High Fat Diet (HFD)-induced obesity is a major contributor to diabetes and cardiovascular disease, but the underlying genetic mechanisms are poorly understood. Here, we use Drosophila to test the hypothesis that HFD-induced obesity and associated cardiac complications have early evolutionary origins involving nutrient-sensing signal transduction pathways. We find that HFD-fed flies exhibit increased triglyceride (TG) fat and alterations in insulin/glucose homeostasis, similar to mammalian responses. A HFD also causes cardiac lipid accumulation, reduced cardiac contractility, conduction blocks and severe structural pathologies, reminiscent of diabetic cardiomyopathies. Remarkably, these metabolic and cardiotoxic phenotypes elicited by HFD are blocked by inhibiting insulin-TOR signaling. Remarkably, reducing insulin-TOR activity by TSC1-2, 4EBP, FOXO) or increasing lipase expression in the myocardium suffices to efficiently alleviate cardiac fat accumulation and dysfunction induced by HFD. We conclude that deregulation of insulin-TOR signaling due to a HFD is responsible for mediating the detrimental effects on metabolism and heart function.

We have introduced a variety of amino acid substitutions into carboxyl-terminal CAIA2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic[Vall2]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the Al position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (==50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAXsignaled modifications may have different contributions to the function of different CAAX-containing proteins.An association with the plasma membrane is critical for Ras transforming activity (1-3), and this association is promoted by a series of three closely linked posttranslational modification steps signaled by the consensus carboxyl-terminal CA1A2X motif (C = cysteine; A = any aliphatic amino acid; X = any amino acid) present in all Ras proteins. Addition of the 15-carbon farnesyl isoprenoid to the cysteine of the CAAX sequence is followed by proteolytic removal of the AAX residues and carboxyl methylation of the now terminal cysteine residue. Mutant Ras proteins lacking either the cysteine or the AAX residues are completely blocked in processing and are cytosolic and completely nontransforming. Posttranslational processing is critical for Ras function, but the precise contribution of each of the three CAAXsignaled processing steps to Ras membrane association and transforming activity remains to be established. The enzymes responsible for Ras processing are now beginning to be characterized. A cytosolic Ras farnesyltransferase activity, identified in both mammalian (4-6) and yeast (7, 8) cells, requires recognition of only the CAAX sequence to farnesylate the cysteine residue. In contrast to farnesyltransferase, the enzymatic activities for the AAX proteolysis (9) and carboxyl methylation (9-12) steps have been detected in the membrane component offractionated cells and tissues.In vitro studies with both synthetic peptides and chimeric Ras proteins have provided details of the sequence requirements for Ras farnesyltransferase modification. The residue at the A1 position can vary, while a much more restricted set of A2 and X residues permits efficient isoprenoid modification (13-15). The X residue also specifies whether the protein is modified by a farnesyl or by a geranylgeranyl group ...

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Reducing insulin/IGF signaling allows for organismal survival during periods of inhospitable conditions by regulating the diapause state, whereby the organism stockpiles lipids, reduces fertility, increases stress resistance, and has an increased lifespan. The Target of Rapamycin (TOR) responds to changes in growth factors, amino acids, oxygen tension, and energy status; however, it is unclear how TOR contributes to physiological homeostasis and disease conditions. Here, we show that reducing the function of Drosophila TOR results in decreased lipid stores and glucose levels. Importantly, this reduction of dTOR activity blocks the insulin resistance and metabolic syndrome phenotypes associated with increased activity of the insulin responsive transcription factor, dFOXO. Reduction in dTOR function also protects against age-dependent decline in heart function and increases longevity. Thus, the regulation of dTOR activity may be an ancient "systems biological" means of regulating metabolism and senescence, that has important evolutionary, physiological, and clinical implications.