Molecular mechanisms in the brain involved in the anorexia of branched-chain amino acid deficiency.

The anterior piriform cortex (APC) of the rat is thought to be the site of indispensable amino acid (IAA) chemosensation in the brain. The branched-chain amino acids, including leucine, are among the IAA that are recognized in the APC. The behavioral outcome of IAA deficiency is an anorectic response. The specific transduction mechanisms by which IAA deficiency and repletion activate the APC are not fully understood, but clearly phosphorylation of proteins, increases in intracellular calcium, and expression of the immediate early gene c-fos, which are among the earliest events occurring after the initial drop in the concentration of the limiting IAA, cause stimulation in the APC. Subsequently, several neurotransmitter systems, including those for norepinephrine, GABA, serotonin, dopamine and nitric oxide, are activated in the APC of rats that have consumed an IAA-imbalanced diet. These systems appear to modulate the output cells from the APC, glutamatergic pyramidal cells that send neural signals to activate subsequent relays in the brain. Ultimately, the feeding circuits of the brain carry out the anorectic response. Continued consumption of a diet containing an IAA imbalance causes a conditioned taste aversion to the diet in all animals that have been studied. Such learning involves synaptic reorganization, requiring both degradation and synthesis of protein, along with alterations in genomic activity.

Augmented insulinotropic action of arachidonic acid through the lipoxygenase pathway in the obese Zucker rat.

OBJECTIVE: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose-stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. RESEARCH METHODS AND PROCEDURES: Islets were isolated from 10- to 12-week-old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12-lipoxygenase metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), was measured by enzyme immunoassay. RESULTS: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (<11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA-induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12-lipoxygenase, esculetin (0.5 microM), inhibited AA-stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12-HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. DISCUSSION: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12-lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.

Increased intracellular calcium in rat anterior piriform cortex in response to threonine after threonine deprivation.

The anterior piriform cortex (APC) may serve as the chemosensor for amino acid (AA) deficiency in rats. To investigate the mechanism by which the APC recognizes a limiting indispensable AA (IAA), we examined changes in intracellular calcium ([Ca2+]i) in APC slices after culture in medium with or without threonine (Thr) or lysine (Lys). The addition of 1 or 10 mM Thr to slices previously incubated in Thr-devoid medium resulted in a significant and sustained increase in [Ca2+]i compared to control slices; an effect not seen when isoleucine, another IAA, was added. Similar results were seen when lysine, but not threonine, was added to slices incubated in lysine-devoid medium. The rise in [Ca2+]i resulting from the addition of the limiting IAA to deficient slices may be linked to enhanced activity of the appropriate AA transporter. This is suggested by preliminary findings that serine, a small neutral AA that uses the same transporter as threonine, gave rise to an enhanced response in the Thr-deficient slice.

A low protein diet lowers islet insulin secretion but does not alter hyperinsulinemia in obese Zucker (fa/fa) rats.

The effects of feeding a low protein diet on pancreatic insulin secretion early in life in genetically lean and obese Zucker rats were studied. Four-week-old lean (Fa/Fa) and obese (fa/fa) Zucker rats were fed either a 5% protein or a 20% protein diet for 3 wk. Rats were killed at 7 wk of age. Pancreatic islets were isolated and insulin response of islets was measured in a 60-min static incubation under one of the following conditions: 2.7 mmol/L glucose +/- 10 mmol/L arginine, 8.3 mmol/L glucose +/- 10 mmol/L arginine, and 16.7 mmol/L glucose. Serum insulin was significantly lower (P < or = 0.05) in lean, but not in obese rats fed low protein compared with those fed the normal protein diet. Interestingly, obese rats fed the low protein diet had the highest plasma glucose concentrations (P < or = 0.05) among the four groups. Feeding the low protein diet reduced insulin secretion of islets from lean rats by 70%, and from obese rats by 30 to 50% compared with rats fed the normal protein diet. Insulin content of islets was reduced significantly in lean rats fed the low protein diet (by 70%) and in obese rats (by 50%). A short-term protein deficiency early in life reduced in vitro insulin secretion of islets from both lean and obese rats. However, reductions in insulin content and insulin release from islets resulting from low protein feeding were not sufficient to alter hyperinsulinemia in genetically obese Zucker rats.

Effect of culture in vitro with eicosatetraenoic (20:4(n-6) ) and eicosapentaenoic (20:5(n-3) ) acids on fatty acid composition, prostaglandin synthesis and chemiluminescence of rat peritoneal macrophages.

Rat peritoneal macrophages were cultured in either eicosatetraenoic acid (20:4(n-6) ) or eicosapentaenoic acid (20:5(n-3) ) and the effects on phospholipid fatty acids, prostaglandin synthesizing capacity and the ability of the macrophages to show chemiluminescence were examined. Chemiluminescence is an activity resulting from the synthesis of reactive oxygen species. It has been reported that prostaglandins inhibit this activity. The fatty acid profile of the four major phospholipids reflected the fatty acid component of the medium. Macrophages cultured in 20:4(n-6) synthesized twice the prostaglandin produced by controls and those cultured in 20:5(n-3) synthesized 10% that of controls and 5% that of 20:4(n-6)-cultured cells. Macrophages cultured with 20:4(n-6) for 12 h showed half the chemiluminescence of those cultured with 20:5(n-3), while those cultured with 20:4(n-6) for 24 h showed 10% the chemiluminescence of 20:5(n-3)-cultured cells. Addition of the prostaglandin synthase inhibitor, indomethacin, had no effect on chemiluminescence.

Modulation of prostaglandin synthesis in rat peritoneal macrophages with omega-3 fatty acids.

In view of the findings that omega 3 fatty acids inhibit the synthesis of prostaglandins (PG) from arachidonic acid (20:4 omega 6) and that among immunologically active cells, the macrophage is a major producer of PG, we undertook a study of the effect of dietary alpha-linolenic acid (18:3 omega 3) on PG synthesis in the macrophage. Rats were fed purified diets containing either 10% corn oil (CO) or linseed oil (LO), providing either a low (1/32) or high (3.5/1) ratio of 18:3 omega 3 to 18:2 omega 6, respectively, for 6 weeks. Fatty acid analysis of macrophage phospholipids showed that there was an appreciable increase in the percentage of omega 3 fatty acids and a decrease in the omega 6 fatty acids in macrophages from rats fed the LO diet. The changes in fatty acid composition were associated with a significant decrease in the synthesis of prostaglandin E (PGE) by macrophages from rats fed the LO diet. Macrophages from rats fed the 2 dietary oils did not differ in their ability to degrade PG, thus the difference in PG production appeared to be a consequence of decreased synthesis only. The dietarily induced changes in PGE synthesis were readily overcome in vitro by culturing macrophages with complexes of fat-free bovine serum albumin and either 20:4 omega 6 or 20:5 omega 3.

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.

Archaebacteria.

Experimental work published elsewhere has shown that the Archaebacteria encompass several distinct subgroups including methanogens, extreme halophiles, and various thermoacidophiles. The common characteristics of Archaebacteria known to date are these: (1) the presence of characteristic tRNAs and ribosomal RNAs; (2) the absence of peptidoglycan cell walls, with in many cases, replacement by a largely proteinaceous coat; (3) the occurrence of ether linked lipids built from phytanyl chains and (4) in all cases known so far, their occurrence only in unusual habitats. These organisms contain a number of 'eucaryotic features' in addition to their many bacterial attributes. This is interpreted as a strong indication that the Archaebacteria, while not actually eucaryotic, do indeed represents a third separate, line of descent as originally proposed.

Are extreme halophiles actually "bacteria"?

Comparative cataloging of the 16SrRNA of Halobacterium halobium indicates that the organism did not arise, as a halophilic adaptation, from some typical bacterium. Rather, H. halobium is a member of the Archaebacteria, an ancient group of organisms that are no more related to typical bacteria than they are to eucaryotes.

Classification of methanogenic bacteria by 16S ribosomal RNA characterization.

The 16S ribosomal RNAs from 10 species of methanogenic bacteria have been characterized in terms of the oligonucleotides produced by T(1) RNase digestion. Comparative analysis of these data reveals the methanogens to constitute a distinct phylogenetic group containing two major divisions. These organisms appear to be only distantly related to typical bacteria.

An ancient divergence among the bacteria.

The 16S ribosomal RNAs from two species of methanogenic bacteria, the mesophile Methanobacterium ruminantium and the thermophile Methanobacterium thermoautotrophicum, have been characterized in terms of the oligonucleotides produced by digestion with T1 ribonuclease. These two organisms are found to be sufficiently related that they can be considered members of the same genus or family. However, they bear only slight resemblance to "typical" Procaryotic genera; such as Escherichia, Bacillus and Anacystis. The divergence of the methanogenic bacteria from other bacteria may be the most ancient phylogenetic event yet detected--antedating considerably the divergence of the blue green algal line for example, from the main bacterial line.