Addition of biologically active phorbol esters to intact quiescent I3T mouse cells stimulates an extremely rapid (detectable within seconds) phosphorylation of a Mr 80,000 cellular protein (termed "80k"). Phorbol 12,13-dibutyrate enhances 80k phosphorylation in a dose-dependent manner; half-maximal effect is obtained at 32 nM. The possibility that this phosphorylation is related to the activation of Ca'+-activated phospholipid-dependent protein kinase is suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with phospholipase C from Clostridium perfrngen8 or with platelet-derived growth factor, which is a potent activator of endogenous phospholipase C activity, also causes a rapid enhancement of 80k phosphorylation. Moreover, prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which leads to a marked decrease in the number of specific phorbol ester binding sites, prevents the phosphorylation of 80k stimulated by phorbol esters, phospholipase C, and platelet-derived growth factor. These findings provide evidence obtained with intact cells that implicate the stimulation of Ca2'-activated phospholipid-dependent protein kinase in the action of phorbol esters and other growth factors.Tumor promoters are compounds that, although not carcinogenic by themselves, increase the incidence of tumors when applied repeatedly to animals that have received a subthreshold dose of a carcinogen (1). Their mechanism of action is of considerable interest, with attention focusing on their biological effects in various types of cultured cells. In confluent and quiescent mouse 3T3 cells, biologically active phorbol esters bind to a single class of high-affinity sites (2, 3) and rapidly stimulate ion fluxes (4), enhance nucleoside and hexose uptake (5, 6), decrease the affinity of the surface receptors for epidermal growth factor (7-9), induce ornithine decarboxylase activity (10), and subsequently stimulate reinitiation of DNA synthesis when added with other mitogens in serum-free medium (2, 3, 10, 11). Although tumor promoters and hormonal peptides such as vasopressin (12) and bombesin (13) share pathways of action (3, 10, 13), the mechanism whereby occupancy of the phorbol ester receptors leads to the elicitation of their biological responses remains unknown and of considerable importance to gain insight into the molecular events leading to tumor promotion.A type of cyclic nucleotide-independent protein kinase has been discovered that is activated by association with membrane phospholipid& in the presence of Ca2+ (14, 15); the activity of the-membrane-associated enzyme is potently stimulated by unsaturated diacylglycerol (16). Recently, several reports indicate that biologically active phorbol esters can substitute. for diacylglycerol in stimulating the partially purified enzyme incubated in the presence of phospholipids (17-20). Similar observations were made with extracts prepared from Swiss 3T3 cells (our unpublished results). Whether or not these observations in vitro...

Obesity-induced insulin resistance is associated with both ectopic lipid deposition and chronic, low-grade adipose tissue inflammation. Despite their excess fat, obese individuals show lower fatty-acid oxidation (FAO) rates. This has raised the question of whether burning off the excess fat could improve the obese metabolic phenotype. Here we used humansafe nonimmunoreactive adeno-associated viruses (AAV) to mediate long-term hepatic gene transfer of carnitine palmitoyltransferase 1A (CPT1A), the key enzyme in fatty-acid b-oxidation, or its permanently active mutant form CPT1AM, to high-fat diet-treated and genetically obese mice. High-fat diet CPT1A-and, to a greater extent, CPT1AM-expressing mice showed an enhanced hepatic FAO which resulted in increased production of CO 2 , adenosine triphosphate, and ketone bodies. Notably, the increase in hepatic FAO not only reduced liver triacylglyceride content, inflammation, and reactive oxygen species levels but also systemically affected a decrease in epididymal adipose tissue weight and inflammation and improved insulin signaling in liver, adipose tissue, and muscle. Obesity-induced weight gain, increase in fasting blood glucose and insulin levels, and augmented expression of gluconeogenic genes were restored to normal only 3 months after AAV treatment. Thus, CPT1A-and, to a greater extent, CPT1AM-expressing mice were protected against obesity-induced weight gain, hepatic steatosis, diabetes, and obesity-induced insulin resistance. In addition, genetically obese db/db mice that expressed CPT1AM showed reduced glucose and insulin levels and liver steatosis. Conclusion: A chronic increase in liver FAO improves the obese metabolic phenotype, which indicates that AAV-mediated CPT1A expression could be a potential molecular therapy for obesity and diabetes. (HEPATOLOGY 2011;53:821-832)

Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH.