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A process for isolating polymers from their solvents by addition of fluids containing carbon dioxide at temperatures of 30° to 280° C. and under pressures of 1 to 1,000 bar is disclosed. The process is characterized in that the concentration of the polymers in their solvents is between 5% and 80% by weight and the fluid containing carbon dioxide consists of at least 50% by weight carbon dioxide.
1. A process for isolating a polymer from a solution containing a polymer and its solvent comprising
(i) mixing into said solution a fluid containing carbon dioxide at temperatures between 30° and 100° C. and under pressures of 1 to 1,000 bar, causing precipitation of said polymer, and
(ii) collecting said polymer, and
(iii) granulating said polymer,
said solution containing said polymer at an amount of 5 to 80% by weight, said fluid containing at least 50% by weight carbon dioxide.
2. The process of claim 1 wherein mixing is carried out in a static mixer.
3. The process of claim 1 further comprising expanding said solution during or after said mixing through a nozzle into a container or by atomization.
4. The process of claim 1 characterized in that said fluid is mixed with said solution in a twin-fluid nozzle at a weight-ratio of fluid to polymer solution selected to cause said precipitation within the twin-fluid nozzle.
5. The process of claim 1 wherein said polymer is polycarbonate and said solvent is at least one member selected from the group consisting of chlorobenzene and methylene chloride.
6. The process of claim 3 characterized in that a residence time of at least one second is interposed between said mixing and said expansion.
7. The process of claim 2 characterized in that 25% by weight, based on the quantity of polymer and solvent, of fluid is added to the polymer solution before said solution is introduced to said static mixer.
The isolation of polymers from organic solvents is an important process step. In general, 5 to 80% polymer solutions have to be separated in such a way that any aftertreatment of the isolated polymer, for example by residual evaporation or residual drying, can be carried out quickly and simply without any damage to the product.
There has been no shortage of attempts to remove polymer solutions from their organic solvents by various precipitation processes.
Thus, according to U.S. Pat. No. 4,634,761 for example, polymers can be isolated from organic solvents by stirring the organic solution into an aqueous solution, specially adjusting the inflow of aqueous and organic phase and volatilizing the organic solvent in the mixture in such a way that polymer agglomerates suitable for subsequent processing are precipitated. This process has the disadvantage that subsequent drying of the agglomerates is expensive on account of the residual water content.
It is also known that residual impurities can be removed from polymers by means of supercritical fluids.
According to EP 334 314 A2, impurities, such as solvent residues, additives, monomers and oligomers, can be isolated from polymers by an entraining gas, such as carbon dioxide, at temperatures between 30° C. and 120° C. and under pressures above 10 bar. However, this process can only be applied when the treated polymers have already been substantially isolated from the organic solvents.
A process for the removal of methylene chloride from polycarbonate with supercritical carbon dioxide is described in the Article by Yamamoto, Koji in: Kagaku Kogaku 53(12), 1989, pages 914 to 916. More particularly, it is explained how the methylene chloride diffuses as a function of the pressure and the temperature of the supercritical fluid-containing system. However, there are no references to the special working up according to the present invention.
DE 3 840 293 A1 describes a process for the removal of impurities from polymeric plastics and to an apparatus for carrying out the process. This known process is based on working up using special screw technology for polymer melts.
JP 0 116 8730-A2 describes a process with which polycarbonate is freed from solvent residues with supercritical carbon dioxide under pressures of 70 to 500 bar and at temperatures of 3° to 300° C. Once again, the process can only be applied after the polymer has already been substantially isolated from the organic solvent.
The residual isolation of methylene chloride from polycarbonate with carbon dioxide in a fixed bed is described in the Article by Yamamoto Koji et al in: Kagaku Kogaku Ronbunshu 15(3), 1989, pages 673-675.
According to the Article by Beckmann and Porter in: J. Polym. Sci. Part B: Polym. Phys. 25(7), pages 1511-1517, 1987, polycarbonate can be made to crystallize very easily where polycarbonate which has already been substantially isolated (polycarbonate solid) and supercritical carbon dioxide are used.
In applicants' view, the process according to the present invention is neither covered by nor can be logically derived from the prior art.
FIG. 1 illustrates an embodiment of the isolation process of the invention relating to polycarbonate/methylene chloride solutions.
The present invention relates to a process for isolating polymers from their solvents by addition of fluids containing carbon dioxide at temperatures of 30° C. to 280° C. under pressures of 1 bar to 1,000 bar, characterized in that the concentration of the polymers in their solvents is between 5% by weight and 80% by weight and the fluid containing carbon dioxide consists of at least 50% by weight carbon dioxide.
Any polymers which are present in their solvents or which accumulate as solutions during their production are generally suitable. Thus, suitable polymers are polymers from the classes of polycarbonates, polyolefins, polystyrenes, polyhalo-olefins, polyvinyls, polyethers, polyacrylates, polyalkyl methacrylates, polydienes, polyesters, polyester carbonates, polyamides or polyarylenes, such as for example polyarylene sulfides, polyarylene ketones and polyarylene sulfones, and polyimides, polyether esters and polysiloxanes. Conductive polymers and polyelectrolytes are also suitable.
The polymers may be both copolymers and homopolymers; the copolymers may be both block and statistical copolymers. The polymers have typical molecular weights, for example, molecular weights between 10,000 g/mol and 500,000 g/mol, molecular weight distributions and non-uniformities and may still contain oligomers and branched or crosslinked components in typical quantities, for example, up to 10% of total weight.
Of these polymers, preference is attributed to polyester carbonates based on diphenols, terephthalic and/or isophthalic acid, carbonic acid, chain terminators and, optionally, branching agents, in which the ratio of carbonate structures to aromatic dicarboxylic acid ester structures is between 10 mol-%:90 mol-% and 40 mol-%:60 mol-%, based on the total mols of carbonate and dicarboxylic acid ester structures. Polyester carbonates such as these are described, for example, in EP-OS 0 036 080.
Aliphatic thermoplastic polyesters are also preferred, polyalkylene terephthalates being particularly preferred. Polyalkylene terephthalates are, for example, polyalkylene terephthalates based on ethylene glycol, propane-1,3-diol, butane-1,4-diol, hexane-1,6-diol and 1,4-bis-hydroxymethyl cyclohexane (see for example U.S. Pat. Nos. 2,647,885, 2,643,989, 2,534,028, 2,578,660, 2,742,494 and 2,901,466).
Other preferred polymers are thermoplastics polyalkyl (meth)acrylates, more particularly poly-C1-4 -alkyl methacrylates, i.e. for example polymers of methyl, ethyl, propyl or butyl methacrylate. By polymers are meant both homopolymers and copolymers of these methacrylates.
Other suitable polymers are linear and branched polyarylene sulfides containing structural units corresponding to the general formula (-Ar-S-)n, in which Ar- is an aromatic constituent which may include up to 4 substituents and preferably at most two substituents and n is an integer of 10 to 1,000. Unsubstituted p-phenylene sulfides are particularly suitable (see for example U.S. Pat. No. 3,354,129 and EP-OS 0 171 021 and the literature cited in EP-OS 0 171 021).
Other preferred polymers are polyphenylene oxides, more particularly poly(2,4-dialkyl-1,4-phenylene oxides), such as those described for example in DE-OS 2 126 434 or in U.S. Pat. No. 3,306,875.
Preferred polymers for the process according to the invention are, in particular, aromatic polycarbonates based on diphenols. Polycarbonates such as these are known from the literature. Suitable diphenols are described in U.S. Pat. Nos. 3,028,365, 2,999,835, 3,148,172, 3,275,601, 2,991,273, 3,271,367, 3,962,781, 2,970,131 and 2,999,846, in DE-OSS 1 570 703, 2 063 050, 2 063 052, 22 110 956, in FR-PS 1 561 518 and in DE-OSS 3 832 396 (Le A 26 344) and 3 833 953 (Le A 26 397).
Particularly preferred diphenols are 4,4'-dihydroxydiphenyl, 2,2-bis-(4-hydroxyphenyl)-propane, 2,2-bis-(3,5-dimethyl-4-hydroxyphenyl)-propane and 1,1-bis-(4-hydroxyphenyl)-3,3,5-trimethyl cyclohexane.
The polycarbonates preferably have molecular weights Mw (weight average, as determined by scattered light measurement) in the range from 15,000 to 200,000 g/mol and more particularly in the range from 20,000 to 80,000 g/mol.
Of these polycarbonates, which may also be copolycarbonates based on the diphenols mentioned, polycarbonates and copolycarbonates based on 2,2-bis-(4-hydroxyphenyl)-propane with chain terminators p-tert. butyl phenol, cumyl phenol, phenol and isooctyl phenol, copolycarbonates of 2,2-bis-(4-hydroxyphenyl)-propane with 2,2-bis-(3,5-dimethyl-4-hydroxyphenyl)-propane and copolycarbonates of 2,2-bis-(4-hydroxyphenyl)-propane with 1,1-bis-(4-hydroxyphenyl)-3,3,5-trimethyl cyclohexane are particularly suitable.
The process according to the invention may be used with particular advantage for isolating the polycarbonates described as particularly preferred.
The polymers are present in their respective inert organic solvents. The expert knows which organic liquid in which temperature range is a solvent for which polymer.
The process according to the invention for isolating polymers may be applied where the polymers have to be isolated from organic solvents in which they are present in concentrations of 5 to 80% by weight polymer, based on the weight of the polymer/solvent mixture. The polymer solutions preferably contain between 10 and 60% by weight and, more preferably, between 14 and 26% by weight polymer. Suitable organic solvents which are inert to the particular polymer are, in general, organic compounds which are capable of dissolving the particular polymer at temperatures below 240° C.
Accordingly, suitable solvents for polycarbonates are, for example, methylene chloride, monochlorobenzene, acetone, acetonitrile and mixtures of these solvents, more particularly solvent mixtures of methylene chloride and chlorobenzene and also methylene chloride and chlorobenzene alone.
Solvents suitable for polyphenylene sulfides are, for example, N-methyl pyrrolidone and N-methyl caprolactam.
Fluids containing carbon dioxide suitable for the purposes of the invention are fluids which contain at least 50% by weight, based on the total weight of liquid, of pure carbon dioxide. These liquids containing at least 50% by weight carbon dioxide are particularly suitable when the respective constituents of the fluids making up the balance to 100% by weight are gases or gas mixtures which have critical temperatures below 200° C. and critical pressures below 200 bar. Accordingly, gases such as these are, in particular, dinitrogen oxide, methane, nitrogen, propane, difluorochloromethane, trifluoromethane, ethane, ammonia, sulfur hexafluoride and/or sulfur dioxide.
The following are preferably added to the carbon dioxide: sulfur dioxide, propane, nitrogen, ethane, ammonia and/or trifluoromethane. In a particularly preferred embodiment, no more than 30% by weight and, in particular, no more than 10% by weight, based on the total weight of fluid used, of fluids other than carbon dioxide are used. It is preferred above all to use substantially pure carbon dioxide.
The temperature range in which it is favorable to add the fluid containing carbon dioxide to the polymer solution depends upon the polymer and its particular solvent and upon the concentration of the polymer in the solvent. A temperature range in which preferably 10% by weight and, more preferably, at least 14% by weight of the particular polymer is dissolved in the solvent is generally selected. The polymer concentrations should be below 80% by weight, preferably below 60% by weight and, more preferably, between 14 and 26% by weight. Accordingly, typical temperatures are between 30° C. and 280° C. and preferably between 30° C. and 100° C. and typical pressures are in the range from 1 bar to 1,000 bar and preferably in the range from 10 bar to 500 bar. It can be of advantage to work in the region of the critical temperature and the critical pressure of the particular carbon-dioxide-containing fluid.
The quantities of fluid to be used in accordance with the invention depend upon the concentration of the polymer to be isolated in its organic solvent. Typical concentrations are 5 to 5000% by weight carbon-dioxide-containing fluid according to the invention, based on the total weight of polymer and organic solvent.
The fluid containing carbon dioxide is added by mixing. This may be done continuously or discontinuously. Suitable mixers are stirred tanks, autoclaves, mixing elements, such as for example static mixers of the BKM type, twin-fluid nozzles having a residence time and mixing time of at least 20 milliseconds, such as for example solid cone nozzles, hollow cone eccentric nozzles, axial hollow cone nozzles and pneumatic spray nozzles. It can be of advantage to hold the pressure for a certain time after mixing rather than releasing it immediately after mixing. Typical residence times are between 0,1 and 100 seconds and preferably more than 1 second. The polymer precipitates from its organic solvent during or after mixing. The solution is expanded thereafter (after a residence time of at least 20 milliseconds) or even during the precipitation process by reducing the pressure, for example by expansion into a container or by atomization.
Suitable temperatures for the expansion step are, in particular, temperatures in the vicinity of the boiling point of the polymer solvent, so that the fluid containing carbon dioxide volatilizes together with the organic solvent during degassing.
The process according to the invention is described by way of example in the following with reference to the isolation of polycarbonate from methylene chloride:
The polycarbonate solution, for example between 14 and 26% by weight in pure methylene chloride, is initially introduced into a stirred tank under pressures of 1 to 3 bar and at temperatures of 20° to 80° C. Fluid containing carbon dioxide is then introduced into the stirred tank with continuous stirring until an increase in pressure of at least 60 bar is observed. With continued stirring and with continued addition of fluid containing carbon dioxide, polymer solution may then be continuously expanded through an outlet in the bottom of the stirred tank without any reduction in the internal pressure of the stirred tank.
It can be of particular advantage initially to stir a certain quantity, for example 25%, based on the quantity of polymer and solvent, of fluid containing carbon dioxide into the polycarbonate solution either in a stirred tank or in any other mixer. The quantity is selected so that the polycarbonate still does not precipitate from the organic solution. More fluid containing carbon dioxide is then added to the organic polycarbonate solution in a twin-fluid nozzle under pressures of 80 to 250 bar, followed by expansion in a separation vessel. Instead of using a twin-fluid nozzle as the mixing and expansion unit, it is also possible to use a nozzle with no mixing function as the expansion unit and a static mixer, for example of the BKM type, as the mixing unit.
An isolation process of the type described above carried out specifically for polycarbonate/methylene chloride solutions using the process according to the invention is illustrated in FIG. 1. The polycarbonate/methylene chloride solution coming from (1) is optionally pretreated in a mixer M1 (2) with fluid containing carbon dioxide in such quantities that polycarbonate does not precipitate. Another 5 to 500% by weight fluid are then added via a static mixer M2 (3) under pressures of 80 to 300 bar. The three-component system of polycarbonate, methylene chloride and fluid is then heated to temperatures of 60° to 80° C. in the preheater W1 (4) and expanded through a nozzle with no mixing function (one-fluid nozzle) D1 (5) into a separation vessel AB (6) in which pressures of 1 to 10 bar prevail. The polycarbonate powder formed during expansion and atomization may be discharged, for example, through a screw AS (7) at the bottom of the separator AB (6). It can also be of advantage to use a fluidized bed at the bottom of the separator which back-mixes polycarbonate already low in methylene chloride with the polycarbonate powder relatively rich in methylene chloride which is still present in the separator and thus enables the particle size of the powder to be varied. The nozzle D1 (5) is optionally heated to avoid reductions in temperature during expansion. The methylene chloride escapes from the separator together with the carbon dioxide and is fed to the condenser KO (8) in which the solvent condenses and the fluid containing carbon dioxide is separated and returned to the process (for example via the compressor KP (9), the heater WU (10) and the valve VE (11)). As already mentioned, the static mixer M2 (3) and the one-fluid nozzle D1 (5) may be replaced by a twin-fluid nozzle. It can also be of advantage to use a static mixer as the mixing unit M1 (2). In addition, a residence time of 20 milliseconds to 10 seconds between introduction of the fluid containing carbon dioxide and expansion in a nozzle may be necessary to optimize the particle size of the powder.
Additional explanations of FIG. 1:
KO (8) has a pressure of 2 to 50 bar; (12) is a CO2 replenishing facility, (13) is solvent
a) General procedure
600 ml of a 20% by weight polycarbonate/methylene chloride solution are initially introduced into a 1,000 ml capacity autoclave equipped with a stirrer and a bottom outlet. The polycarbonate has a relative viscosity, as measured by gel permeation chromatography at 25° C. and at a polymer concentration of 5 g/liter in methylene chloride, of 1.28. The polymer solution is initially introduced at room temperature. An initial pressure of 10 bar is built up in the autoclave by addition of carbon dioxide and the carbon dioxide is mixed in for 3 minutes at a stirring speed of 400 r.p.m. The bottom outlet is then opened, forcing the polycarbonate/methylene chloride/carbon dioxide solution into a pipe having an internal diameter of 5 mm. The pipe is heated to around 60° C. After a length of about 50 cm, it terminates in a twin-fluid nozzle into which carbon dioxide at 80° C. is introduced under a pressure of 100 bar and expanded into a separation vessel (pressure in the separation vessel: 1 bar). The polycarbonate powder accumulating at the bottom of the vessel is inspected.
b) Test parameters
Nozzle used: mixing nozzle with jet mixing, exit from inner part at same level as exit from jacket, mixing outside the nozzle
Initial pressure in autoclave: 10 bar
Carbon dioxide pressure: 100 bar
The 20% polycarbonate solution is introduced into the jacket space of the nozzle while the carbon dioxide is introduced into the inner space. The feed pipe from the carbon dioxide reservoir to the nozzle is heated to 60° C.
Nozzle used: mixing nozzle with internal mixing otherwise as Example 1. The carbon dioxide and the polycarbonate solution are combined in the nozzle and mixed by means of a static mixer. The initial pressure in the polycarbonate storage vessel is 20 bar.
As Example 1, except that the carbon dioxide is first passed through the nozzle and the polycarbonate solution is then added. Initial pressure in the storage vessel: 20 bar.
A nozzle with jet mixing is used as the mixing nozzle. The polycarbonate is introduced into the inner space of the nozzle while the carbon dioxide is introduced into the jacket space. The nozzle orifice is removed from the outlet of the inner part by means of a check nut. Mixing takes place inside the nozzle. Initial pressure in the storage vessel: 20 bar, otherwise as Example 1.
As Example 1, except that the carbon dioxide is mixed in under a pressure of 200 bar.
As Example 1, except that a mixture of carbon dioxide and nitrogen (composition: 70% by weight carbon dioxide, 30% by weight nitrogen) is mixed in at 80° C./300 bar.
c) Evaluation of the isolating effect
Differential scanning calorimetry and thermogravimetry measurements were carried out on all the samples in the separation vessel. All the samples had a granular structure and were free-flowing.
Residual methylene chloride content:
______________________________________ Example 1: <1% Example 2: 3% Example 3: 3% Example 4: <1% Example 5: <1% Example 6: <1%______________________________________
The polycarbonate powders thus obtained can be residually degassed in known manner, for example in a degassing screw, and processed to granules.
d) Comparison Example
Pure nitrogen having a critical temperature below -140° C. and a critical pressure of about 34 bar was introduced as fluid into the nozzle of Example 1 at a temperature of 60° C. and under a pressure of 300 bar. The polymer product in the separation vessel was speckled, did not have a good consistency and still contained more than 10% by weight methylene chloride.
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text:
Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta are protected against diet-induced obesity.
ABSTRACT The CCAAT/enhancer-binding protein beta (C/EBPbeta) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBPbeta, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBPbeta(-/-) mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBPbeta(-/-) mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBPbeta gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of beta-oxidation genes in the brown adipose tissue. CO(2) production was significantly higher in the C/EBPbeta(-/-) mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBPbeta is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity.
- [Show abstract] [Hide abstract]
ABSTRACT: The unfolded protein response (UPR) is a protective cellular response activated under conditions of endoplasmic reticulum (ER) stress. The hepatic UPR is activated in several forms of liver disease including nonalcoholic fatty liver disease (NAFLD). Recent data defining the role of the UPR in hepatic lipid metabolism have identified molecular mechanisms that may underlie the association between UPR activation and NAFLD. It has become increasingly evident that the IRE1α/Xbp1 pathway of the UPR is critical for hepatic lipid homeostasis, and dysregulation of this evolutionarily conserved pathway is associated with human nonalcoholic steatohepatitis (NASH). Although increasing evidence has delineated the importance of UPR pathway signaling in fatty liver disorders, the regulation of the hepatic UPR in normal physiology and fatty liver disorders remains incompletely understood. Understanding the role of the UPR in hepatic lipid metabolism may lead to the identification of novel therapeutic targets for the treatment of NAFLD.Seminars in Liver Disease 11/2013; 33(4):321-9. · 8.27 Impact Factor
Article: Transcription factor Smad3 is required for the inhibition of adipogenesis by retinoic acid.[Show abstract] [Hide abstract]
ABSTRACT: The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the up-regulation of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression. C/EBPbeta then acts to up-regulate the expression of adipogenic factors such as C/EBPalpha, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPbeta transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPbeta of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPbeta but rather stimulates the expression of the transforming growth factor beta-effector protein Smad3, which can interact with C/EBPbeta via its Mad homology 1 domain and can interfere with C/EBPbeta DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBPbeta DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBPbeta DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.Journal of Biological Chemistry 02/2010; 285(17):13274-84. · 4.65 Impact Factor
Article: Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice.[Show abstract] [Hide abstract]
ABSTRACT: The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARgamma binding site in the promoter of Pck1 (PPARE(-/-)). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE(-/-) mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE(-/-) mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.The Journal of Lipid Research 06/2010; 51(6):1452-63. · 4.39 Impact Factor
Page 1
Mice With a Deletion in the Gene for CCAAT/Enhancer-Binding
Protein β are Protected Against Diet-Induced Obesity
Carrie A. Millward1, Jason D. Heaney2, David S. Sinasac2, Eric C. Chu1, Ilya R. Bederman1,
Danielle A. Gilge1, Stephen F. Previs1, and Colleen M. Croniger1
1Department of Nutrition, Case Western Reserve University, Cleveland, Ohio
2Department of Genetics, Case Western Reserve University, Cleveland, Ohio.
The CCAAT/enhancer-binding protein β (C/EBPβ) is required for adipocyte differentiation and
maturation. We have studied the role of the transcription factor, C/EBPβ, in the development of diet-
induced obesity. Mice with a deletion in the gene for C/EBPβ (C/EBPβ−/−) and wild-type mice were
fed a high-fat diet (60% fat) for 12 weeks. The C/EBPβ−/− mice lost body fat, whereas the wild-type
mice increased their total body fat on a high-fat diet. The C/EBPβ−/− mice had lower levels of blood
triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the
wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet.
Deletion of C/EBPβ gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA
carboxylase, and fatty acid synthase and increasing the expression of β-oxidation genes in the brown
adipose tissue. CO2 production was significantly higher in the C/EBPβ−/− mice as was the level of
uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/
EBPβ is an important regulator in controlling lipid metabolism and in the development of diet-
induced obesity.
In the last 20 years, the prevalence of obesity has greatly increased by ~10–15% (1). With the
increase of obesity, there is an increased risk of certain diseases such as diabetes, hypertension,
coronary heart disease, and even some types of cancer (2–4). The development of obesity is a
consequence of both environmental and genetic factors. The CCAAT/enhancer-binding protein
(C/EBP) family of transcription factors has been shown to coordinate expression of genes
involved in adipocyte differentiation (5–8). During adipogenesis, the C/EBP family functions
in a transcriptional cascade. C/EBPβ and C/EBPδ are expressed early in development and
activate the transcription of peroxisome proliferator–activated receptor γ (PPARγ). This results
in induced expression of the C/EBPα gene. C/EBPβ can also activate C/EBPα independently
of PPARγ (9,10). These transcription factors activate lipogenic genes necessary for terminal
differentiation of the adipocyte.
Much understanding of the roles for the C/EBP family of transcription factors has been
elucidated from gene-targeted deletion studies in mice. The mice with a deletion for the C/
EBPα gene (C/EBPα−/−) lack the development of adipose tissue and die in the perinatal period
with a profoundly fatty liver (7). Mice with a deletion for the C/EBPβ gene (C/EBPβ−/−) have
adipocytes but fail to accumulate lipid in the cells even though they have normal levels of
PPARγ and C/EBPα (8). Mice with a double gene deletion for C/EBPβ and C/EBPδ have a
significant decrease in the weight of epididymal white adipose tissue (WAT), suggesting that
Address correspondence and reprint requests to Colleen M. Croniger, Department of Nutrition, Case Western Reserve University,
Cleveland, OH 44106. E-mail: E-mail: email@example.com.
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Published in final edited form as:
Diabetes. 2007 January ; 56(1): 161–167. doi:10.2337/db06-0310.
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both transcription factors have a major role in the development of adipose tissue and thus
obesity (8).
In addition to a role in adipose tissue development, the members of the C/EBP family are
involved in a broad range of physiological processes. These range from the acute-phase
response to regulation of glucose and lipid metabolism (11). C/EBPβ−/− mice have two
phenotypes, A and B. Mice with the B phenotype die in the perinatal period because of
hypoglycemia and a failure to mobilize their glycogen (12). Mice with the A phenotype survive
to adulthood, but exhibit fasting hypoglycemia, with reduced plasma insulin, blood lipids, free
fatty acids (FFAs) and impaired hepatic glucose production. The C/EBPβ−/− mice have a
blunted response to glucagon and adrenaline primarily due to altered levels of hepatic cAMP
production and reduced activity of protein kinase A (6,12). These results suggest that deletion
of the C/EBPβ gene affects both glucose homeostasis and lipid homeostasis.
In this study, we wanted to extend the previous studies for the role of C/EBPβ in lipid
homeostasis. The main aim of this study was to determine whether the deletion of the gene for
C/EBPβ would protect the mice from developing obesity and fatty liver on a high-fat diet.
The C/EBPβ−/− mice were obtained for this study by breeding heterozygous female mice with
a targeted deletion in the gene of C/EBPβ (C/EBPβ+/−) with heterozygous male mice. The
generation of the C/EBPβ−/− mice and their genetic background has been described previously
by Screpanti et al. (13). Briefly, embryonic stem cell clones from the CCE cell line (derived
from the 129/Sv/Ev strain) carrying the mutation were injected into C57BL/6J blastocysts and
were transplanted into the uteri of F1 (CBA × C57BL/6J) foster mothers. Male chimeras were
mated to MF1 females, and offspring heterozygous for the mutant allele were intercrossed to
obtain homozygous mice. Adult male and female mice were 8–12 weeks old at the time of their
use. Mice containing the gene for C/EBPβ (C/EBPβ+/+) are designated as wild-type animals
and are used for controls throughout the study. Screening for the C/EBPβ−/− mice was
performed by Southern blot analysis as outlined previously (6). All experimental protocols
were approved by the Case Western Reserve University Institutional Animal Care and Use
Dietary studies
Eight-week-old wild-type and C/EBPβ−/− mice were housed three to five mice per
microisolator cage and were maintained on a fixed 12-h light/dark cycle. Animals had free
access to water and were fed normal mouse chow (Teklad F6 8664; Harlan Teklad, Madison,
WI) or low-fat diet D12450B or high-fat diet D12492 (Research Diets, New Brunswick, NJ).
The chow diet contained 27 kcal% protein, 17 kcal% fat, and 57 kcal% carbohydrate. The low-
fat diet (D12450B) contained 20 kcal% protein, 10 kcal% fat, and 70 kcal% carbohydrate. The
high-fat diet (D12492) contained 20 kcal% protein, 60 kcal% fat, and 20 kcal% carbohydrate.
For the high-fat studies, the mice were fed the diet for a total of 12 weeks. The low-fat– and
chow-fed animals were fed for 12 weeks and used as controls as indicated. Food intake
measurements were measured as grams of food consumed per kilogram body weight over a
24-h period of mice that were individually housed.
Insulin measurements
Insulin was measured in mice that were fed a high-fat diet for 12 weeks. The mice were fasted
for 18 h and injected intraperitoneally with 2 g glucose/kg body wt. Blood was sampled from
the tail vein and assayed for insulin at 0 and 30 min with an Ultrasensitive Mouse Insulin
ELISA enzyme immunoassay from Mercodia (Lincoln Park, MO).
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Biochemical analysis
After an overnight fast, mice were killed, and a heart puncture was performed to collect the
blood. Plasma was generated using Microtainer plasma separator tubes from Becton Dickinson
(Franklin Lakes, NJ). The measurements of cholesterol, β-hydroxybutyrate, FFA, and
triglyceride were performed by Veterinary Diagnostic Services (Marshfield Laboratories,
Marshfield, WI). The determination of liver triglycerides was performed using the Triglyceride
(GPO) reagent set from Pointe Scientific (Lincoln Park, MI). Insulin was determined with an
Ultrasensitive Mouse Insulin ELISA enzyme immunoassay from Mercodia.
Body composition
A bolus of labeled water, 2H2O, was administered by intraperitoneal injection to fasted mice.
After allowing for isotope equilibration (2 h), a blood sample was collected by the tail vein,
and the 2H enrichment of body water was measured using gas chromatography–mass
spectrometry (14). Body water was calculated from the isotope dilution, and the fat mass was
determined as previously described (15).
CO2 production
The rate of CO2 production was determined using doubly labeled water (16–18). Fed wild-
type and C/EBPβ−/− mice were intraperitoneally injected with a bolus of 2H2O and H218O to
achieve initial labeling of ~2% 2H and 0.25% 18O. Blood was sampled at 2, 48, and 72 h after
injection. CO2 production was determined from the difference between the elimination of 2H
and 18O from body water (17). The 2H-labeling was determined after exchange with acetone
(14). The 18O enrichment of water was determined after the generation of trimethylphosphate
(17). The rate of CO2 production (moles per day) was calculated using the equation: CO2
production rate = 0.5 × N1 (k18O – k2H), where the fractional biological decay constants
(k18O and k2H) are in day−1 and N1 is the body water pool (moles) calculated as [(V2H +
V18O)/2]. Data are expressed as means ± SE.
Histology of liver
Liver tissues from fasted mice were fixed in 10% formalin (Sigma, St. Louis, MO) at 4°C. The
tissues were embedded in paraffin and stained with hematoxylin and eosin by the Pathology
Core Facility (Case Western Reserve University, Cleveland, OH). Pictures were taken at ×40
Real-time RT-PCR analysis
Total RNA was isolated with RNeasy Mini Isolation kit from Qiagen (Valencia, CA). The
single-strand cDNA was synthesized from 2 µg total RNA with random hexamer primers and
MMTV reverse transcriptase (Ambion, Austin, TX). Real-time PCRs were performed as
described previously (19). Standard curves were generated by diluting wild-type liver cDNA
for Fig. 3 and Fig. 6 and by diluting wild-type muscle cDNA for Fig. 4 (1:4, 1:8, 1:16, 1:32,
1:64, and 1:128 in H2O). One microliter of the dilution was used for PCR. PCR was performed
in a 25-µl volume using Syber Green PCR Core reagent mix (Applied Biosystems, Foster City,
CA) as a source of Taq, buffer, and dNTPs. Primers were used at a final concentration of 0.2
µmol/l, and the sequence of the primers used for RT-PCR are in Table 1. PCR was performed
in the Chromo4 Cycler (MJ Research) with 40 cycles (15 min at 95°C; followed by 40 cycles
of 30 s at 95°C, 30 s at 60°C, 45 s at 72°C, and 30 s at 73°C [optimal read]; followed by a 5
min extension at 72°C and a melting curve). The relative amounts of the mRNAs were
determined by linear regression from the standard curves derivative maximum method, with
Opticon Monitor 3 software (MJ Research), and 18S rRNA was used as a loading control. The
RT-PCR reactions were run on RNA isolated from wild-type or C/EBPβ−/− mice that had been
fed a high-fat diet for 12 weeks. For mRNA expressed as mRNA fold over wild type, the gene
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expression from wild-type mice was set arbitrarily at 1 and the gene expression for the C/
EBPβ−/− mice is represented as fold difference relative to wild-type mice gene expression. The
gene expression was measured for both fed and fasted conditions in separate RT-PCRs with
wild-type mice gene expression set at 1 under both conditions. The number of mice for each
gene tested is four.
Statistical analysis
Results are expressed as means ± SE. Student's t tests for paired data were used. Differences
were considered significant if P < 0.05.
Reduced accumulation of body fat in C/EBPβ− mice fed a high-fat diet
Both wild-type and C/EBPβ−/− mice were fed either the high-fat diet or low-fat diet for 12
weeks. The animals were weighed weekly, and food intake was measured for a 24-h period
weekly (Fig. 1). After 12 weeks on a low-fat diet, wild-type mice had a 17.6% body wt increase,
whereas C/EBPβ−/− mice had only 11.1% body wt increase (Fig. 1B and C). However, wild-
type mice fed a high-fat diet had a statistically significant increase in body weight of 27%,
whereas the C/EBPβ−/− mice had only a 13% increase in body weight (P < 0.03). The wild-
type mice consumed less food on the high-fat diet compared with the C/EBPβ−/− mice even
though the wild-type mice gained more weight (Fig. 1A) (P < 0.05).
To verify whether the increase in body weight was due to an increase in total body lipid, the
percentage body fat was measured using the 2H2O method (Fig. 2A). In this experiment, mice
were fed a control chow diet, low-fat diet, or high-fat diet for 12 weeks. The wild-type mice
had no difference in total body lipid after being fed a low-fat diet. However, after exposure on
a high-fat diet for 12 weeks, the wild-type mice had an increase in total body lipid from 27.4
to 36.7% (P < 0.04). The total body lipid for the C/EBPβ−/− mice did not change significantly
in response to alteration in diet. The C/EBPβ−/− mice had significantly less total body lipid
compared with the wild-type mice on all diets tested (P < 0.04). The C/EBPβ−/− mice dropped
total body lipid from 14% on the low-fat diet to 10% on the high-fat diet. Therefore the amount
of total body fat was reduced by 29% in the C/EBPβ−/− mice, whereas the wild-type mice
increased their total body fat by 34% on a high-fat diet.
Brown adipose tissue (BAT) and WAT were isolated and weighed for wild-type and C/
EBPβ−/− mice after 12 weeks of either a low-fat or high-fat diet. The wild-type mice had an
increased amount of both WAT and BAT on a high-fat diet, whereas the C/EBPβ−/− mice only
had a marginal increase in the weight of WAT only. The wild-type mice have a 1.93-fold
increase in the weight of the WAT and a 2.2-fold increase in the weight of the BAT on a high-
fat diet. The C/EBPβ−/− mice have a 2.9-fold increase of WAT weight on a high-fat diet but
no increase in the weight of the BAT on the high-fat diet (Fig. 2B and C).
Increased CO2 production in C/EBPβ−/− mice
Because the C/EBPβ−/− mice consumed more calories than the wild-type mice on a high-fat
diet yet remained resistant to diet-induced obesity, we investigated the CO2 production of the
mice using the doubly labeled water technique (2H2 18O) (Fig. 3A). We found that the C/
EBPβ−/− mice produced 1.6-fold more CO2 compared with the wild-type mice (C/EBPβ−/− 5.8
± 0.748 and wild type 3.6 ± 0.29 mol CO2 · kg−1 · day−1)(P < 0.04), suggesting increased
energy expenditure.
To determine which tissues are contributing to the increased CO2 production, we measured
gene expression for the fatty acid oxidation genes and expressed the relative mRNA levels
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normalized by 18S rRNA as fold over wild-type values for each tissue. The genes tested were
the liver-specific form of carnitine palmitoyltransferase (CPT) 1 (CPT-1a), the muscle-specific
form of CPT-1 (CPT-1b), long acyl-CoA dehydrogenase (LCAD), and acyl-CoA oxidase
(AOX) in fed mice after 12 weeks of a high-fat diet. In the C/EBPβ−/− mice, the liver, WAT,
and muscle have similar values of fatty acid oxidation genes for most of the genes tested. In
the livers of the C/EBPβ−/− mice, the rate-limiting step of fatty acid oxidation, CPT-1a, is
expressed normally. However, there was 0.5- and 0.6-fold less hepatic gene expression for
LCAD and AOX, respectively, compared with wild-type mice (Fig. 3B). We tested CPT-1a
and CPT-1b in the WAT because both isoforms are expressed in adipose tissues. The level of
CPT-1a mRNA expression in WAT is reduced 0.66-fold in the C/EBPβ−/−, whereas the levels
of CPT-1b are equally expressed compared with wild-type mice (Fig. 3D). The muscle-specific
form of CPT-1, CPT-1b, was elevated by 1.3-fold in the C/EBPβ−/− mice, yet in the muscle of
the C/EBPβ−/− mice, we found the same level of LCAD and AOX gene expression compared
with wild-type mice.
The increased CO2 production in the C/EBPβ−/− mice may be explained by increased fatty acid
oxidation gene expression in the BAT (Fig. 3C). The amount of CPT-1a in the BAT is elevated
by 1.5-fold but is not statistically significant (P = 0.12). The expression of CPT-1b in the BAT
of C/EBPβ−/− mice is equal to the gene expression in wild-type mice. However, in the BAT of
the C/EBPβ−/− mice, there was an increase of 2.0-fold more LCAD gene expression and 1.4-
fold more AOX expression compared with wild-type mice. Therefore the BAT tissue has
increased fatty acid oxidation gene expression that may contribute to the resistance of obesity
on high-fat diet.
Increased expression of uncoupling protein-1 and −3 in the muscle of C/EBPβ−/− mice
To conclude whether energy was lost through uncoupling proteins (UCPs), we measured the
levels of UCP-1 and UCP-3 gene expression in the BAT, WAT, and muscle of fed wild-type
and C/EBPβ−/− mice after 12 weeks of a high-fat diet. We found that the C/EBPβ−/− mice had
a significant increase of UCP-1 gene expression in muscle tissue compared with wild-type
mice (Fig. 4) (P < 0.03). The level of UCP-3 gene expression was significantly increased in
muscle of C/EBPβ−/− mice as well (P = 0.04). However, the contribution of the increased gene
expression is minimal compared with the primary site of UCP-1 mRNA expression found in
the BAT. The C/EBPβ−/− mice have increased levels of UCP-1 mRNA, but the level is not
statistically significant (P = 0.07). The WAT has very little mRNA expressed for UCP-1 and
Metabolic consequences of the high-fat diet
An increase in adiposity often results in an increase of triglyceride accumulation in the liver.
To test the role of C/EBPβ in the accumulation of hepatic triglycerides, we isolated livers from
wild-type and C/EBPβ−/− mice after consuming the high-fat diet for 12 weeks and fasted
overnight. Hematoxylin and eosin staining was done on paraffin-embedded sections of the
liver. The wild-type mice had an accumulation of macrovesicular and microvesicular fat
droplets, whereas the C/EBPβ−/− mice had no visible lipid droplets (Fig. 5).
To confirm that the droplets seen in the livers from wild-type mice were due to an accumulation
of triglyceride, we measured the triglyceride content in the livers. The wild-type mice had 107
± 17.85 mg/g liver triglycerides, whereas the C/EBPβ−/− mice had 27.57 ± 9.11 mg/g liver
triglycerides (Table 2) (P < 0.05).
We were also interested in whether the C/EBPβ−/− mice had lipid accumulating in the blood.
We isolated plasma after an overnight from mice fed the high-fat diet for 12 weeks. The C/
EBPβ−/− mice had lower levels of blood glucose, triglyceride, FFAs, and cholesterol. All values
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were statistically significant (P < 0.05). Therefore the C/EBPβ−/− mice are resistant to the
deleterious effects of a high-fat diet. They have lower blood triglycerides (C/EBPβ 37.33 ± 2.8
mg/dl and wild type 63.75 ± 5.94) and lower liver triglycerides, even though the C/EBPβ−/−
mice have consumed more of the high-fat diet.
The levels of β-hydroxybutyrate were also greatly reduced in the C/EBPβ−/− mice. In Table 2,
the C/EBPβ−/− mice have 1.7 ± 0.23 mmol/l β-hydroxybutyrate compared with 2.1 ± 0.229
mmol/l in wild-type mice (P < 0.05). We measured hepatic gene expression of the ketogenic
genes, ketothiolase, and hydroxymethylglutaryl (HMG)-CoA lyase in fasted mice (Fig. 6B).
The C/EBPβ−/− mice have less ketothiolase gene expression (0.2-fold of wild type) and less
HMG-CoA lyase gene expression (0.6-fold of wild type) (P < 0.05).
Reduced hepatic lipogenic gene expression in C/EBPβ−/− mice on high-fat diet
The reduced levels of triglycerides in the liver and FFAs in the blood of the C/EBPβ−/− mice
suggest that the mice may have reduced fatty acid synthesis. To establish whether the lipogenic
and glycolytic genes are affected, we performed RT-PCR. We measured the key genes for
glucose uptake, lipogenesis, and gluconeogenesis in livers of C/EBPβ−/− and wild-type mice
in both the fasted and fed states after 12 weeks of a high-fat diet (Fig. 6A and B). The gene
expression for glucose uptake, GLUT2, and glucokinase was greatly reduced. The gene
expression for the regulator of lipogenesis, sterol regulatory element binding protein-1
(SREBP-1), was also greatly reduced in both the fed and fasted states. The target genes for the
SREBP-1 transcription factor, acetyl CoA carboxylase-1 (ACC-1), fatty acid synthase (FAS),
and sterol CoA desaturase-1 (SCD-1) were significantly reduced. We measured the gene
expression of the rate-limiting step in the gluconeogenic pathway, the cytosolic form of
PEPCK-C, and determined that the mRNA levels of PEPCK-C were slightly elevated in fed
mice and significantly reduced in the fasted C/EBPβ−/− mice.
Insulin regulates glucose uptake and fatty acid synthesis. To ascertain whether the C/
EBPβ−/− mice had decreased insulin concentration on a high-fat diet, we measured their insulin
levels after an intraperitoneal injection of glucose (2 g/kg body wt). The levels of insulin at 0
and 30 min are 0.5 and 1.1 ng/ml, respectively, for C/EBPβ−/− and wild-type mice. The
concentration of insulin was induced equally in response to an injection of glucose for the C/
EBPβ−/− and wild-type mice (data not shown).
The balance of energy intake and expenditure has been well documented in studies on obesity.
In genetic models of mice susceptible to obesity, such as db/db mice, the lack of leptin
production results in hyperphagia and obesity (20). Environmental factors such as a high-fat
diet in the C57BL/6J inbred mouse strain have focused on the mechanism of the environmental
influence for obesity (21,22). To better understand the mechanism for the development of
obesity, we focused on the role of a transcription factor C/EBPβ. We studied the effect of
deleting C/EBPβ on energy storage and the development of obesity from a high-fat diet. We
found the C/EBPβ−/− mice consumed more food than wild-type mice, yet were protected
against increased total body lipid and against lipid accumulation in the liver when fed a high-
fat diet. This decreased energy storage indicates an increase of energy metabolism. The C/
EBPβ−/− mice had higher CO2 production, suggesting that the C/EBPβ−/− mice have increased
flux through the tricarboxylic acid cycle and increased energy expenditure. The BAT in the C/
EBPβ−/− mice had significantly increased levels of fatty acid oxidation genes, LCAD and AOX.
The β-oxidation of very long–chain fatty acids occurs in peroxisomes, and the rate-limiting
enzyme of peroxisomal fatty acid β-oxidation is AOX (23,24). In the C/EPBβ−/− mice, the
BAT has 1.4-fold increased gene expression of AOX and 2.0-fold increase of LCAD gene
expression over wild-type gene expression. Peroxisomal β-oxidation produces an increase of
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short- and medium-chain fatty acids that can now then enter the mitochondria for β-oxidation
(25). The increase of LCAD gene expression would also contribute to the increased overall
oxidation of fatty acids on a high-fat diet. The size of the BAT in the C/EBPβ−/− mice remains
unchanged after high-fat feeding. These data suggest that increased metabolic activity in the
BAT protects the C/EBPβ−/− mice against obesity. The excess energy in the triglycerides from
the high-fat diet is lost in a futile cycle in the BAT of the C/EBPβ−/− mice. Future studies will
focus on the role of C/EBPβ in oxidation of fatty acids.
Other mechanisms for increased energy expenditure are UCPs. UCP-1 is well accepted as the
mechanism for nonshivering thermogenesis in BAT, and its expression is induced by cold
exposure and fatty acid administration (26). In most tissues, oxidation of fuels results in ATP
production by oxidative phosphorylation in the mitochondria (27). The presence of UCP-1 in
the mitochondrial membrane produces a leak in the proton gradient, which results in a loss of
ATP synthesis as heat (28). Transgenic mice that overexpress UCP-1 in the skeletal muscle
have 98% higher oxygen consumption and were resistant to diet-induced obesity on a high-fat
diet similar to the phenotype in the C/EBPβ−/− mice (29). We have shown that the C/
EBPβ−/− mice had significantly higher levels of UCP-1 and UCP-3 gene expression in the
skeletal muscle and an increase of UCP-1 in the BAT that was approaching significance. This
increased UCP-1 gene expression may contribute to the overall increased energy expenditure
and resistance to obesity on a high-fat diet in the C/EBPβ−/− mice.
Alterations in food intake and overall metabolism require peripheral metabolic signals to the
brain such as leptin and cytokines from the adipose tissue and peptide YY and ghrelin from
the gastrointestinal tract. Signaling in the brain by metabolic sensors such as neuropeptide Y
and melanocortin peptides are also required for brain-mediated responses to nutritional
challenges (30). These may be altered in the C/EBPβ−/− mice but have not yet been tested.
Possible regulation by the transcription factor C/EBPβ for the reduced triglyceride formation
in the blood and liver may be the reduced gene expression of key genes in the fatty acid synthesis
pathway in both the liver and adipose tissues. Carmona et al. (31) have reported reduced gene
expression of FAS in BAT of C/EBPβ−/− mice at 21 and 4°C during a cold challenge. We have
shown that ACC, FAS, and SREPB gene expression is greatly reduced in the livers of C/
EBPβ−/− mice on a high-fat diet. C/EBPβ can bind directly to the cAMP responsive element–
binding protein regulatory region (CRE) in promoters of various genes. Tae et al. (32) have
reported that C/EBPβ is required for the full induction of ACC gene expression by cAMP
through the CRE region in the promoter. SREBP is a key regulator of hepatic lipogenesis.
Overexpression studies of SREBP-1a in cultured cells or animal livers have resulted in
activation of lipogenic genes (33). Selective overexpression of SREBP-1c in adipose tissue
resulted in animals that developed insulin-resistant hypoglycemia and fatty livers (34). In the
C/EBPβ−/− mice, the lower gene expression of SREBP and the deletion of C/EBPβ contribute
to the reduced ACC and FAS gene expression.
In conclusion, the data presented in this study indicate that the C/EBPβ−/− mice are protected
from obesity and fatty liver on a high-fat diet because of reduced hepatic expression of lipogenic
genes, ACC, and FAS and increased CO2 production from increased metabolism in the BAT
and muscle. The experiments presented indicate that C/EBPβ plays a highly integrative role
in the regulation of whole-body energy metabolism and is required for the development of fatty
liver during obesity. The C/EBPβ gene may be a potential target for drug therapy in the
prevention of fatty liver due to obesity.
ACC, acetyl CoA carboxylase
Millward et al. Page 7
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AOX, acyl-CoA oxidase
BAT, brown adipose tissue
C/EBPβ, CCAAT/enhancer-binding protein β
CPT, carnitine palmitoyltransferase
CRE, cAMP responsive element–binding protein regulatory region
FAS, fatty acid synthase
FFA, free fatty acid
HMG, hydroxy-methylglutaryl
LCAD, long acyl-CoA dehydrogenase
PRARγ, peroxisome proliferator–activated receptor γ
SCD-1, sterol CoA desaturase-1
SREBP-1, sterol regulatory element–binding protein-1
UCP, uncoupling protein
We are grateful to Dr. Valeria Poli and Dr. Richard Hanson for the generous gift of the C/EBPβ−/− mice.
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and promoter I of acetyl-CoA carboxylase. J Biol Chem 1995;270:21487–21494. [PubMed:
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34. Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, Goldstein JL, Brown MS.
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36. Clapham JC, Coulthard VH, Moore GB. Concordant mRNA expression of UCP-3, but not UCP-2,
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Millward et al. Page 10
Diabetes. Author manuscript; available in PMC 2009 May 4.
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C/EBPβ−/− mice are resistant to increased weight on high-fat diet. A: Food consumed was
measured over a 24-h period and normalized by kg body wt for wild-type and C/EBPβ−/− mice.
The values are the means ± SE for 11–14 mice per group for food consumption. Body weights
were measured for low-fat diets (B) and high-fat diets (C). The wild-type initial (□ 8 weeks
old) and wild-type final (■ 20 weeks old) body weights and the C/EBPβ−/− initial (▨ 8 weeks)
and C/EBPβ−/− final ( 20 weeks) body weights are shown. The values are the means ± SE
for five to eight mice per group. *P < 0.03 compared with wild-type initial body weights; aP
< 0.04 compared with wild-type initial body weight; **P < 0.03 compared with wild-type final
body weight.
Millward et al.Page 11
Diabetes. Author manuscript; available in PMC 2009 May 4.
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Reduced body fat on high-fat diet. A: The percentage of body fat was measured for wild-type
and C/EBPβ−/− mice on control chow, low-fat, or high-fat diets (*P < 0.04 compared with
wild-type low-fat and control diet; **P < 0.04 compared with wild-type mice). Fat pads were
isolated for BAT (B) and WAT (C) after 12 weeks of either a low-fat or high-fat diet. *P <
0.05 compared with wild-type mice on low-fat diet; **P < 0.05 compared with C/EBPβ−/−
mice on low-fat diet. The values are the means ± SE for three to six mice per group.
Millward et al.Page 12
Diabetes. Author manuscript; available in PMC 2009 May 4.
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C/EBPβ−/− mice have increased energy expenditure. A: CO2 production for fed wild-type and
C/EBPβ−/− mice on a high-fat diet for 12 weeks. The values are the means ± SE for six mice
per group for CO2 production. *P < 0.05. Real-time PCR quantification for β-oxidation gene
expression in liver (B), BAT (C), WAT (D), and muscle (E) of mice fasted overnight after 12
weeks of high-fat diet. *P < 0.05 compared with wild-type mice. The values are the means ±
SE for four mice per group normalized with 18s RNA and expressed as fold difference over
wild-type mice in that tissue.
Millward et al.Page 13
Diabetes. Author manuscript; available in PMC 2009 May 4.
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Analysis of UCP gene expression. Real-time PCR quantification in fed mice on a high-fat diet
for UCP-1 and UCP-3 gene expression in WAT, BAT, and muscle. The values are the means
± SE for four mice per group normalized with 18s rRNA. *P < 0.05.
Millward et al.Page 14
Diabetes. Author manuscript; available in PMC 2009 May 4.
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Page 15
The C/EBPβ−/− mice are resistant to fatty liver. Hematoxylin and eosin–stained sections of
wild-type and C/EBPβ−/− livers after 12 weeks on high-fat diet.
Millward et al.Page 15
Diabetes. Author manuscript; available in PMC 2009 May 4.
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text:
Similar Artists: Yoshinori Monta & Junko Ohashi | Emmanuel Moire et Anne-Laure G | Igrata i Golemiq | 21st Street | Thomas Bergersen and Brian Tyler
Sorry, there aren't any upcoming shows for jeta faqolli, +add one?
423 upcoming shows @ 66 different spots
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107 ALL AGES shows!
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If you buy used kegs from any other source than distributors or breweries, they may be ill gotten ... don't sweat it.
I picked up mine off CL for$50 a piece. The guy had bought them off CL planning to convert to keggles, himself.
Until retired kegs are permanently marked, you'll never really know what you are buying.
Get them and clean them up. Enjoy your brew day...
"Make sure that the beer - four pints a week - goes to the troops under fire before any of the parties in the rear get a drop." -Winston Churchill to his Secretary of War, 1944
So I went over to the guys house to buy one or two of these kegs for our group. Turns out all of the kegs (except one) were full of dry concrete mix and weighed well over 150 lbs. I bought the one empty one for $25and will try to convert it.
Mick Arnett
North Liberty, Iowa
"Beer will change the world. I don't know how, but it will."
Wow; talk about leaving out a detail. There are always kegs on Craigslist; just keep an eye out for a good deal.


text:
COMMISSIONER'S OFFICE – Suspended N.Y. Yankee RHP Alfredo Aceves (Scranton/Wilkes-Barre-IL) 50 games after a second positive test for a drug of abuse in violation of the Minor League Drug Prevention and Treatment Program.
American League
CHICAGO WHITE SOX – Released LHP Scott Downs.
CLEVELAND INDIANS – Agreed to terms with 1B Michael Papi on a minor league contract.
HOUSTON ASTROS – Agreed to terms with SSs Juan Pineda and Ozziel Sanchez on minor league contracts.
LOS ANGELES ANGELS – Optioned RHP David Carpenter to Arkansas (TL).
NEW YORK YANKEES – Optioned INF Yangervis Solarte to Scranton/Wilkes-Barre (IL). Selected the contract of INF Zelous Wheeler from Scranton/Wilkes-Barre. Designated INF Dean Anna for assignment. Agreed to terms with RHP Edgmer Escalona on a minor league contract.
OAKLAND ATHLETICS – Designated LHP Jeff Francis for assignment. Reinstated LHP Eric O'Flaherty from the 60-day DL.
TAMPA BAY RAYS – Optioned LHP Jeff Beliveau to Durham (IL). Recalled 1B Vince Belnome from Durham.
National League
COLORADO ROCKIES – Optioned LHP Christian Friedrich to Colorado Springs (PCL). Reinstated 3B Nolan Arenado from the 15-day DL.
LOS ANGELES DODGERS – Optioned INF Carlos Triunfel to Albuquerque (PCL). Recalled INF Erisbel Arruebarrena from Albuquerque.
MIAMI MARLINS – Assigned OF Brent Keys outright to Jacksonville (SL). Placed RHP Tom Koehler on paternity leave. Reinstated LHP Brad Hand from the 15-day DL.
MILWAUKEE BREWERS – Sent RHP Jim Henderson to the AZL Brewers for a rehab assignment.
ST. LOUIS CARDINALS – Agreed to terms with RHPs Sebastian Nunez and Junior Fernandez, SS Starlin Balbuena and 2B Esequeil Delgado on minor league contracts.
SAN FRANCISCO GIANTS – Sent SS Ehire Adrianza to Fresno (PCL) for a rehab assignment.
WASHINGTON NATIONALS – Assigned 1B/3B Greg Dobbs outright to Syracuse (IL).
American Association
GARY SOUTHSHORE RAILCATS – Signed INF Donald Blunt.
ST. PAUL SAINTS – Signed RHP Mackenzie King. Released LHP Cole Nelson.
Frontier League
WASHINGTON WILD THINGS – Signed OF Jarod Jamerson.
NFL – Suspended Indianapolis WR LaVon Brazill for the 2014 season and Miami DE Dion Jordan and Kansas City OL Rokevious Watkins four games for violating the league's substance-abuse policy.
WINNIPEG BLUE BOMBERS – Released DB Korey Banks.
ARIZONA COYOTES – Named Darcy Regier senior vice president and assistant general manager and signed him to a multi-year contract.
BUFFALO SABRES – Signed D Tyson Strachan to a one-year contract.
CAROLINA HURRICANES – Agreed to terms with D Tim Gleason on a one-year contract and RW Jared Staal and D Michal Jordan on one-year, two-way contracts.
DALLAS STARS – Named Derek Laxdal coach of Texas (AHL).
TORONTO MAPLE LEAFS – Signed F Petri Kontiola to a one-year contract.
HAMILTON BULLDOGS – Signed F T.J. Hensick to a one-year contract.
READING ROYALS – Agreed to terms with F Kevin Walrod.
MLS – Suspended FC Dallas F Fabian Castillo four games and fined him an undisclosed amount for his actions during the June 11 game.
DARTMOUTH – Named women's rowing coach Wendy Bordeau senior associate athletics director.
EAST CAROLINA – Named Jeff Palumbo assistant baseball coach and recruiting coordinator.
MEMPHIS – Named Blake Smart men's golf coach.
PRINCETON – Named Brad Dexter men's assistant hockey coach.
RUTGERS – Named Mike O'Koren and Greg Vetrone men's assistant basketball coaches.
TCU – Named Ben Stapp women's assistant coach.


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There is a Joseph Rust of Nobleborough Maine who is son of Capt. Joseph Rust of Ipswich. He married Jane Weeks (dau. of John Weeks). Seems he would be the brother of Jonathan. (a "Lot Rust" in the area is their son). Interesting that Simeon is Capt. Joseph's age. Capt. Joseph was given a land grant after the Rev. War. which is why he migrated to Maine. I wonder if he and Simeon didn't come together to the area -? Simeon married a "Huston" who was one of the first families to have settled in the region.
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Democrats are paying heavily for their political failures - and for ditching the working stiff.
Yasmine Arrington was in high school when she founded the program ScholarCHIPS.
Ellen Cantarow reported on Israel and the West Bank from 1979 to 2009 for the Village Voice, Mother Jones, Inquiry, and Grand Street, among other publications. For the past four years she has been writing about the toll the oil and gas industries are taking on the environment. She views corporate penetrations of American land for fracking as colonization that is turning the US into a petro-state with impacts similar to those in, among other Third World countries, Nigeria.


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More Gas Tanks Vandalized In Sioux Falls
by Jill Johnson
January 17, 2013 10:44 AM
Police are looking for the person or persons responsible for several vandalisms in Sioux Falls. Police say they've gotten seven different reports of cars receiving damage to their fuel tanks.
Emily Sorenson says she noticed there was a problem Saturday morning.
Sorenson said, "When I was trying to turn on my vehicle I kinda felt like, it smelt like it was flooded."
Her car simply wouldn't start. Then, she noticed the puddle surrounding her car.
"It's a huge area of gas laying on the ground. You can tell it soaked into the asphalt," said Sorenson.
But it wasn't until the fire department came, that she found out what actually happened.
Sorenson said, "When I went to have it towed, that's when the fire department came because of the gas spill and they climbed underneath and took a look at it."
They discovered that someone had punctured holes into her fuel tank. And she isn't the only victim.
Since Jan. 8, police have recieved seven reports similar to Sorenson's, all vehicles in apartment complex's on the southwest side of town.
Vandalisms were reported on the 3000 block of South Louise Avenue, the 4700 block of South Oxbow Avenue, the 4200 block of South Theodore Avenue and the 4900 Block South Klein Avenue. Other reports include one on the 3300 Block of West 53rd and two on the 3300 block of West Valhalla.
Police say they can only speculate that it's all in an effort to steal gas and how they're collecting it is also just a guess.
So far, all of the vehicles that have been targeted have been larger vehicles and officials at Vern Eide say it could be because it allows for easier access.
Vern Eide Service Manager Gregg Allison said, "It's easier for them to craw underneath and it gives room in between the ground and the gas tank and the drill."
Allison says they have the task of fixing Sorenson's car, along with three other fuel tanks. Made of plastic, he says, it's an easy crime to commit.
"All you need is a cordless drill, looks like a 3/8 inch drill bit. It would take you all of about 20 seconds to drill through there," said Allison.
However, it's not an easy fix.
Allison said, "This fuel tank alone is about $1,100 without the labor to install it."
But it's not the money Sorenson is concerned about, she says the vandalisms are putting others at risk.
Sorenson said, "Even just the slightest static electricity could cause a spark. That's my concern. I just don't want anybody to get hurt."
Allison at Vern Eide says the fuel tanks that they've seen are being punctured on the bottom. However, one of them was drilled on the side. Not all of the gas had drained out of the tank and the man driving the vehicle didn't realize something was wrong until he got to the gas station and was putting fuel in his car.
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Helena Opid Modrzejewska, or Madame Modjeska as she was called, is a name most Nevadans wouldn't recognize. But, perhaps, it should be, as Nevadans, who are recalling key historical dates and individuals important in commemorating this year's 150th …Read More »
American Legion Community Outreach Volunteers: From military personnel stationed at NAS Fallon, veterans, retirees and community members, they all work together to make this a meaningful Thanksgiving for Churchill County seniors. Every Thanksgiving a …Read More »
On Friday, the Nevada 150 Commission received official verification the state set the record for the "Most People Singing A State Song at Once" with 45,553 Nevadans participating. As one of the celebrations' culminating events, #NevadaSings provided …Read More »
He was larger in life to many people who knew him, but for the family and friends of Virgil Getto, he was known as a down-to-earth man who left a legacy to his community. Getto, 90, a former state assemblyman and state senator, died Nov. 6 after a le …Read More »
For those who knew him in the state Legislature and of his leadership in rural Nevada politics, he was known as Mr. Getto. For those who worked with him in agriculture, he was a friend, a diligent, hard-working rancher. Yet, for those who knew him in …Read More »
A well-traveled, 72-year-old World War II veteran made a triumphant return the annual Veterans Day parade in Reno on Tuesday. A remodeled 1942 Dodge WC-54 3/4-Ton Army Ambulance, owned by the Department of Veterans Affairs and refurbished by the Neva …Read More »
In just four days, on Tuesday, Nov. 11, we will be commemorating Veterans Day, the annual federal holiday which honors America's military veterans who have served us so heroically and unflinchingly in times of peril. Army Gen. George S. Patton Jr., …Read More »
Gov. Brian Sandoval was re-elected to a second term Tuesday in a landslide against little-known Democrat Bob Goodman by one of the biggest margins in state history as a GOP wave rolled across Nevada and the nation. Sandoval's pick for lieutenant gove …Read More »
LAS VEGAS — Republicans riding a nationwide conservative wave blasted past their own wildest expectations Tuesday, seizing a broad majority in the Nevada Assembly and turning the legislative balance of power on its head. Republicans took 25 seats in …Read More »


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Book your room at Days Inn Milan/Cedar Point South
Double Room, 2 Double Beds, SmokingMax Occupancy: 5 guests (up to 4 children)
Standard Room, 1 Queen BedMax Occupancy: 4 guests (up to 3 children)
Double Room, 2 Double BedsMax Occupancy: 5 guests (up to 4 children)
Standard Room, 1 Queen BedMax Occupancy: 4 guests (up to 3 children)
Upgraded, 1 Queen Bed, Non SmokingMax Occupancy: 4 guests (up to 3 children)
Efficiency, Non SmokingMax Occupancy: 6 guests (up to 5 children)
Upgraded, Room, 1 King Bed, Non SmokingMax Occupancy: 2 guests (up to 1 child)
Standard Room, 1 King Bed, SmokingMax Occupancy: 2 guests (up to 1 child)
Hotel with an indoor pool, in Milan (Milan - Norwalk)
This hotel is located in Milan (Milan - Norwalk), close to Milan Historical Museum and The Edison Birthplace. Area attractions also include Thunderbird Hills Golf Courses and Keys Golf Course.
Hotel Features
At Days Inn Milan/Cedar Point South, recreational amenities include an indoor pool and a spa tub. The hotel also features complimentary wireless Internet access and a 24-hour business center.
Room Amenities
Plasma televisions come with cable channels. Guestrooms also feature complimentary wireless Internet access, air conditioning, and coffee/tea makers.Information missing or incorrect? Tell us!
Days Inn Milan/Cedar Point South
Family Friendly Amenities
- Indoor pool
- Free Wi-Fi
- Rollaway/extra beds available
- Laundry facilities
- Arcade/game room
- Cribs/infant beds (complimentary)
Hotel Amenities
In addition to an indoor pool, Days Inn Milan/Cedar Point South provides a spa tub and a fitness center. Public areas are equipped with complimentary high-speed wireless Internet access. Business-related amenities consist of a 24-hour business center and meeting rooms. A complimentary breakfast is offered each morning. 2 meeting rooms are available. This Milan hotel also offers an arcade/game room, complimentary newspapers in the lobby, and laundry facilities. Complimentary self parking is available on site.
- Number of meeting rooms - 2
- Meeting rooms
- Free Wi-Fi
- 24-hour business center
- Fitness facilities
- Total number of rooms - 66
- Number of floors - 3
- Number of buildings/towers - 1
- 24-hour front desk
- Arcade/game room
- ATM/banking
- Complimentary newspapers in lobby
- Free breakfast
- Express check-out
- Laundry facilities
- Free self parking
- Spa tub
- Indoor pool
Available in all rooms: Free Wi-Fi
Available in some public areas: Free Wi-Fi
Room Amenities
- Complimentary newspaper
- Coffee/tea maker
- Private bathroom
- Hair dryer
- Rollaway/extra beds available
- Free Wi-Fi
- Connecting/adjoining rooms available
- Cribs/infant beds (complimentary)
- Air conditioning
- Cable TV service
- Plasma TV
Where to Eat
Guests are offered a complimentary continental breakfast each morning.
Nearby Things to Do
An indoor pool and a spa tub are on site. Other recreational amenities include a fitness center.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Golfing nearby
- Personal motorized watercraft on site
Hotel Policies
Check-in time starts at 3 PM
Check-out time is 11 AM
Payment types
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- Rollaway bed fee: USD 10 per night
We should mention
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.