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In type 1 diabetes mellitus, the impaired testosterone synthesis in the testes leads to androgen deficiency. The long-term application of high gonadotropin doses for its correction decreases the sensitivity of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors in Leydig cells to the endogenous gonadotropins. The aim of this work was to study the effect of a 3-day treatment of male Wistar rats with streptozotocin type 1 diabetes with the 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido) phenyl)thieno[2,3-d]pyrimidine-6-carboxamide allosteric LH/hCG receptor agonist (TP03, 15 mg/kg/day) on steroidogenic effects of a relatively low-dose hCG (10 IU/rat, single dose, s.c.). Pretreatment of diabetic rats with TP03 enhanced the stimulatory effect of hCG on testosterone levels, slightly modifying its effects on the expression of steroidogenic proteins (Star, Cyp11a1, Cyp17a1) and LH/hCG receptor (Lhr) genes. Thus, in type 1 diabetes, TP03 increases the steroidogenic effect of low-dose hCG, at the same time as maintaining its effect on the gene expression of LH/hCG receptor and steroidogenesis enzymes in the testes.

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.

Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. It was assumed that activation of TRPC6 channels leads directly to the Ca2+ influx into cells. Another Ca2+ influx pathway, a store-operated Ca2+ entry (SOCE) via ORAI channels, was also shown to be disturbed during the diabetes development. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca2+ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca2+ imaging indicated the involvement of ORAI and sodium-calcium exchanger (NCX) in Ca2+ entry induced upon store depletion. Ca2+ inflow by NCX is a result of pronounced TRPC6-mediated Na+ influx leading moreover to the membrane depolarization and ORAI suppression. In rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of SOCE in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca2+ influx such that TRPC6 channels lost their sensitivity to Ca2+ store depletion and ORAI-mediated Ca2+ entry is suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account in the pharmacological treatment of the early stages of diabetic nephropathy.

Functional disorders in obesity are largely due to a decrease in tissue sensitivity to insulin and leptin. One of the ways to restore it is inhibition of protein phosphotyrosine phosphatase 1B (PTP1B) and T-cell protein phosphotyrosine phosphatase (TCPTP), negative regulators of the insulin and leptin signaling. Despite progress in the development of inhibitors of these phosphatases, commercial preparations based on them have not been developed yet, and the mechanisms of action are poorly understood. The aim of the work was to study the effect of new derivatives of 4-oxo-1,4-dihydrocinnoline (PI04, PI06, PI07) on the activity of PTP1B and TCPTP, as well as to study the effect of their five-day administration (i.p., 10 mg/kg/day) to Wistar rats with diet-induced obesity on body weight and fat, metabolic and hormonal parameters, and gene expression of phosphatase and insulin and leptin receptors in the liver. It has been shown that PI04 is a mild, low selective inhibitor of both phosphatases (PTP1B, IC50=3.42(2.60-4.51) μM; TCPTP, IC50=4.16(3.49-4.95) μM), while PI06 and PI07 preferentially inhibit PTP1B (IC50=3.55 (2.63-4.78) μM) and TCPTP (IC50=1.45(1.18-1.78) μM), respectively. PI04 significantly reduced food intake, body weight and fat, attenuated hyperglycemia, normalized glucose tolerance, basal and glucose-stimulated levels of insulin and leptin, and insulin resistance index. Despite the anorexigenic effect, PI06 and PI07 were less effective, having little effect on glucose homeostasis and insulin sensitivity. PI04 significantly increased the expression of the PTP1B and TCPTP genes and decreased the expression of the insulin and leptin receptor genes. PI06 and PI07 had little effect on these indicators. Thus, PI04, the inhibitor of PTP1B and TCPTP phosphatases, restored metabolic and hormonal parameters in obese rats with greater efficiency than inhibitors of PTP1B (PI06) and TCPTP (PI07). This indicates the prospect of creating mixed PTP1B/TCPTP inhibitors for correction of metabolic disorders.Funktsional'nye narusheniia pri ozhirenii vo mnogom obuslovleny snizheniem chuvstvitel'nosti tkaneĭ k insulinu i leptinu. Odnim iz puteĭ ee vosstanovleniia iavliaetsia ingibirovanie proteinfosfotirozinfosfatazy 1B (PTP1B) i T-kletochnoĭ proteinfosfotirozinfosfatazy (TCPTP) — negativnykh reguliatorov insulinovogo i leptinovogo signalinga. Nesmotria na progress v razrabotke ingibitorov étikh fosfataz kommercheskie preparaty na ikh osnove ne razrabotany, a mekhanizmy deĭstviia malo izucheny. Tsel' raboty sostoiala v issledovanii vliianiia novykh proizvodnykh 4-okso-1,4-digidrotsinnolina (PI04, PI06, PI07) na aktivnost' PTP1B i TCPTP, a takzhe v izuchenii vliianiia ikh piatidnevnogo vnutribriushinnogo vvedeniia (10 mg/kg/sutki) krysam Wistar s indutsirovannym dietoĭ ozhireniem na massu tela i zhira, metabolicheskie i gormonal'nye pokazateli, ékspressiiu genov fosfataz i retseptorov insulina i leptina v pecheni. Pokazano, chto PI04 iavliaetsia miagkim nizkoselektivnym ingibitorom obeikh fosfataz (PTP1B, IC50=3,42(2,60–4,51) mkM; TCPTP, IC50=4,16(3,49–4,95) mkM), v to vremia kak PI06 i PI07 predpochtitel'no ingibiruiut PTP1B (IC50=3,55(2,63–4,78) mkM) i TCPTP (IC50=1,45(1,18–1,78) mkM) sootvetstvenno. PI04 znachimo snizhal potreblenie korma, massu tela i zhira, oslablial giperglikemiiu, normalizoval tolerantnost' k gliukoze, bazovye i stimulirovannye gliukozoĭ urovni insulina i leptina, indeks insulinovoĭ rezistentnosti. Nesmotria na anoreksigennyĭ éffekt, PI06 i PI07 byli menee éffektivnymi, slabo vliiaia na gliukoznyĭ gomeostaz i chuvstvitel'nost' k insulinu. PI04 sushchestvenno povyshal ékspressiiu genov PTP1B i TCPTP i snizhal ékspressiiu genov retseptorov insulina i leptina. PI06 i PI07 slabo vliiali na éti pokazateli. Takim obrazom, PI04 — ingibitor fosfataz PTP1B i TCPTP — s bol'sheĭ éffektivnost'iu v sravnenii s ingibitorami PTP1B (PI06) i TCPTP (PI07) vosstanavlival metabolicheskie i gormonal'nye pokazateli u krys s ozhireniem, chto ukazyvaet na perspektivnost' sozdaniia smeshannykh PTP1B/TCPTP-ingibitorov dlia korrektsii metabolicheskikh rasstroĭstv.

Human chorionic gonadotropin that is widely used for improving spermatogenesis. The effect of chorionic gonadotropin is mediated through luteinizing hormone receptor. Treatment with gonadotropin is associated with undesirable effects due to hyperactivation of testosterone production and luteinizing hormone receptor desensitization. A promising alternative could be low-molecular-weight agonists of luteinizing hormone receptors, but their effects on spermatogenesis have not been investigated. Here we analyzed the effect of a thieno[2,3-d]pyrimidines (TP), 4-((3-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno [2,3-d]pyrimidine-4-yl) phenyl)carbamoyl)pyridine 1-oxide (TP22), an allosteric agonist of luteinizing hormone receptors, on the seminiferous tubules and spermatogenic cells in 4- and 18-month-old male rats and in animals with diabetes mellitus. TP22 and gonadotropin were administered in daily doses of 15 mg/kg and 20 U/rat for 5 days. Blood testosterone level, morphology of the seminiferous tubules, and the number of germ cells in them were estimated. Being comparable by the efficiency to gonadotropin, TP22 increased the testosterone level in all the studied groups of rats and restored epithelium thickness in the seminiferous tubules and the number of spermatogonia and pachytenic spermatocytes that are reduced in aging and diabetes, but, unlike gonadotropin, did not suppress the expression of luteinizing hormone receptor. The efficacy of TP22 as a stimulator of testicular spermatogenesis has been demonstrated both under normal conditions and in age-related and diabetes-associated reproductive dysfunctions.

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) regulate testicular steroidogenesis by interacting with the orthosteric site located in the extracellular domain of the LH receptor (LHR). The use of hCG and LH in medicine is fraught with side effects caused by hyperactivation of LH-dependent cascades and the development of resistance of target cells to endogenous gonadotropins due to a decrease in the activity and expression of LHR. An alternative to gonadotropins is low-molecular-weight compounds which interact with the LHR transmembrane allosteric site. The goal of this work was to study the relationship between the steroidogenic effects of hCG and thieno[2,3-d]pyrimidine derivatives (TPDs) with an activity of LHR agonists and their ability to influence the expression of the LHR-encoding Lhr gene both in vitro, when acting on the primary culture of rat Leydig cells, and in vivo, when administered to male rats. hCG stimulated testosterone production with a high efficiency at an early stage of its effect on Leydig cells (after 30 min) and upon a single administration to male rats (after 3 h), exceeding TPDs in activity by this parameter. After 1–3 h of acting on Leydig cells and long-term administration to male rats, the steroidogenic effect of hCG decreased and became comparable to that of TPDs. In the Leydig cells and rat testes, hCG suppressed Lhr gene expression, which was partially restored in vivo on days 7–10 being accompanied by a slight increase in the steroidogenic effect of hCG, though to a significantly smaller extent than on the first day. The treatment with TP03, which was the most active of the TPDs studied, had a little effect on Lhr gene expression in Leydig cells but significantly increased it in the rat testes after long-term administration of the drug. The steroidogenic effect of TP03 positively correlated with Lhr gene expression. When TP03 was applied to aging rats, its steroidogenic effect decreased, most likely, due to the absence of its stimulatory effect on LHR expression. Thus, after long-term administration, TPDs exert a moderately expressed, stable over time, stimulatory effect on testosterone production but do not decrease LHR expression, which prevents testicular resistance to endogenous gonadotropins under conditions of steroidogenesis stimulation.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG), due to binding to the LH/hCG receptor, activate the adenylyl cyclase (AC) system which regulates testosterone (T) production. Long-term administration of LH and hCG causes desensitization of this system and attenuates the steroidogenic response, thereby necessitating the search for new agonists of the LH/hCG receptor. The aim of the work was to explore, in comparison with hCG, the stimulatory effects of the previously developed thieno[2,3-d]pyrimidines, TP03 and TP04, and a new thieno[2,3-d]pyrimidine derivative, 5-amino-N-(tert-butyl)-4-(3-(4-aminopyrimidine-5-carboxamido)-phenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP37), on AC activity in rat testicular membranes, as well as on T production and gene expression of the LH/hCG receptor and the key testicular steroidogenic proteins under conditions of a single and 3-day administration to male rats. hCG increased AC activity in testicular membranes more efficiently compared to thieno[2,3-d]pyrimidines, and after a single injection (50 and 100 IU/rat) was superior to TP03 and TP04 (15–50 mg/kg) in its steroidogenic effect. After a 3-day administration, the steroidogenic effect of hCG was attenuated compared to that for TP03 and TP04. After 3 days of treatment with gonadotropin, testicular expression of genes encoding the StAR protein and cytochrome P450scc was considerably increased, but expression of the Lhr and Cyp17a1 genes encoding LH/hCG receptor and cytochrome P450-17α was suppressed. TP03 and TP04 slightly increased StAR gene expression but did not affect expression of other genes. TP37, which was active in vitro, after a short stimulation of T production, suppressed the steroidogenic function at a dose of 50 mg/kg, probably due to its degradation and the ability to suppress Cyp17a1 gene expression. Our data indicate significant differences in the mechanisms underlying the effect of gonadotropins and thieno[2,3-d]pyrimidines with an activity of LH/hCG receptor agonists on testicular steroidogenesis. We also demonstrate that long-term administration of thieno[2,3-d]pyrimidines to stimulate T production does not attenuate steroidogenesis and induces no LH resistance.

Leptin is one of the major regulators of the reproductive system which targets all components of the hypothalamic—pituitary—gonadal axis. In type 2 diabetes mellitus (DM2), functions of the male reproductive system are impaired, which may be due to altered activity of the testicular leptin system. The aim of this work was to study the leptin level and the functional state of the leptin signaling system in the testes of male Wistar rats with different degrees of severity of DM2 induced by a high-fat diet and low-dose streptozotocin treatment. It was found that rats with highly severe DM2 (D-HS group) manifested marked hyperleptinemia, decreased plasma testosterone level, lower sperm count, and higher number of the abnormally shaped sperm accompanied by hyperglycemia, impaired glucose tolerance, insulin resistance, and increased levels of glycated hemoglobin and free fatty acids. The testes of this rat group had an increased leptin level, decreased number of leptin receptors, and attenuated phosphorylation of the effectory components of leptin signaling—transcription factor STAT3 at Tyr705 and Akt-kinase at Ser473. In rats with moderately severe DM2 (D-MS group), metabolic and hormonal disturbances, as well as a decrease in the plasma testosterone level and sperm abnormalities, were less pronounced. The plasma leptin level significantly exceeded that in control only after glycemic load. In the D-MS group’s testes, no statistically significant differences in levels of leptin and its receptor, as well as in STAT3 and Akt-kinase phosphorylation, were revealed compared to control. Thus, pathological changes in steroidogenesis and spermatogenesis in male rats with DM2 depend on the severity of metabolic and hormonal disturbances and the functional state of testicular leptin signaling, the severity of changes in which correlates positively with systemic hyperleptinemia.

The effectiveness of high-dose metformin (MF) for the treatment of endocrine and oncological diseases has been shown in recent years. However, the use of high-dose MF may be associated with lactic acidosis and liver dysfunctions. The goal of the work was to study the effect of long-term (10-day) oral administration of a relatively high dose of MF (600 mg/kg per day) to yellow C57Bl/6J (Ay/a) Agouti line mice with melanocortin-type obesity on liver function, which was evaluated based on the hepatocyte morphology and steatosis severity, the expression of inflammatory and apoptotic factors, and the aminotransferase activity, as well as on the plasma lactate level in the animals. In Agouti mice, MF (600 mg/kg per day) caused a decrease in body and fat mass, led to reduced hyperglycemia, hyperinsulinemia, and hyperleptinemia, and restored sensitivity to glucose and insulin. At the same time, small- and large-drop fatty and hydropic degeneration were attenuated in the liver of Agouti line mice treated with MF. The expression of proinflammatory IL-1β and proapoptotic Bax protein and the Bax/Bcl-2 ratio did not differ from the control C57Bl/6J (a/a) mice. The activity of alanine aminotransferase was normalized in the blood of Agouti mice treated with MF, and the lactate level increased, but to a moderate degree. It was concluded that high-dose MF did not induce lactic acidosis in Agouti mice. At the same time, it restored the liver functions impaired by melanocortin obesity. This allows the use of the high-dose MF to be considered a possible means to treat obesity and metabolic disorders associated with hepatic steatosis.