Zhong-Zhou Yang

Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis.

Protein kinase B is implicated in many crucial cellular processes, such as metabolism, apoptosis and cell proliferation. In contrast to Pkbα and Pkbβ-deficient mice, Pkbγ-/- mice are viable, show no growth retardation and display normal glucose metabolism. However, in adult Pkbγ mutant mice, brain size and weight are dramatically reduced by about 25%. In vivo magnetic resonance imaging confirmed the reduction of Pkbγ-/- brain volumes with a proportionally smaller ventricular system. Examination of the major brain structures revealed no anatomical malformations except for a pronounced thinning of white matter fibre connections in the corpus callosum. The reduction in brain weight of Pkbγ-/- mice is caused, at least partially, by a significant reduction in both cell size and cell number. Our results provide novel insights into the physiological role of PKBγ and suggest a crucial role in postnatal brain development.

Life with a single isoform of akt : Mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies

ABSTRACT To address the issues of isoform redundancy and isoform specificity of the Akt family of protein kinases in vivo, we generated mice deficient in both Akt2 and Akt3. In these mice, only the Akt1 isoform remains to perform essential Akt functions, such as glucose homeostasis, proliferation, differentiation, and early development. Surprisingly, we found that Akt2−/−Akt3−/− and even Akt1+/−Akt2−/−Akt3−/− mice developed normally and survived with minimal dysfunctions, despite a dramatic reduction of total Akt levels in all tissues. A single functional allele of Akt1 appears to be sufficient for successful embryonic development and postnatal survival. This is in sharp contrast to the previously described lethal phenotypes of Akt1−/−Akt2−/− mice and Akt1−/−Akt3−/− mice. However, Akt2−/−Akt3−/− mice were glucose and insulin intolerant and exhibited an ∼25% reduction in body weight compared to wild-type mice. In addition, we found substantial reductions in relative size and weight of the brain and testis in Akt2−/−Akt3−/− mice, demonstrating an in vivo role for both Akt2 and Akt3 in the determination of whole animal size and individual organ sizes.

Dosage-Dependent Effects of Akt1/Protein Kinase Bα (PKBα) and Akt3/PKBγ on Thymus, Skin, and Cardiovascular and Nervous System Development in Mice

ABSTRACT Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1− / − Akt3 +/ − mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1 +/ − Akt3 − / − mice survive normally. Double knockout (Akt1 − / − Akt3 − / −) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

PKBα is required for adipose differentiation of mouse embryonic fibroblasts

Protein kinase Bα (PKBα) is a key regulator of metabolism, proliferation and differentiation. We have explored the role of PKBα in adipogenesis using wild-type and PKBα-knockout mouse embryonic fibroblasts (MEFs) and show that lack of PKBα prevents MEF differentiation into adipocytes. Expression of ectopic PKBα in PKBα-deficient cells restores adipogenesis. We identified 80 genes whose expression was upregulated in wild-type MEFs during adipogenesis but whose expression was significantly reduced in PKBα-deficient MEFs under the same conditions. Significantly, the regulator of adipogenesis Krüppel-like transcription factor 15 gene expression was downregulated in PKBα-deficient MEFs but could be restored by expressing an active PKBα in the deficient cells. The level of lipocalin 2, renin 1 and receptor-activity-modifying protein 3 genes expressed by adipose cells was also decreased in PKBα-deficient MEFs, and are inhibited by LY294002 treatment during early adipocyte differentiation of 3T3-L1 cells. The results underscore an essential role for PKBα in the transcriptional program required for adipogenesis.

Epithelium-Mesenchyme Interactions Control the Activity of Peroxisome Proliferator-Activated Receptor β/δ during Hair Follicle Development

ABSTRACT Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARβ/δ- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARβ/δ protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARβ/δ-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARβ/δ during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

ABSTRACT Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.

NDR Kinases Are Essential for Somitogenesis and Cardiac Looping during Mouse Embryonic Development.

Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double null mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double null embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double null embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double null embryos. In addition, Ndr1/2-double null embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double null embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double null embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.