The mobilization of stored lipid by hormones is a fundamental function of fat cells, and there is strong evidence that perilipin (Plin), a lipid droplet scaffold, and adipose tissue triglyceride lipase (Atgl), a triglyceride-specific lipase, play critical roles. Previous work suggested that Abhd5, a protein activator of Atgl, coordinates with Plin in controlling basal and stimulated lipolysis; however, the underlying mechanism is controversial. Growing evidence indicates that hormone-stimulated lipolysis involves protein trafficking and specific protein-protein interactions at the surface of lipid droplets (LDs), 2 and there is strong evidence that perilipin A (Plin), a lipid droplet scaffold protein, plays a central role in orchestrating interactions among lipolytic effector proteins (1, 2). For example, phosphorylated Plin provides a docking site by which phosphorylated hormone-sensitive lipase (HSL) gains access to substrates at the surface of LDs (3-5). Adipose triglyceride lipase (Atgl, also known as desnutrin, Pnpla2) also participates in protein kinase A (PKA)-stimulated lipolysis (6, 7), and Plin is thought to be involved in this regulation (8); however, the mechanisms involved are not understood, and proposed models that address this regulation disagree.Unlike HSL, Atgl is not a direct target of PKA phosphorylation, and therefore, the mechanism of its activation must be indirect. One model whereby Plin could indirectly regulate Atgl activity is by controlling availability of its co-activator, Abhd5 (also known as CGI-58), in a manner that depends on PKA-dependent phosphorylation of Plin (3). According to this "sequestration-release" model, Plin sequesters Abhd5 in the basal state, thereby preventing activation of Atgl and suppressing basal lipolysis. PKA activation leads to Plin phosphorylation, release of Abhd5, and subsequent activation of Atgl. This mechanism is supported by biochemical and dynamic imaging experiments demonstrating that Abhd5 binds Plin in the unstimulated state, and PKA-mediated phosphorylation of Plin leads to rapid release of Abhd5 from Plin (3, 9 -11). Additionally, knockdown of Abhd5 reduces both basal and stimulated lipolysis (11, 12), although whether this effect involves interactions with Plin has been questioned (12).Although these data are consistent with the general model, there are no data demonstrating that 1) the interaction of Abhd5 with Plin and Atgl is mutually exclusive (that is, Plin suppresses the interaction of Abhd5 with Atgl) or that 2) phosphorylation of Plin promotes the physical interaction of Abhd5 with Atgl. In fact, an alternative model has been described whereby Atgl regulates basal but not stimulated lipolysis (1). According to this model, basal lipolysis is stimulated by a ternary complex containing Plin, Abhd5, and Atgl. Furthermore, the model proposes that Abhd5 is not involved in PKA-stimulated activation of Atgl because it is released into the cytoplasm upon Plin phosphorylation.In the experiments detailed below, we examined the functional interact...

Obesity. 2006;14:1905-1913. Objective: Human adenovirus 36 (Ad-36) increases adiposity and reduces serum lipids in chicken, mouse, and nonhuman primate models, and it is linked to obesity in seroepidemiological studies in humans. Involvement of the central nervous system (CNS) or adipose tissue in the mechanism of Ad-36-induced adiposity is unknown. The effects of Ad-36 on adiposity and on the neuroendocrine system were investigated in a rat model. Research Methods and Procedures: Five-week-old male Wistar rats were inoculated intraperitoneally with Ad-36 or medium. Results: Despite similar food intakes, infected rats attained significantly greater body weight and fat pad weight by 30 weeks post-inoculation. Epididymal-inguinal, retroperitoneal, and visceral fat pad weights of the infected group were greater by 60%, 46%, and 86%, respectively (p Ͻ 0.00001). The fasting serum insulin level and homeostasis model assessment index indicated greater insulin sensitivity in the infected group. Visceral adipose tissue expression of glycerol 3-phosphate dehydrogenase, peroxisome proliferatoractivated receptor ␥, and CCAAT/enhancer-binding protein ␣ and ␤ was markedly increased in the infected animals compared with controls. Ad-36 decreased norepinephrine levels significantly in the paraventricular nucleus in infected vs. control rats (mean Ϯ standard error, 8.9 Ϯ 1.1 vs. 12.8 Ϯ 1.2 pg/g protein; p Ͻ 0.05). Ad-36 markedly decreased serum corticosterone in infected vs. control rats (mean Ϯ standard error, 97 Ϯ 41.0 vs. 221 Ϯ 111 ng/mL; p Ͻ 0.005). Discussion: The results suggest that the pro-adipogenic effect of Ad-36 may involve peripheral as well as central effects. The male Wistar rat is a good model for the elucidation of metabolic and molecular mechanisms of Ad-36-induced adiposity.

Objective: Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process. Design and Results: Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-b and peroxisome proliferator-activated receptor g2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted. Conclusion: Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

OBJECTIVE-Experimental infection of rats with human adenovirus type 36 (Ad-36) promotes adipogenesis and improves insulin sensitivity in a manner reminiscent of the pharmacologic effect of thiozolinediones. To exploit the potential of the viral proteins as a therapeutic target for treating insulin resistance, this study investigated the ability of Ad-36 to induce metabolically favorable changes in human adipose tissue. RESEARCH DESIGN AND METHODS-We determinedwhether Ad-36 increases glucose uptake in human adipose tissue explants. Cell-signaling pathways targeted by Ad-36 to increase glucose uptake were determined in the explants and human adipose-derived stem cells. Ad-2, a nonadipogenic human adenovirus, was used as a negative control. As a proof of concept, nondiabetic and diabetic subjects were screened for the presence of Ad-36 antibodies to ascertain if natural Ad-36 infection predicted improved glycemic control.RESULTS-Ad-36 increased glucose uptake by adipose tissue explants obtained from nondiabetic and diabetic subjects. Without insulin stimulation, Ad-36 upregulated expressions of several proadipogenic genes, adiponectin, and fatty acid synthase and reduced the expression of inflammatory cytokine macrophage chemoattractant protein-1 in a phosphotidylinositol 3-kinase (PI3K)-dependent manner. In turn, the activation of PI3K by Ad-36 was independent of insulin receptor signaling but dependent on Ras signaling recruited by Ad-36. Ad-2 was nonadipogenic and did not increase glucose uptake. Natural Ad-36 infection in nondiabetic and diabetic subjects was associated with significantly lower fasting glucose levels and A1C, respectively. CONCLUSIONS-Ad-36 proteins may provide novel therapeutic targets that remodel human adipose tissue to a more metabolically favorable profile.

Objective: Human adenovirus Ad-36 causes adiposity in animal models and shows association with human obesity. Ad-36 enhances differentiation of 3T3-L1 and human preadipocytes, without cell lysis, a characteristic that may contribute to its adipogenic effect observed in vivo. Ad-2, another human adenovirus is nonadipogenic in animals and in 3T3-L1 cells and shows no correlation with human obesity. The objective of this study was to determine the adipogenic roles of viral mRNA and DNA, which may explain the differential effects of Ad-36 and Ad-2 on preadipocyte differentiation. Methods: This study determined the duration of selected Ad-36 gene expression in 3T3-L1 cells, and the effect on preadipocytes differentiation, when Ad-36 gene expression was attenuated by Cidofovir, an antiadenoviral agent. Results:The results showed that Ad-36, but not Ad-2, expresses viral mRNA. Ad-36 gene expression peaked at 2-4 days postinoculation and very low levels persisted after day 7. Despite the viral mRNA expression, Ad-36 infection of 3T3-L1 cells was abortive as indicated by a progressive decrease in viral DNA quantity. Attenuation of Ad-36 mRNA expression by Cidofovir reduced the adipogenic effect of the virus. Conclusion: In conclusion, viral mRNA expression, although transient, is a prerequisite for enhancing differentiation of preadipocytes by Ad-36. Viral DNA replication was not required for the effect. This is the first evidence for the role of gene expression of an adipogenic human virus in enhancing preadipocytes differentiation. This study provides the basis for further understanding novel regulatory modulators of preadipocytes differentiation.

Several metabolic abnormalities are associated with relative excess or deficiency of adipose tissue. Identifying the regulators of adipogenic differentiation is critical for its successful manipulation. Ad36, a human adenovirus, is a novel factor that promotes adipogenesis. We exploited the adipogenic potential of Ad36 to reveal exogenous modifiers of adipogenesis in rodent preadipocyte cell line in the presence or absence of differentiation inducers methyl‐isobutyl‐xanthine, dexamethasone, and insulin (M, D, and I; MDI). A nonadipogenic human adenovirus Ad2 was used as a negative control for viral infection. First, we confirmed that, Ad36, but not Ad2, increases lipid accumulation in the presence or absence of MDI. Time‐course studies for expression of key genes of adipogenic cascade showed that it is Ad36, but not Ad2, which downregulated preadipocyte marker gene Wnt10b, and upregulated expression of early (C/EBPΔ and C/EBPβ), intermediate (PPARγ2), and late genes (aP2 and G3PDH) of adipogenic cascade even in the absence of MDI. In the presence of MDI, onset of expression of adipogenic genes coincided for Ad36 and control groups, but the expressions were significantly greater for the Ad36 group. Next, we observed that attenuation of Ad36 mRNA expression by an antiadenoviral agent reduced 3T3‐L1 differentiation, indicating that viral mRNA expression is required for the process. Furthermore, with or without MDI or its components, Ad36 significantly increased lipid accumulation in 3T3‐L1 cells. Cell confluency at the time of Ad36 infection positively influenced lipid accumulation. The results reveal that Ad36 is an MDI‐independent exogenous regulator of the adipogenic process. Elucidating the molecular pathways involved may reveal novel regulatory controls of adipogenesis.

Experimental infection with human adenovirus Ad‐36, not Ad‐2, increases adiposity in animals and natural infection shows association with human obesity. In rodent and human adipocyte progenitor cultures, Ad‐36 induces but Ad‐2 reduces lipid accumulation despite viral gene expression. To identify the mechanism for anti‐lipogenic effect, the effects of Ad‐2 on time course of adipogenic gene expression cascade up to 7 days post infection was determined in absence or presence of adipogenic inducers‐ methyl isobutyl xanthine, dexamethasone and insulin (MDI). Ad‐36 was used as an adipogenic positive control. Ad‐2 infected groups had significantly lower lipid accumulation even in the presence of MDI. By acting downstream of C/EBPβ, Ad‐2 appears to abort the adipogenic gene expression cascade initiated by MDI and thus inhibit lipid accumulation. The findings provide novel insight in control of down regulation of cellular pathways of adipogenesis. Funded by NIH R‐01 DK066164. Changes in gene expression and lipid accumulation by Ad‐36 and Ad‐2 compared to uninfected control groups, in 3T3‐L1 preadipocytes expressing CAR‐receptor for efficient viral entry. p < .05 for all changes noted. ND: no difference vs uninfected control.

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