Proteomic analysis of livers from fat-fed mice deficient in either PKCδ or PKCε identifies Htatip2 as a regulator of lipid metabolism.

Insulin resistance contributes to the development of Type 2 diabetes, and is associated with lipid oversupply. Deletion of isoforms of the lipid-activated protein kinase C (PKC) family, PKCδ or PKCε, improves insulin action in fat-fed mice, but differentially affects hepatic lipid metabolism. To investigate the mechanisms involved, we employed an in vivo adaptation of SILAC to examine the effects of a fat diet together with deletion of PKCδ or PKCε on the expression of liver proteins. We identified a total of 3359 and 3488 proteins from the PKCδ and PKCε knockout study groups, respectively, and showed that several enzymes of lipid metabolism were affected by the fat diet. In fat-fed mice, 23 proteins showed changes upon PKCδ deletion while 19 proteins were affected by PKCε deletion. Enzymes of retinol metabolism were affected by the absence of either PKC. Pathway analysis indicated that monosaccharide metabolism was affected only upon PKCδ deletion, while isoprenoid biosynthesis was affected in a PKCε-specific manner. Certain proteins were regulated inversely, including HIV-1 tat interactive protein 2 (Htatip2). Overexpression or knockdown of Htatip2 in hepatocytes affected fatty acid storage and oxidation, consistent with a novel role in mediating the differential effects of PKC isoforms on lipid metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000971 (http://proteomecentral.proteomexchange.org/dataset/PXD000971).

Protein Kinase C Epsilon Deletion in Adipose Tissue, but Not in Liver, Improves Glucose Tolerance.

Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCɛ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCɛ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCɛ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCɛ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCɛ activation in this tissue should be interpreted with caution. However, PKCɛ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.

A proteome map of murine heart and skeletal muscle.

The balance of hypertrophy and atrophy is critical for the adaptation of cardiac and skeletal muscle mass to the demands of the environment and when deregulated can cause disease. Here we have used a proteomics approach to generate protein reference maps for the mouse heart and skeletal muscle, which provide a molecular basis for future functional and pathophysiological studies. The reference map provides information on molecular mass, pI, and literature data on function and localization, to facilitate the identification of proteins based on their migration in 2-D gels. In total, we have identified 351 cardiac and 284 skeletal muscle protein spots, representing 249 and 214 different proteins, respectively. In addition, we have visualized the protein pattern of mouse heart and skeletal muscle at defined conditions comparing knockout (KO) animals deficient in the sarcomeric protein titin (a genetic atrophy model) and control littermates. We found 20 proteins that were differently expressed linking titin's kinase region to the heat-shock- and proteasomal stress response. Taken together, the established reference maps should provide a suitable tool to relate protein expression and PTM to cardiovascular and skeletal muscle disease using the mouse as an animal model.

Cardiac hypertrophy and reduced contractility in hearts deficient in the titin kinase region.

BACKGROUND: Titin is a giant protein crucial for the assembly and elasticity of the sarcomere. Recently, titin has been linked to signal transduction through its kinase domain, which has been proposed to sense mechanical load. We developed a knockout in which expression of M-line-deficient titin can be induced in adult mice and investigated the role of the titin kinase region in cardiac function. METHODS AND RESULTS: Isolated heart experiments revealed that in titin M-line-deficient mice, the contractile response to beta-adrenergic agonists and extracellular calcium is reduced. However, the Ca2+ sensitivity and cooperativity of activation of skinned cardiac muscle were unchanged. In knockout mice, calcium transients showed a reduced rate of calcium uptake, and expression analysis showed reduced levels of calmodulin, phospholamban, and SERCA2. Ultimately, knockout mice developed cardiac hypertrophy and heart failure, which involves protein kinase C signal transduction but not the mitogen-activated protein kinase pathway. CONCLUSIONS: The titin kinase region emerges as a regulator of contractile function through effects on calcium handling and hypertrophy through protein kinase signal transduction. These novel functions of titin might provide a rationale for future therapeutic approaches to attenuate or reverse symptoms of heart failure.