Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Pro- and anti-epileptic roles of microglia.

Microglia are brain-resident immune cells that contribute to the maintenance of brain homeostasis. In the epileptic brain, microglia show various activation phenotypes depending on the stage of epileptogenesis. Therefore, it remains unclear whether microglial activation acts in a pro-epileptic or anti-epileptic manner. In mesial temporal lobe epilepsy, one of the most common form of epilepsies, microglia exhibit at least two distinct morphologies, amoeboid shape and ramified shape. Amoeboid microglia are often found in sclerotic area, whereas ramified microglia are mainly found in non-sclerotic area; however, it remains unclear whether these structurally distinct microglia share separate roles in the epileptic brain. Here, we review the roles of the two distinct microglial phenotypes, focusing on their pro- and anti-epileptic roles in terms of inflammatory response, regulation of neurogenesis and microglia-neuron interaction.

Modulation of rat behaviour by using a rat-like robot.

In this paper, we study the response of a rat to a rat-like robot capable of generating different types of behaviour (stressful, friendly, neutral). Experiments are conducted in an open-field where a rat-like robot called WR-4 is put together with live rats. The activity level of each rat subject is evaluated by scoring its locomotor activity and frequencies of performing rearing (rising up on its hind limbs) and body grooming (body cuddling and head curling) actions, whereas the degree of preference of that is indicated by the robot-rat distance and the frequency of contacting WR-4. The moving speed and behaviour of WR-4 are controlled in real-time based on the feedback from rat motion. The activity level and degree of preference of rats for each experimental condition are analysed and compared to understand the influence of robot behaviour. The results of this study show that the activity level and degree of preference of the rat decrease when exposed to a stressful robot, and increase when the robot exhibit friendly behaviour, suggesting that a rat-like robot can modulate rat behaviour in a controllable, predictable way.

Role of KLF15 in regulation of hepatic gluconeogenesis and metformin action.

OBJECTIVE: An increase in the rate of gluconeogenesis is largely responsible for the hyperglycemia in individuals with type 2 diabetes, with the antidiabetes action of metformin being thought to be achieved at least in part through suppression of gluconeogenesis. RESEARCH DESIGN AND METHODS: We investigated whether the transcription factor KLF15 has a role in the regulation of gluconeogenesis and whether KLF15 participates in the antidiabetes effect of metformin. RESULTS: Here we show that KLF15 regulates the expression of genes for gluconeogenic or amino acid-degrading enzymes in coordination with the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha. Liver-specific ablation of KLF15 in diabetic mice resulted in downregulation of the expression of genes for gluconeogenic or amino acid catabolic enzymes and in amelioration of hyperglycemia. Exposure of cultured hepatocytes to metformin reduced the abundance of KLF15 through acceleration of its degradation and downregulation of its mRNA. Metformin suppressed the expression of genes for gluconeogenic or amino acid-degrading enzymes in cultured hepatocytes, and these effects of metformin were attenuated by restoration of KLF15 expression. Administration of metformin to mice inhibited both the expression of KLF15 and glucose production in the liver, the latter effect also being attenuated by restoration of hepatic KLF15 expression. CONCLUSIONS: KLF15 plays an important role in regulation of the expression of genes for gluconeogenic and amino acid-degrading enzymes and that the inhibitory effect of metformin on gluconeogenesis is mediated at least in part by downregulation of KLF15 and consequent attenuation of the expression of such genes.

Detection and prediction of drowsiness by reflexive eye movements.

A reliable predictor of drowsiness using objective measures is desirable for machine and vehicle operations in which human errors may cause fatal accidents. We have evaluated the Vestibulo-Ocular Reflex (VOR) as a possible predictor of drowsiness. The VOR is a compensatory eye movement that stabilizes retinal image during head motion, and is inevitably induced by vibration in a car running on the road. We employed an uneventful driving simulation (DS) featuring vibration stimulation to induce both drowsiness and VOR in healthy human subjects. VOR performance was characterized by its gain and variability, and evaluated in relation to the subjects' drowsiness. A significant decrease in VOR gain and increase in variability accompanied subjective sleepiness, with the changes occurring before subjects became aware of sleepiness. From this finding, we developed a reliable method (88.9% accuracy) to predict oncoming sleepiness using changes in VOR performance as a cue.

Comparative enzymatic analysis of azoreductases from Bacillus sp. B29.

We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23 kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 degrees C (AzrB) and 55 degrees C (AzrC), and retained activities up to 55 degrees C (AzrB) and 50 degrees C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV).

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.

Role of hepatic STAT3 in the regulation of lipid metabolism.

Regulation of hepatic gene expression is largely responsible for the control of nutrient metabolism. We previously showed that the transcription factor STAT3 regulates glucose homeostasis by suppressing the expression of gluconeogenic genes in the liver. However, the role of STAT3 in the control of lipid metabolism has remained unknown. We have now investigated the effects of hepatic overexpression of STAT3, achieved by adenovirus-mediated gene transfer, on glucose and lipid metabolism in insulin-resistant diabetic mice. Forced expression of STAT3 reduced blood glucose and plasma insulin concentrations as well as the hepatic abundance of mRNA for phosphoenolpyruvate carboxykinase. However, it also increased the plasma levels of triglyceride and total cholesterol without affecting those of low density lipoprotein- or high density lipoprotein-cholesterol. The hepatic abundance of mRNAs for fatty acid synthase and acetyl-CoA carboxylase, both of which catalyze the synthesis of fatty acids, was increased by overexpression of STAT3, whereas that of mRNAs for sterol regulatory element-binding proteins 1a, 1c, or 2 was unaffected. Moreover, the amount of mRNA for acyl-CoA oxidase, which contributes to beta-oxidation, was decreased by forced expression of STAT3. These results indicate that forced activation of STAT3 signaling in the liver of insulin-resistant diabetic mice increased the circulating levels of atherogenic lipids through changes in the hepatic expression of genes involved in lipid metabolism. Furthermore, these alterations in hepatic gene expression likely occurred through a mechanism independent of sterol regulatory element-binding proteins.

An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp.: functional expression and enzymatic characterization.

The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.

Restoration of glucokinase expression in the liver normalizes postprandial glucose disposal in mice with hepatic deficiency of PDK1.

Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase-dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes-like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.

An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region.

Aquabirnaviruses, represented by Infectious pancreatic necrosis virus (IPNV), have been isolated from epizootics in salmonids and a variety of aquatic animals in the world; six genogroups of aquabirnaviruses have been identified. In comparisons of nucleotide sequences of the VP2/NS junction region, maximum nucleotide diversities of 30.8 % were observed among 93 worldwide aquabirnavirus isolates. A phylogenetic tree revealed the existence of a new genogroup, VII, for Japanese aquabirnavirus isolates from marine fish and molluscan shellfish. Nucleotide diversities between genogroups VII and I-VI were 18.7 % or greater. At the nucleotide level, Japanese IPNV isolates from epizootics in salmonids were nearly identical to a genogroup I strain from the USA or Canada. It is suggested that Japanese IPNV isolates belonging to genogroup I were originally introduced from North American sources, whereas Japanese aquabirnavirus isolates of genogroup VII were from marine aquatic animals indigenous to Japan.

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins.

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.

An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.

The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2'. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea. The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The k(cat)/ K(m) ratio increased with basic residues at P4 or P6, mainly due to a lower K(m) rather than an increase in k(cat). On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli-expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.

Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions.

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.