Amino acid transporters and nutrient-sensing mechanisms: new targets for treating insulin-linked disorders?

The IIS (insulin/IGF (insulin-like growth factor) signalling) cascade has an important role in regulating normal development and physiology, as evidenced by its effects in a host of major human diseases including cancer, Type 2 diabetes and neurodegeneration. Recently, it has become clear that multiple types of local nutrient-sensing mechanisms have an impact on cellular insulin-sensitivity through the downstream kinase TOR (target of rapamycin). In vivo analysis in flies has surprisingly highlighted PATs (proton-assisted amino acid transporters) as having a uniquely potent role in regulating IIS/TOR activity and growth, potentially via a novel signalling mechanism. Other molecules such as the heterodimeric amino acid transporter, CD98, which provides the principal route for cellular uptake of leucine, an amino acid implicated in regulating TOR, also appear to have important effects. As our understanding of how nutrient sensing has an impact on IIS/TOR increases, novel targets to modulate aberrant IIS in disease are likely to emerge, which could complement current strategies designed to block kinases in this pathway.

Cationic amino acid transport through system y+L in erythrocytes of patients with lysinuric protein intolerance.

We test the hypothesis that lysinuric protein intolerance (LPI), a rare autosomal recessive defect of cationic amino acid transport, results from the absence of the recently described y+L amino acid transporter. We compare fluxes of lysine (1 microM) into erythrocytes of normal subjects with those of patients homozygous for the LPI mutation. No significant differences in fluxes through system y+L in normal or LPI cells were found, excluding the possibility that system y+L cannot be expressed in patients with LPI. Reasons for supposing that there may be tissue-specific processing of two recently described genes encoding the y+L transporter are discussed. Polymerase chain reaction measurement of expression of these two genes in an erythroleukemic cell line suggests that alternatively there may be an as-yet-unidentified additional member of this gene family.

Dipeptide transport in brush-border membrane vesicles (BBMV) prepared from human full-term placentae.

The uptakes of the tritiated, hydrolysis-resistant cationic (d-Phe-L-Lys), neutral (D-Phe-L-Ala) and anionic (D-Phe-L-Glu) peptides into human full-term placental brush-border membrane vesicles (BBMV) were time-dependent and into an osmotically-active space. Uptakes of D-Phe-L-Lys and D-Phe-L-Glu were temperature-dependent. Uptake of D-Phe-L-Lys was electroneutral (either cation exchange or anion co-transport), whereas D-Phe-L-Ala and D-Phe-L-Glu were both stimulated by an increasingly inside-positive membrane potential (explained by either cation exchange or anion co-transport, or translocation alone, respectively). Uptake of D-Phe-L-Ala was stimulated (approximately 50 per cent) by an inwardly-directed proton gradient (pHin = 7.4, pHout = 5.5), whereas D-Phe-L-Glu was unaffected, and D-Phe-L-Lys uptake was inhibited (approximately 50 per cent) but was unaffected by the organic cation-exchange inhibitors 1,1-diethyl-2,2-cyanine (decynium22) and 5-(N-methyl-N-isobutyl)amiloride (MIBA). Over the concentration range studies, the peptides did not self-inhibit, and the only cross-inhibition was by D-Phe-L-Glu on D-Phe-L-Lys uptake (estimated K(I) 24.2 +/- 1.36 mM), suggesting very low affinity transporter(s). Under conditions favouring its transport by PepT1, D-Phe-L-Glu uptake was unaffected by diethylpyrocarbonate (DEPC); neither D-Phe-L-Ala nor D-Phe-L-Lys was inhibited by DEPC under maximally proton-stimulated conditions of uptake. We conclude that Pep-T-like transporters are not responsible for peptide uptake into human placental BBMV; while the molecular identity of the transporter(s) involved remains unclear, we hypothesize that they could be similar to the as yet unidentified epithelial basolateral peptide transporter(s).

Reduction of central epinephrine concentrations is consistent with the continued occurrence of ovulation in rats treated with an inhibitor (LY 134046) of phenylethanolamine N-methyltransferase.

Groups of 4-day cyclic rats were injected (i.p.) with LY 134046 (50 mg/kg), a central inhibitor of phenylethanolamine N-methyltransferase, or saline at 09.00, 13.00 and 19.00 h on the day of proestrus. The incidence of ovulation was examined the following estrous morning. There was no difference in the number of ova in drug-treated animals compared to saline-treated controls. In other groups of 4-day cyclic rats, LY 134046 or saline was injected daily at 10.00 h for 5 consecutive days from proestrus to proestrus inclusive. The animals were decapitated the following day and ova were counted. Epinephrine concentrations were determined by radioenzymatic assay in the mediobasal hypothalamus (MBH) and the medial preoptic area (MPOA). All saline-treated controls and 10/14 of the drug-treated animals had ovulated, while epinephrine concentrations in the MBH and MPOA had been reduced by 95.8 and 94.7%, respectively, compared to saline-treated controls. These experiments suggest that a significant surge of luteinizing hormone occurs to initiate ovulation even after a severe reduction in central epinephrine concentration has taken place.

Evidence that central epinephrine neurons participate in the control and regulation of neuroendocrine events during the estrous cycle.

Although evidence has shown that central epinephrine (E) neurons play an essential role in the control of preovulatory gonadotropoin surge in rats, their function and site(s) of action are unknown. These experiments were performed in an attempt to identify any changes in E concentration or activity that might take place in areas of the brain known to receive adrenergic axon terminals and to be associated with increased output of gonadotropins (LH, FSH, and PRL) during the estrous cycle. E concentrations were measured by radioenzymatic assay, and E activity was assessed by the linear rate of decline of E (RDE) which occurs 2 h after administration of the centrally active E synthesis inhibitor, SKF 64139. During the proestrous critical period (1500-1700 h), significant increases in both concentration and RDE occurred in the medial preoptic area (mPOA) accompanied by a smaller but significant increase in the RDE in the mediobasal hypothalamus (MBH); 4 h later (2100-2300 h), significant increases in both concentration and RDE were seen in the MBH. At estrus, although E concentrations were generally higher in both the mPOA and MBH than on other days of the cycle, the concentration and RDE in the MBH increased significantly between 1500-1700 h, while RDE in the mPOA increased again between 1700-1900 h. There were no significant changes in either E concentration or RDE in the mPOA or MBH at metestrus or diestrus or in the perifornical area at any of the times studied. Thus, these findings may be associated with the output of gonadotropins over the periovulatory period.

Adrenaline concentration and turnover in the arcuate nucleus and median eminence during the critical period in the rat.

Previous work has shown a relatively high turnover of adrenaline in the mediobasal hypothalamus during the critical period (15.00-17.00 h) of the proestrous rat. We now report that this high level of adrenergic activity can be detected in the median eminence (turnover rate 1.62 +/- 0.36 pg/micrograms protein/h) rather than the arcuate nucleus (turnover rate 0.18 +/- 0.32 pg/micrograms protein/h). In addition the median eminence was isolated as medial and lateral components and determination of catecholamine concentrations revealed a greater proportion of adrenaline (A) (59%) in the lateral median eminence whereas a larger proportion of dopamine (60%) was found in medial median eminence.

Effects of manipulating serotonin on the incidence of ovulation in the rat.

Previous studies have shown that while depletion of brain serotonin by the administration of p-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, blocks the daily surge of LH in oestrogen-treated ovariectomized rats, restoration of serotonin synthesis by treatment with its immediate precursor, 5-hydroxytryptophan (5-HTP), at a critical time of day, reinstates the surge. The present study indicates that the experimental procedure involving serotonin depletion and its subsequent replenishment may also be used to control the preovulatory LH surge and ovulation in intact cyclic rats provided that (1) the PCPA is administered subcutaneously rather than intraperitoneally nad (2) the 5-HTP is given in conjunction with carbidopa, a peripheral decarboxylase inhibitor: the latter observation providing further evidence for a central role for serotonin in the control of ovulation. These precautions were unnecessary when oestrogen was administered at the same time as the PCPA. It appears that PCPA administered intraperitoneally results in a suppression of the preovulatory rise in oestrogen secretion (and may have additional deleterious effects at the level of the ovaries) and that 5-HTP, in the absence of supplementary oestrogen, may block ovulation by peripheral action after conversion to serotonin. This study indicates the need for caution when using pharmacological 'cocktails' to investigate neuroendocrine events underlying ovulation when the experiments are carried out in the presence of the ovaries.