Analysis of the effect of a novel therapeutic for type 2 diabetes on the proteome of a muscle cell line.

Elevated serum retinol-binding protein (RBP) concentration has been implicated in the development of insulin resistance and type 2 diabetes. Two series of small molecules have been designed to lower serum levels by reducing secretion of the transthyretin-RBP complex from the liver and enhancing RBP clearance through the kidney. These small molecules were seen to improve glucose and insulin tolerance tests and to reduce body weight gain in mice rendered diabetic through a high fat diet. A proteomics study was conducted to better understand the effects of these compounds in muscle cells, muscle being the primary site for energy expenditure. One lead compound, RTC-15, is seen to have a significant effect on proteins involved in fat and glucose metabolism. This could indicate that the compound is having a direct effect on muscle tissue to improve energy homeostasis as well as a whole body effect on circulating RBP levels. This newly characterized group of antidiabetic compounds may prove useful in the treatment and prevention of insulin resistance and obesity.

The effect of retinol binding protein on the proteome of C2C12 muscle cells.

BACKGROUND: Retinol binding protein (RBP) and its membrane receptor, STRA6, are vital for the management of vitamin A in the body. Recently, elevated serum RBP levels have been implicated as a contributing factor to the development of insulin resistance and type 2 diabetes. However, conflicting opinions exist as to how these increased levels can cause insulin resistance. METHODS: In order to better understand the influences of RBP, a proteomic study was devised to determine the direct effect of RBP on a mouse muscle cell line, because the muscle is the principal site of insulin induced glucose uptake. C2C12 cells were treated with RBP for 16 h and the proteome analysed for alterations in protein abundance and phosphorylation by 2-DE. RESULTS: A number of changes were observed in response to retinol binding protein treatment, of which the most interesting were decreased levels of the phosphatase, protein phosphatase 1 ß. This phosphatase is responsible for regulating glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes involved in glycogen storage and utilization. Retinol binding protein treatment resulted in increased phosphorylation and inhibition of glycogen synthase, with detrimental effects on insulin stimulated glycogen production in these cells. CONCLUSION: The results indicate that RBP may have a negative effect on energy storage in the cell and could contribute to the development of insulin resistance in muscle tissue. Understanding how retinol binding protein influences insulin resistance may reveal novel strategies to target this disease.

A novel phospholipid-based drug formulation, VP025, modulates age- and LPS-induced microglial activity in the rat.

BACKGROUND: A common change that occurs with age in the central nervous system is an increase in microglial-associated inflammation. This is usually coupled with an increase in the concentration of the inflammatory cytokine interleukin-1beta (IL-1beta) in the hippocampus and an inhibition in long-term potentiation. OBJECTIVES: To assess the effects of a novel preparation of phospholipid nanoparticles incorporating phosphatidylglycerol, VP025, on inflammatory changes in hippocampus of aged and lipopolysaccharide (LPS)-treated rats. METHODS/RESULTS: We report that a possible initial target cell of the putative anti-inflammatory actions of VP025 may be macrophages, as VP025 is engulfed by, and has the capacity to alter the activity of, these cells. VP025 reversed the increase in IFN-gamma concentration in supernatant taken from peritoneal macrophages harvested from LPS-treated rats. In addition, markers of microglial activity, major histocompatibility complex class II (MHC II) mRNA expression, CD40 expression and IL-1beta concentration were increased, and CD200 expression was reduced, in the hippocampus of these rats. VP025 reversed changes in CD40, IL-1beta and CD200 in aged rats, and also restored long-term potentiation in aged and LPS-treated rats. CONCLUSIONS: We conclude that VP025 has the ability to modulate the activity of macrophage, microglia and neurons in response to stressors such as ageing and LPS treatment.

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice.

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

The deficit in long-term potentiation induced by chronic administration of amyloid-beta is attenuated by treatment of rats with a novel phospholipid-based drug formulation, VP025.

Amyloid-beta (Abeta) peptides, the primary component of the amyloid plaques in Alzheimer's disease (AD), exert profound effects on neurons in vitro and negatively impact on neuronal function in vivo. One of the consequences of increased Abeta in the brain, either as a result of overexpression of the precursor amyloid precursor protein in transgenic mice, or injection into the brain is a decrease in one form of synaptic plasticity, long-term potentiation (LTP) in the hippocampus. Here we investigated the effect of infusion of Abeta for 28 days on LTP in dentate gyrus of rats and demonstrate that it was profoundly decreased compared with control-treated rats. We show that this effect is accompanied by increased activity of caspase 3, which is an indicator of cell stress. Significantly these changes were attenuated in animals which were pretreated with particles incorporating phosphatidylglycerol (VP025) and the evidence indicated that even when treatment was given 2 weeks after the start of the Abeta infusion, VP025 was capable of attenuating Abeta-induced changes. The evidence suggests that activation of caspase 3 was mediated by an Abeta-induced increase in sphingomyelinase, with the subsequent production of ceramide which is known to have a detrimental effect on neuronal function.

Neuroprotective actions of eicosapentaenoic acid on lipopolysaccharide-induced dysfunction in rat hippocampus.

Eicosapentaenoic acid (EPA) protects hippocampus from age-related and irradiation-induced changes that lead to impairment in synaptic function; the evidence suggests that this is due to its anti-inflammatory effects, specifically preventing changes induced by the proinflammatory cytokine, interleukin-1beta (IL-1beta). In this study, we have investigated the possibility that EPA may prevent the effects of lipopolysaccharide (LPS) administration, which have been shown to lead to deterioration of synaptic function in rat hippocampus. The data indicate that treatment of hippocampal neurones with EPA abrogated the LPS-induced increases in phosphorylation of the mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), the transcription factor, c-Jun and the mitochondrial protein, Bcl-2. In parallel, we report that intraperitoneal administration of LPS to adult rats increases phosphorylation of JNK, c-Jun and Bcl-2 in hippocampal tissue and that these changes are coupled with increased IL-1beta concentration. Treatment of rats with EPA abrogates these effects and also blocks the LPS-induced impairment in long-term potentiation in perforant path-granule cell synapses that accompanies these changes. We propose that the neuroprotective effect of EPA may be dependent on its ability to inhibit the downstream consequences of JNK activation.

Neuroprotective effect of eicosapentaenoic acid in hippocampus of rats exposed to gamma-irradiation.

Exposure to irradiation leads to detrimental changes in several cell types. In this study we assessed the changes induced in hippocampus by exposure of rats to whole body irradiation; the findings reveal that irradiation leads to apoptotic cell death in hippocampus, and as a consequence, long term potentiation in perforant path-granule cell synapses is markedly impaired. The evidence is consistent with the view that irradiation induced an increase in reactive oxygen species and that this leads to stimulation of the stress-activated protein kinase, JNK, and activation of the transcription factor, c-Jun. Consequent upon activation of JNK, a cascade of cell signaling events was stimulated that ultimately resulted in apoptosis, as suggested by parallel increases in cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end labeling staining. Treatment of rats with eicosapentaenoic acid inhibited the irradiation-induced increase in reactive oxygen species production and the subsequent cellular signaling events, suggesting that oxidative stress triggered apoptotic cell death in the hippocampus of rats exposed to irradiation. Significantly, when the compromise in cell viability induced by irradiation was prevented by eicosapentaenoic acid, long term potentiation was sustained in a manner similar to that in the sham-treated control group.

Apoptotic changes in the aged brain are triggered by interleukin-1beta-induced activation of p38 and reversed by treatment with eicosapentaenoic acid.

Among the several changes that occur in the aged brain is an increase in the concentration of the proinflammatory cytokine interleukin-1beta that is coupled with a deterioration in cell function. This study investigated the possibility that treatment with the polyunsaturated fatty acid eicosapentaenoic acid might prevent interleukin-1beta-induced deterioration in neuronal function. Assessment of four markers of apoptotic cell death, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end staining, revealed an age-related increase in each of these measures, and the evidence presented indicates that treatment of aged rats with eicosapentaenoate reversed these changes as well as the accompanying increases in interleukin-1beta concentration and p38 activation. The data are consistent with the idea that activation of p38 plays a significant role in inducing the changes described since interleukin-1beta-induced activation of cytochrome c translocation and caspase-3 activation in cortical tissue in vitro were reversed by the p38 inhibitor SB203580. The age-related increases in interleukin-1beta concentration and p38 activation in cortex were mirrored by similar changes in hippocampus. These changes were coupled with an age-related deficit in long term potentiation in perforant path-granule cell synapses, while eicosapentaenoate treatment was associated with reversal of age-related changes in interleukin-1beta and p38 and with restoration of long term potentiation.