Obesity is frequently associated with systemic insulin resistance, glucose intolerance, and hyperlipidemia. Impaired insulin action in muscle and paradoxical diet/ insulin-dependent overproduction of hepatic lipids are important components of obesity, but their pathogenesis and inter-relationships between muscle and liver are uncertain. We studied two murine obesity models, moderate high-fatfeeding and heterozygous muscle-specific PKC-l knockout, in both of which insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but conserved in liver. In both models, activation of hepatic sterol receptor element binding protein-1c (SREBP-1c) and NFkB (nuclear factorkappa B), major regulators of hepatic lipid synthesis and systemic insulin resistance, was chronically increased in the fed state. In support of a critical mediatory role of aPKC, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP-1c and NFkB, and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Moreover, in high-fat-fed mice, impaired insulin signaling to IRS-1-dependent phosphatidylinositol 3-kinase, PKB/Akt and aPKC in muscle and hyperinsulinemia were largely reversed. In obesity, conserved hepatic aPKC-dependent activation of SREBP-1c and NFkB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic insulin resistance. Accordingly, hepatic aPKC is a potential target for treating obesityassociated abnormalities. Supplementary key words atypical protein kinase C • high fatObesity, particularly when accompanied by systemic insulin resistance, glucose intolerance, and hyperlipidemia (i.e., a "metabolic syndrome") is a global health problem and a frequent forerunner of type 2 diabetes mellitus. Whereas both exogenous/diet-induced and genetically determined obesity can produce insulin resistance and metabolic syndrome features, vice versa, systemic insulin resistance can produce obesity and metabolic syndrome features. However, mechanisms underlying lipid abnormalities and insulin resistance in these situations, and the critical interplay between muscle and liver, are poorly understood.The high-fat-fed (HFF) mouse model is useful for studying diet-induced obesity-related insulin resistance. In our experience, feeding mice a Western-type 20% milk highfat diet for 3-4 weeks leads to diminished insulin activation of phosphatidylinositol (PI) 3-kinase (PI3K) effectors, atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt) in muscle (1, 2), with little or no effect on hepatic aPKC and PKB/Akt activation (1). In this HFF model, we have observed no increases in basal (unstimulated) activities of conventional (a,b2) or novel (y,y) PKCs in liver, despite observing increases in muscle (unpublished). Accordingly, our HFF model may partly differ from others wherein higher dietary fat was used, thereby activating hepatic novel ...

Studies have suggested that the expression, translocation, and function of a4b2 nicotinic receptors may be modulated by a4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on a4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human a4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type b2 subunits into SH-EP1 cells. Cells were pre-incubated with 32 Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated a4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two a4 protein bands, a major band of 71/75 kDa and a minor band of 80/ 85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the a4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.

Neuronal nicotinic receptor α4 subunits associated with nicotinic α4β2 receptors are phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human α4 and/or β2 subunits were incubated with 32 Pi, and PKA or PKC were activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing α4β2 receptors or only α4 subunits were used to identify free α4 subunits, and α4 subunits present in immature α4β2 complexes and mature α4β2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of α4 subunits associated with mature pentamers was 3 times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free α4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature α4β2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated β2 subunits associated with mature α4β2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation α4β2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of α4β2 receptors. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2010 February 18. Published in final edited form as:Neuroscience. (Zoli et al., 1995). These α4β2 receptors participate in numerous biochemical and physiological processes, have been implicated in several neurological and behavioral disorders including nocturnal frontal lobe epilepsy and Alzheimer's disease, and may be responsible for the rewarding and addictive effects of nicotine (Picciotto et al., 2001;Tapper et al., 2004).Studies have suggested that the expression and function of α4β2 receptors are regulated posttranslationally through phosphorylation/dephosphorylation mechanisms involving both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (Rothhut et al., 1996;Eilers et al., 1997;Gopalakrishnan et al., 1997;Fenster et al., 1999;Jeanclos et al., 2001;Nashmi et al., 2003;Exley et al., 2006). Initial studies using M10 fibroblasts stably transfected with chicken α4 and β2 su...

No abstract