Amino acid homeostasis is a target of metformin therapy.

OBJECTIVE: Unexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect. METHODS: We used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material. RESULTS: In cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective doses in vivo and in vitro observed in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells. CONCLUSIONS: Metformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.

Effect of chronic administration of 17ß-estradiol on the vasopressor responses induced by the sympathetic nervous system in insulin resistance rats.

Several studies have demonstrated that the underlying mechanism of insulin resistance (IR) is linked with developing diseases like diabetes mellitus, hypertension, metabolic syndrome, and polycystic ovary syndrome. In turn, the dysfunction of female gonadal hormones (especially 17ß-estradiol) may be related to the development of IR complications since different studies have shown that 17ß-estradiol has a cardioprotector and vasorelaxant effect. This study aimed was to determine the effect of the 17ß-estradiol administration in insulin-resistant rats and its effects on cardiovascular responses in pithed rats. Thus, the vasopressor responses are induced by sympathetic stimulation or i.v. bolus injections of noradrenaline (α1/2), methoxamine (α1), and UK 14,304 (α2) adrenergic agonist were determined in female pithed rats with fructose-induced insulin resistance or control rats treated with: 1) 17ß-estradiol or 2) its vehicle (oil) for 5 weeks. Thus, 17ß-estradiol decreased heart rate, prevented the increase of blood pressure induced by ovariectomy, but with the opposite effect on sham-operated rats; and decreased vasopressor responses induced by i.v. bolus injections of noradrenaline on sham-operated (control and fructose group) and ovariectomized (control) rats, and those induced by i.v. bolus injections of methoxamine (α1 adrenergic agonist). Overall, these results suggest 17ß-estradiol has a cardioprotective effect, and its effect on vasopressor responses could be mediated mainly by the α1 adrenergic receptor. In contrast, IR with ovariectomy 17ß-estradiol decreases or loses its cardioprotector effect, this could suggest a possible link between the adrenergic receptors and the insulin pathway.

Spinal TASK-1 and TASK-3 modulate inflammatory and neuropathic pain.

This study assessed the participation of spinal TWIK-related acid-sensitive K+ channels 1 and 3 (TASK-1 and TASK-3) in inflammatory (formalin test) and neuropathic (spinal nerve ligation, SNL) pain in rats. Intrathecal pre-treatment (-10 min) with the TASK-1 blocker ML365 or TASK-3 blocker PK-THPP, but not vehicle, enhanced in a dose-dependent manner 1% formalin-induced acute and long-lasting secondary mechanical allodynia and mechanical hyperalgesia in rats. In contrast, intrathecal pre-treatment with terbinafine, an activator of TASK-3, reduced formalin-induced flinching and allodynia/hyperalgesia. Both blockers and terbinafine had similar effects on female and male rats. In addition, intrathecal injection of ML365 or PK-THPP blocked the terbinafine-induced antiallodynic effect in neuropathic rats, but they did not modify baseline withdrawal threshold in naïve or sham-operated rats. TASK-1 and TASK-3 mRNA and protein were expressed in L4 and L5 dorsal root ganglia (DRG) and dorsal and ventral spinal cord of naïve animals. Interestingly, formalin injection increased TASK-1 expression in ipsilateral L5 DRG, but not in the spinal cord. Moreover, formalin injection transiently enhanced TASK-3 expression in ipsilateral L5 DRG and dorsal spinal cord. In contrast, SNL down-regulated TASK-3 expression in the ipsilateral L4 and L5 DRG but not in dorsal or ventral spinal cord, while SNL did not modify TASK-1 expression at any tissue. The pharmacological and molecular results suggest that TASK-1 and TASK-3 have a relevant antinociceptive role in inflammatory and neuropathic pain.

Fructose-Induced Insulin Resistance as a Model of Neuropathic Pain in Rats.

Peripheral neuropathy is one of the main complications of diabetes. The pathogenesis of this affectation is not completely understood. Several studies refer to hyperglycemia as the principal cause of diabetic neuropathy. Nonetheless, there are changes in the expression of insulin receptor during the progress of diabetic neuropathy, suggesting that this disorder begins before high glucose blood levels are established. In this study, we investigated fructose-induced insulin resistance as a model of neuropathic pain. Insulin resistance was induced by 15% fructose in drinking water for 16 weeks. Fructose slightly enhanced blood glucose levels. In contrast, chronic fructose increased insulin plasma levels and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index. Moreover, fructose induced hyperalgesia (to 0.5% formalin) and tactile allodynia. Interestingly, gabapentin and metformin, but not diclofenac, reversed in a dose-dependent manner fructose-induced tactile allodynia. Fructose enhanced activating factor transcription 3 (ATF3), but not caspase-3 and α2δ-1 subunit, in individual L4 and L5 dorsal root ganglia (DRG) and sciatic nerve. Chronic fructose also increased anoctamin-1 and ASIC3 whereas it reduced insulin receptor-ß, α5GABAA receptors and TASK-3 channels protein expression in DRG and sciatic nerve. In contrast, fructose did not change TRPV1 channel protein expression. Treatment with metformin for 4 weeks reversed some of the fructose-induced changes in protein expression. Taken together, these data suggest that insulin resistance induced by fructose reproduces several aspects of neuropathic-like pain. Our data also suggest that nociceptive hypersensitivity in this model is due to the modulation of several ionic channels at the primary afferent neurons.

Pharmacological evaluation of metformin and N-benzylbiguanide, a novel analogue of metformin, on the vasopressor responses to adrenergic system stimulation in pithed rats with fructose-induced insulin resistance.

Metformin has been associated with cardioprotection, vasorelaxation and normalization of endothelial function during type 2 Diabetes Mellitus. However, few studies have analysed its effects on vascular adrenergic system. Our study has evaluated the vasopressor responses induced by sympathetic stimulation or by i.v. bolus injections of the agonists noradrenaline (α1/2), methoxamine (α1) and UK 14,304 (α2) in rats with fructose-induced insulin resistance chronically pretreated with either metformin or EGL-6M (N-benzylbiguanide), a novel analogue of metformin. Rats were treated with fructose (15%) or tap water (control) during 16 weeks. Next, both groups were treated daily during 4 weeks with: (1) vehicle; (2) metformin (50mg/kg); or (3) EGL-6M (50mg/kg). Blood glucose and plasma insulin were determined before and after administration of glucose during oral glucose tolerance test. Animals treated with fructose showed hyperinsulinemia and insulin resistance, which were decreased by metformin and EGL-6M. In animals treated with fructose, the vasopressor responses induced by: (1) sympathetic stimulation were decreased; (2) noradrenaline were increased; and (3) methoxamine and UK 14,304 remained unaffected compared with control group. In control animals, metformin failed to modify the vasopressor responses analysed, while EGL-6M increased the vasopressor responses to sympathetic stimulation. In rats treated with fructose, metformin decreased vasopressor response to noradrenaline but did not modify the sympathetic stimulation responses. EGL-6M increased the vasopressor responses to sympathetic stimulation without modifying those to noradrenaline, methoxamine or UK 14,304. Collectively, these data suggest that EGL-6M is capable to increase insulin sensitivity and the vasopressor sympathetic outflow in rats.

Pharmacological evidence that NaHS inhibits the vasopressor responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats.

It has been reported that i.v. administration of NaHS, a donor of H2S, elicited dose-dependent hypotension although the mechanisms are not completely understood. In this regard, several mechanisms could be involved including the inhibition of the vasopressor sympathetic outflow. Thus, this study was designed to determine the potential capability of NaHS to mediate inhibition of the vasopressor responses induced by preganglionic sympathetic stimulation. For this purpose, Wistar rats were anaesthetised, pithed and cannulated for drug administration. In animals pre-treated with gallamine, the effect of i.v. infusion of NaHS (310 and 560µg/kgmin) or its vehicle (phosphate buffer) was determined on the vasopressor responses induced by: (1) sympathetic stimulation (0.03-10Hz); (2) i.v. bolus injections of exogenous noradrenaline (0.03-3µg/kg); or (3) methoxamine (1-100µg/kg). The vasopressor responses induced by preganglionic sympathetic stimulation were dose-dependently inhibited by i.v. infusion of NaHS (310 and 560µg/kgmin), but not by vehicle, particularly at high frequencies. In marked contrast, the vasopressor responses to exogenous noradrenaline or methoxamine were not inhibited by the above doses of NaHS or its vehicle. The above results, taken together, demonstrate that NaHS inhibited the vasopressor responses induced by preganglionic sympathetic outflow by a prejunctional mechanism. This is the first evidence demonstrating this effect by NaHS that may contribute, at least in part, to the hypotension induced by NaHS.

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17ß-estradiol on isolated canine basilar and internal carotid arteries.

Progesterone and 17ß-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17ß-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17ß-estradiol on KCl- and/or PGF2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl2 was also determined. In both arteries progesterone (5.6-180 µM) and 17ß-estradiol (1.8-180 µM): (1) produced concentration-dependent relaxations of KCl- or PGF2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10 µM), cycloheximide (10 µM), SQ 22,536 (100 µM) or ODQ (30 µM), potassium channel blockers and ICI 182,780 (only for 17ß-estradiol). In the basilar artery the vasorelaxation induced by 17ß-estradiol was slightly blocked by tetraethylammonium (10mM) and glibenclamide (KATP; 10 µM). In both arteries, progesterone (10-100 µM), 17ß-estradiol (3.1-31 µM) and nifedipine (0.01-1 µM) produced a concentration-dependent blockade of the contraction to CaCl2 (10 µM-10mM). These results suggest that progesterone and 17ß-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca(2+) channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17ß-estradiol in the internal carotid artery; meanwhile KATP channels play a minor role on the effect of 17ß-estradiol in the basilar artery.

Pharmacological evidence that 5-HT1A/1B/1D, α2-adrenoceptors and D2-like receptors mediate ergotamine-induced inhibition of the vasopressor sympathetic outflow in pithed rats.

The sympathetic nervous system that innervates the peripheral circulation is regulated by several mechanisms/receptors. It has been reported that prejunctional 5-HT1A, 5-HT1B, 5-HT1D, D2-like receptors and α2-adrenoceptors mediate the inhibition of the vasopressor sympathetic outflow in pithed rats. In addition, ergotamine, an antimigraine drug, displays affinity at the above receptors and may explain some of its adverse/therapeutic effects. Thus, the aims of this study were to investigate in pithed rats: (i) whether ergotamine produces inhibition of the vasopressor sympathetic outflow; and (ii) the major receptors involved in this effect. For this purpose, male Wistar pithed rats were pre-treated with gallamine (25 mg/kg; i.v.) and desipramine (50 µg/kg) and prepared to stimulate the vasopressor sympathetic outflow (T7-T9; 0.03-3 Hz) or to receive i.v. bolus of exogenous noradrenaline (0.03-3 µg/kg). I.v. continuous infusions of ergotamine (1 and 1.8 µg/kgmin) dose-dependently inhibited the vasopressor responses to sympathetic stimulation but not those to exogenous noradrenaline. The sympatho-inhibition elicited by 1.8 µg/kg min ergotamine was (i) unaffected by saline (1 ml/kg); (ii) partially antagonised by WAY 100635 (5-HT1A; 30 µg/kg) and rauwolscine (α2-adrenoceptor; 300 µg/kg), and (iii) dose-dependently blocked by GR 127935 (5-HT1B/1D; 100 and 300 µg/kg) or raclopride (D2-like; 300 and 1000 µg/kg), The above doses of antagonists did not modify per se the sympathetically-induced vasopressor responses. The above results suggest that ergotamine induces inhibition of the vasopressor sympathetic outflow by activation of prejunctional 5-HT1A, 5-HT1B/1D, α2-adrenoceptors and D2-like receptors in pithed rats.