Ingestion of lean meat elevates muscle inositol hexakisphosphate kinase 1 protein content independent of a distinct post-prandial circulating proteome in young adults with obesity.

BACKGROUND: We have recently shown that a novel signalling kinase, inositol hexakisphosphate kinase 1 (IP6K1), is implicated in whole-body insulin resistance via its inhibitory action on Akt. Insulin and insulin like growth factor 1 (IGF-1) share many intracellular processes with both known to play a key role in glucose and protein metabolism in skeletal muscle. AIMS: We aimed to compare IGF/IP6K1/Akt signalling and the plasma proteomic signature in individuals with a range of BMIs after ingestion of lean meat. METHODS: Ten lean [Body mass index (BMI) (in kg/m2): 22.7 ±â€¯0.4; Homeostatic model assessment of insulin resistance (HOMAIR): 1.36 ±â€¯0.17], 10 overweight (BMI: 27.1 ±â€¯0.5; HOMAIR: 1.25 ±â€¯0.11), and 10 obese (BMI: 35.9 ±â€¯1.3; HOMAIR: 5.82 ±â€¯0.81) adults received primed continuous L-[ring-13C6]phenylalanine infusions. Blood and muscle biopsy samples were collected at 0 min (post-absorptive), 120 min and 300 min relative to the ingestion of 170 g pork loin (36 g protein and 5 g fat) to examine skeletal muscle protein signalling, plasma proteomic signatures, and whole-body phenylalanine disappearance rates (Rd). RESULTS: Phenylalanine Rd was not different in obese compared to lean individuals at all time points and was not responsive to a pork ingestion (basal, P = 0.056; 120 & 300 min, P > 0.05). IP6K1 was elevated in obese individuals at 120 min post-prandial vs basal (P < 0.05). There were no acute differences plasma proteomic profiles between groups in the post-prandial state (P > 0.05). CONCLUSIONS: These data demonstrate, for the first time that muscle IP6K1 protein content is elevated after lean meat ingestion in obese adults, suggesting that IP6K1 may be contributing to the dysregulation of nutrient uptake in skeletal muscle. In addition, proteomic analysis showed no differences in proteomic signatures between obese, overweight or lean individuals.

Inositol Hexakisphosphate Kinase 2 is a Presymptomatic Biomarker for Amyotrophic Lateral Sclerosis.

OBJECTIVE: Inositol hexakisphosphate kinase 2 (InsP6K2), an enzyme that converts inositol hexakisphosphate (InsP6) to diphosphoinositol pentakisphosphate (InsP7), induces cell death. InsP6K2 is abundant in the central nervous system, especially anterior horn cells of spinal cord. To identify the role of InsP6K2 in amyotrophic lateral sclerosis (ALS), we investigated the expression levels of InsP6K2 in transgenic mice expressing mutant superoxide dismutase-1 (SOD1) (mSOD1 Tg mice). METHODS: The specimens of spinal cords were obtained from mSOD1 Tg mice and age-matched wild-type mice. We investigated the expression of InsP6K2 at the gene and protein levels of the spinal cord in mSOD1 Tg and wild-type mice. RESULTS: The gene expression levels of InsP6K2 in mSOD1 Tg mice was significantly higher than that in wild-type mice before ALS symptoms developed. In immunohistochemistry and western blotting results showed that InsP6K2 translocated from the nucleus to the cytoplasm in mSOD1 Tg mice. CONCLUSION: These findings suggest that InsP6K2 activates in mSOD1 Tg mice before the onset of ALS. Therefore, InsP6K2 might be a presymptomatic biomarker for ALS.

Phosphomevalonate kinase is a cytosolic protein in humans.

In the past decade, a predominant peroxisomal localization has been reported for several enzymes functioning in the presqualene segment of the cholesterol/isoprenoid biosynthesis pathway. More recently, however, conflicting results have been reported raising doubts about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study, we have determined the subcellular localization of human phosphomevalonate kinase using a variety of biochemical and microscopic techniques, including conventional subcellular fractionation studies, digitonin permeabilization studies, immunofluorescence, and immunoelectron microscopy. We found an exclusive cytosolic localization of both endogenously expressed human phosphomevalonate kinase (in human fibroblasts, human liver, and HEK293 cells) and overexpressed human phosphomevalonate kinase (in human fibroblasts, HEK293 cells, and CV1 cells). No indication of a peroxisomal localization was obtained. Our results do not support a central role of peroxisomes in isoprenoid biosynthesis.

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells.

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from Catharanthus roseus suspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis-Menten kinetics with respect to ATP and their specific substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n = 3) is always less than 2.5% and in between determinations (n = 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay.

Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell.

The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids. The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter. Polyphosphatase, a polyphosphate depolymerase, was similarly expressed by using the arabinose-inducible PBAD promoter. The ability of cells containing these constructs to produce active enzymes only when induced was confirmed by polyphosphate extraction, enzyme assays, and RNA analysis. The inducer concentrations giving optimal expression of each enzyme were determined. Experiments were performed in which ppk was induced early in growth, overproducing PPK and allowing large amounts of polyphosphate to accumulate (80 mumol in phosphate monomer units per g of dry cell weight). The ppx gene was subsequently induced, and polyphosphate was degraded to inorganic phosphate. Approximately half of this polyphosphate was depleted in 210 min. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells and was secreted into the medium, leading to a down-regulation of the phosphate-starvation response. In addition, the steady-state polyphosphate level was precisely controlled by manipulating the degree of ppx induction. The polyphosphate content varied from 98 to 12 mumol in phosphate monomer units per g of dry cell weight as the arabinose concentration was increased from 0 to 0.02% by weight.