucp1-/- mice to the in skeletal muscle change be in expression of and in fat as a of of an in by increased levels of be by that the adaptation period was to for in to type and Ucp1-/- mice to 4 and at 4 for term cold adaptation to the of these in skeletal muscle and tissue by to the increased thermogenic in body fat be between Ucp1+/+ and Ucp1-/- mice not to the term cold no in be in inguinal fat by change in phospholamban expression be in however, in inguinal fat the in total phospholamban was as well as a in the of with an for the phosphorylated phospholamban in both total AMP kinase and in phosphorylated AMP kinase also occurred in of Ucp1-/- It is apparent that are between and phospholamban that include in the of phospholamban and the of phospholamban to that in the of by and J. PubMed Google of in at to between phospholamban and and these are the are for and The that these of are in cold-adapted Ucp1-/- mice that they be associated with and molecular that the thermogenic of the be to the and of these molecular to the regulation of thermogenesis in white blot analysis of cycling in the muscle and inguinal tissue from and Ucp1-/- mice on the type and Ucp1-/- mice to cold by temperature 2 mice at 4 for mice at for the period of in at for and for to to the of phospholamban to is increased in inguinal fat of Ucp1-/- between was by was with and showed that the of total phosphorylated is increased in inguinal fat of Ucp1-/- of phosphorylated was also in Ucp1-/- of and phosphorylated phosphorylated to for activity was in the of brown and inguinal fat from Ucp1-/- and Ucp1+/+ mice The activity was of total activity in skeletal muscle and of activity in brown of activity was in both skeletal muscle and brown fat of Ucp1-/- mice with type mice of the activity in brown fat was by and of the total activity in and to by that a of the in muscle of Ucp1-/- mice is a that cannot be by the activity be from as the of the of activity is in Ucp1-/- was in to be that the that inguinal fat of type Ucp1-/- mice activity the of activity is increased Because activity was in the of not be from the activity of the which is increased in these activity in of cold-adapted and Ucp1-/- brown activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to is activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to activity levels are between at was in on an reticulum from a of to is in a that be to thermogenesis in inguinal fat include and mitochondrial of the also that inguinal fat an enhanced of as for a tissue Mice with a Ucp1 gene are sensitive to an acute exposure to the cold at 4 but are to temperature the temperature is an to alternative mechanisms of thermogenesis that are of UCP1-based nonshivering thermogenesis in brown the of shivering to body temperature in mice upon acute exposure that shivering be for the maintenance of body temperature, and is an that not is not The physiological that the capacity of UCP1-deficient mice to adapt to the cold is to oxygen mice oxygen consumption cold mice show a in oxygen and which are to the show a and the other of the mice, which cannot adapt to the are to oxygen consumption J. 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