Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis

C/EBPalpha is highly expressed in liver and regulates many genes that are preferentially expressed in liver. Because C/EBPalpha-null mice die soon after birth, it is impossible to analyze the function of C/EBPalpha in the adult with this model. To address the function of C/EBPalpha in adult hepatocytes, liver-specific C/EBPalpha-null mice were produced using a floxed C/EBPalpha allele and the albumin-Cre transgene. Unlike whole body C/EBPalpha-null mice, mice lacking hepatic C/EBPalpha expression did not exhibit hypoglycemia, nor did they show reduced hepatic glycogen in adult. Expression of liver glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase remained at normal levels. However, these mice exhibited impaired glucose tolerance due in part to reduced expression of hepatic glucokinase, and hyperammonemia from reduced expression of hepatic carbamoyl phosphate synthase-I. These mice also had reduced serum cholesterol and steatotic livers that was exacerbated with aging. This phenotype could be explained by increased expression of hepatic lipoprotein lipase and reduced expression of microsomal triglyceride transfer protein, apolipoproteins B100, and A-IV. These data demonstrate that hepatic C/EBPalpha is critical for ammonia detoxification and glucose and lipid homeostasis in adult mice.     

C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.     

Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene.

To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-).delta(-/-) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPbeta(-/-).delta(-/-) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPbeta(-/-).delta(-/-) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-).delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrated that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta.     

c-Myc is a critical target for c/EBPalpha in granulopoiesis

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.     

Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1alpha or PGC-1beta coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1alpha and PGC-1beta were deficient. These data show that PGC-1alpha is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis.     

Essential role of insulin receptor substrate 1 in differentiation of brown adipocytes

The most widely distributed members of the family of insulin receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of IRS-1 in adipocyte biology, we established brown adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals. Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and lipid accumulation (10 to 40%) compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO cells showed decreased expression of adipogenic marker proteins, such as peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha (C/EBPalpha), fatty acid synthase, uncoupling protein-1, and glucose transporter 4. The differentiation deficit in the KO cells could be reversed almost completely by retrovirus-mediated reexpression of IRS-1, PPARgamma, or C/EBPalpha but not the thiazolidinedione troglitazone. Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various stages of the differentiation process revealed a strong and transient activation in IRS-1, IRS-2, and phosphotyrosine-associated PI 3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed impaired phosphotyrosine-associated PI 3-kinase activation, all of which was associated with IRS-2. Akt phosphorylation was reduced in parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase with LY294002 blocked differentiation of wild-type cells. Thus, IRS-1 appears to be an important mediator of brown adipocyte maturation. Furthermore, this signaling molecule appears to exert its unique role in the differentiation process via activation of PI 3-kinase and its downstream target, Akt, and is upstream of the effects of PPARgamma and C/EBPalpha.     

Ablation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and brown adipose tissues

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) improves insulin sensitivity during aging. Compared to wild-type (WT) mice, old Ghsr(-/-) mice have reduced fat and preserve a healthier lipid profile. Old Ghsr(-/-) mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS-R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS-R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age-associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS-R antagonist abolishes ghrelin's effects and increases UCP1 expression. Hence, GHS-R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging-associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.