The functional activity of hypothalamic signaling systems in rats with neonatal diabetes mellitus treated with metformin

The effect of the two-month metformin treatment (200 mg/kg/day) of rats with the neonatal model of type 2 diabetes mellitus on the functional activity of hypothalamic signaling systems was studied. It was shown that metformin treatment restored the sensitivity of hypothalamic adenylyl cyclase signaling system to agonists of the type 4 melanocortin receptor and the type 2 dopamine receptor but did not influence significantly the functions of the insulin signaling system. These data suggest new targets and mechanisms of metformin action in the CNS, which may mediate its restoring effect on energy homeostasis impaired in diabetic pathology.     

Restoration of hypothalamic signaling systems as a cause of improved metabolic parameters in rats with neonatal diabetes model during treatment with bromocriptine mesylate

One approach to correction of diabetes mellitus 2 type (DM2) and its complications is the use of bromocriptine mesylate (BCM), a selective agonist of the dopamine receptor type 2 (DA₂R). However, the effectiveness and mechanisms of the action of BCM in the treatment of severe forms of DM2 forms currently not understood. The purpose of this study was to investigate the influence of 4-week treatment of male rats with neonatal DM2 model using BCM (300 mg/kg daily) on their metabolic parameters and on the activity of the adenylyl cyclase signaling system (ACSS) in the hypothalamus. Exposure to BCM restored glucose tolerance and glucose utilization by exogenous insulin, normalized lipid metabolism, and lowered triglycerides and atherogenic cholesterol levels, which are elevated in DM2. In the hypothalamus of diabetic rats treated with BCM, the regulation of ACSS by agonists of melanocortin receptors type 4 (MC₄R), DA₂R, and serotonin 1B-subtype receptors and expression of the Mc4r gene encoding MC₄R were restored. Furthermore, BCM treatment did not influence the insulin levels in the blood and its production by pancreatic β-cells. The data indicate that the use of BCM to correct severe forms of experimental DM2 holds promise and show that the therapeutic potential of this drug is based on its ability to restore signaling systems of the hypothalamus that are sensitive to monoamines and peptides of the melanocortin family, which are responsible for the control of energy metabolism and insulin sensitivity.     

Peptidergic signaling brain systems in diabetes mellitus

One of the key causes of development of diabetes mellitus (DM) and its complications is change in the functional activity of hormonal signaling systems regulated by hormones of different natures, as is shown by literature data and by the results of our study on animal models of DM and on human DM of types 1 and 2. The brain peptidergic systems regulated by melanocortin receptor agonists, neuropeptide Y, glucagon-like peptide-1, kisspeptines, and somatostatin, play an important role in etiology and pathogenesis of DM. However, the data on interrelations between the functional state of these systems and the development of DM and its complications are scarce and contradictory. The changes in the peptidergic systems are usually the result of metabolic and functional disregulations caused by DM but in some cases may themselves become the cause of DM, as is shown in the case of the melanocortin signaling system. This review is focused on functioning of the brain peptidergic systems in DM and on the possible role of changes of their activity in development of the disease. The hypothesis of central genesis of type 2 DM, which based on data on the generation of insulin resistance and disturbances of carbohydrate and lipid metabolism in response to changes of functional activity of the brain signaling systems regulated by neuropeptides, is discussed.     

The effect of prolonged intranasal administration of serotonin on the activity of hypothalamic signaling systems in male rats with neonatal diabetes

The functioning of the serotonergic system of the brain is impaired in type II diabetes (T2D), and this leads to metabolic and hormonal dysfunction. The elevation of serotonin level in the CNS is one of the approaches for correcting of the serotonergic system of the brain. The aim of the present work was to investigate the effect of intranasal serotonin (InS) administration for 5 weeks at a daily dose of 20 μg on the metabolic parameters and functional activity of adenylate cyclase signaling system (ACSS) sensitive to peptide hormones and biogenic amines in the hypothalamus of male rats with neonatal T2D. Neonatal model of T2D was induced by injecting streptozotocin (70 mg/kg) into 5-day-old rat pups. Four-month-old animals with apparent T2D manifestations were divided into two groups: an untreated group (D0, n = 6) and a group that received InS treatment (DIS, n = 6). InS administration to diabetic rats restored ACSS regulation by the agonists of type 2 dopamine receptors (DA₂R) and type 4 melanocortin receptors (MC₄R) and enhanced the inhibitory effect of serotonin on adenylate cyclase activity. Elevated expression of genes encoding DA₂R, MC₄R, and serotonin receptor of the 1B subtype (5-HT_1BR) was among the main causes of this change. The relative activity of signaling cascades involving various types of serotonin (G_s-coupled 5-HT_4,6,7R/G_i-coupled 5-HT₁R), dopamine (DA₁R/ DA₂R), and melanocortin (MC₃R/MC₄R) receptors involved in ACSS regulation was also altered in the animals of the DIS group. InS administration restored hormonal regulation in the hypothalamus, improved glucose tolerance, and increased the sensitivity of tissues to insulin. The data obtained show that the elevation of serotonin level in the CNS is a promising approach for the recovery of hypothalamic signaling pathways in T2D and correction of the metabolic disturbances dependent on these pathways.     

A change of hormonal regulation of adenylyl cyclase in epididymal adipose tissue of rats with experimental models of diabetes mellitus

One of the key causes of diabetes mellitus (DM) and its complications are hormonal disturbances in functioning of hormonal signaling systems, including the adenylyl cyclase signaling system (ACSS). The goal of this work was to study the functional state and hormonal sensitivity of ACSS in the epididymal adipose tissue of male rats in the 7-month model of mild type 1 DM (DM1), in the 18-month neonatal model of type 2 DM (DM2), and in the taken for comparison model of the 30 day-longs acute DM1. It is shown for the first time that in adipocytes from the epididymal fat of rats with the studied DM models the basal AC activity and its stimulation by forskolin were decreased, which indicates a weakening of the catalytic functions of the enzyme adenylyl cyclase (AC). Stimulation of AC by guanine nucleotides in DM changed to the lesser extent, which argues in favor of preservation of functions of heterotrimeric Gs-proteins in the epididymal fat. In rats with DM1 the sensitivity of AC of adipocytes to agonists of β-adrenergic receptors (β-AR), activators of lipolysis, remained practically unchanged, while in animals with DM2 the AC stimulating effects of β-AR-agonists were reduced or completely blocked, like in the case of β₃-AR-agonists BRL-37344 and CL-316243. In adipocytes of rats with DM1 the AC inhibitory effect of N⁶-cyclopentyladenosine, agonist of type 1 adenosine receptors (Aden₁R), an inhibitor of lipolysis, was attenuated, whereas in DM2 this effect was completely preserved. Thus, in the epididymal adipose tissue of rats with DM1 the antilipolytic AC cascades including Aden₁R were decreased and the stimulation of AC by β-AR-agonists was preserved, whereas in rats with DM2 the β-AR-mediated AC cascades activating lipolysis were reduced, but Aden₁R-mediated AC cascades inhibiting lipolysis did not change. The changes of hormonal regulation of ACSS in adipocytes from the epididymal fat lead to disturbances of the metabolic status of animals with DM1 and DM2 and should be considered in the diagnostics and treatment of DM and its complications.     

Increased hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.     

The effect of long-term diabetes mellitus induced by treatment with streptozotocin in 6-week-old rats on functional activity of the adenylyl cyclase system

In type 1 diabetes mellitus (DM), changes occurring in the adenylyl cyclase signaling system (ACSS) are one of the key causes of complications of the disease. Since type 1 DM has been most often diagnosed in childhood and adolescence, the study of changes in ACSS in the early development of the disease is a genuine problem. For this, we developed a prolonged model of type 1 DM, which was induced by treatment of 6-week-old rats with moderate doses of streptozotocin (1.5M-DM), and studied the functional state of ACSS in the brain, myocardium, and testes of rats with this model of the disease 7 months after its start. The 1.5M-DM model was compared with the model that was induced by streptozotocin treatment of adult, 5-month-old animals (5M-DM). It was shown that, in 1.5M-DM, in the tissues of diabetic rats, the functional activity of ACSS sensitive to biogenic amines and polypeptide hormones was significantly changed. In rats with 1.5M-DM, the adenylate cyclase (AC) inhibitory effects of somatostatin (in all studied tissues), noradrenaline (in the myocardium and the brain), and agonists of type 1 serotonin receptor (in the brain) were weakened to the greatest degree. In the brain, the AC-stimulating effects of relaxin, isoproterenol, and agonists of G_s-protein-coupled serotonin receptors also decreased; in the myocardium, the corresponding effects of GppNHp, relaxin, and β-adrenergic agonists declined; and, in the testes, the AC effects of GppNHp and chorionic gonadotropin declined. When comparing the 1.5M-DM and 5M-DM models, the most pronounced differences between them were found in the effect of DM on hormonal regulation of ACSS in the brain, this being true both for AC-stimulating effects of dopamine and PACAP-38 and for AC-inhibiting effects of bromocriptine and somatostatin. These results indicate significant changes in hormonal regulation of the nervous, cardiovascular, and reproductive systems in rats with early induction of type 1 DM, in some cases more severe changes as compared with late model of 5M-DM. These changes may be the basis for development of diabetic cardiomyopathy, cognitive deficiency, and hypogonadotropic states, which are often detected in children and adolescents with type 1 DM.     

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase

The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.     

Effect of intranasal insulin and serotonin on functional activity of the adenylyl cyclase system in myocardium, ovary, and uterus of rats with prolonged neonatal model of diabetes mellitus

The disturbances in hormonal signaling systems, adenylyl cyclase system (ACS) in particular, occur at the early stages of diabetes mellitus (DM) being one of the key causes of its complications. Since the correlation between the severity of DM and severity of disturbances in ACS is established, studying ACS activity can be used for monitoring DM and its complications and evaluating the effectiveness of their treatment. Recently, intranasal insulin (I-I) and the drugs increasing brain serotonin level, thus effectively restoring CNS functions, have begun to be used for the treatment of type 2 DM. However, the mechanisms of their action on peripheral tissues and organs at DM are not understood. The aim of this work was to study an influence of I-I and intranasal serotonin (I-S) on the functional activity of ACS in myocardium, ovary and uterus of rats with a neonatal model of type 2 DM. In the tissues of diabetic rats the changes in the regulation of adenylate cyclase (AC) by guanine nucleotides and hormones acting on enzyme in stimulatory and inhibitory manner were found, and these changes were characterized by receptor and tissue specificity. In diabetic rats I-I restored AC-stimulating effects of isoproterenol in the myocardium, that of guanine nucleotides and gonadotropin in the ovaries and relaxin in the uterus, as well as AC-inhibiting effects of somatostatin in all tissues and norepinephrine in the myocardium. Treatment with I-S led to a partial recovery of AC-inhibiting effect of norepinephrine in the diabetic myocardium, but did not affect the regulation of AC by other hormones. These data indicate that I-I normalizes the functional activity of ACS in the myocardium and in the tissues of reproductive system of female rats with neonatal DM, whereas the effect of I-S on ACS in the studied tissues is less pronounced. These results should be considered for the design and optimization of the strategy of I-I and I-S application for the treatment of DM and its complications.     

The expression of MC4Rs in D1R neurons regulates food intake and locomotor sensitization to cocaine

While it is known that mice lacking melanocortin 4 receptor (MC4R) expression develop hyperphagia resulting in early‐onset obesity, the specific neural circuits that mediate this process remain unclear. Here, we report that selective restoration of MC4R expression within dopamine‐1 receptor‐expressing neurons [MC4R/dopamine 1 receptor (D1R) mice] partially blunts the severe obesity seen in MC4R‐null mice by decreasing meal size, but not meal frequency, in the dark cycle. We also report that both acute cocaine‐induced anorexia and the development of locomotor sensitization to repeated administration of cocaine are blunted in MC4R‐null mice and normalized in MC4R/D1R mice. Neuronal retrograde tracing identifies the lateral hypothalamic area as the primary target of MC4R‐expressing neurons in the nucleus accumbens. Biochemical studies in the ventral striatum show that phosphorylation of DARPP‐32^Thr‐34 and GluR1^Ser‐845 is diminished in MC4R‐null mice after chronic cocaine administration but rescued in MC4R/D1R mice. These findings highlight a physiological role of MC4R‐mediated signaling within D1R neurons in the long‐term regulation of energy balance and behavioral responses to cocaine.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

Design and bioactivities of melanotropic peptide agonists and antagonists: Design based on a conformationally constrained somatostatin template

Summary α-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (α-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained α-melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the μ opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.     

Role of hypothalamic interleukin-1beta (IL-1beta) in regulation of energy homeostasis by melanocortins

In the current study we sought to determine whether hypothalamic IL-1beta is regulated by melanocortin signaling and if melanocortin-induced changes in energy balance are dependent on IL-1beta. A melanocortin agonist, MTII, increased hypothalamic IL-1beta mRNA levels by two-fold, whereas a melanocortin antagonist, SHU9119, blunted lipopolysaccharide (LPS)-mediated increase of hypothalamic IL-1beta content. Pharmacological or genetic disruption of IL-1 receptor signaling prevented MTII-mediated reductions in locomotor activity, but did not reduce MTII-induced anorexia. These data suggest a potential role for central melanocortins in mediating the decrease of ambulation characteristic of the 'sickness' response.     

Activation of the melanocortin-4 receptor causes enhanced excitation in presympathetic paraventricular neurons in obese Zucker rats

Sympathetic nerve activity is increased in obesity-related hypertension. However, the central mechanisms involved in the increased sympathetic outflow remain unclear. The hypothalamic melanocortin system is important for regulating energy balance and sympathetic outflow. To understand the mechanisms by which the melanocortin systems regulates sympathetic outflow, we investigated the role of melanocortin 4 receptors (MC4R) in regulating presympathetic paraventricular nucleus (PVN) neurons. We performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the rostral ventrolateral medulla in brain slices from obese zucker rats (OZRs) and lean zucker rats (LZRs). The MC4R agonists melanotan II (MTII) and α-melanocyte-stimulating hormone (α-MSH) increased the firing activity and depolarized the labeled PVN neurons from both LZRs and OZRs in a concentration-dependent manner. MTII produced significant greater increase in the firing activity in OZRs than in LZRs. Blocking MC4R with the specific antagonist SHU9119 had no effect on the basal firing rate but abolished the MTII-induced increase in the firing rate in both OZRs and LZRs. Furthermore, intracellular dialysis of guanosine 5'-O-(2-thodiphosphate), but not bath application of kynurenic acid and bicuculline, eliminated the MTII-induced increase in firing activity. In addition, MTII had no effect on the frequency and amplitude of glutamatergic excitatory postsynaptic currents and GABAergic inhibitory postsynaptic currents in labeled PVN neurons. Collectively, our findings suggest that MC4R contributes to the elevated excitability of PVN presympathetic neurons, which may be involved in obesity-related hypertension.     

Hypothalamic insulin signaling is required for inhibition of glucose production

Circulating insulin inhibits endogenous glucose production. Here we report that bidirectional changes in hypothalamic insulin signaling affect glucose production. The infusion of either insulin or a small-molecule insulin mimetic in the third cerebral ventricle suppressed glucose production independent of circulating levels of insulin and of other glucoregulatory hormones. Conversely, central antagonism of insulin signaling impaired the ability of circulating insulin to inhibit glucose production. Finally, third-cerebral-ventricle administration of inhibitors of ATP-sensitive potassium channels, but not of antagonists of the central melanocortin receptors, also blunted the effect of hyperinsulinemia on glucose production. These results reveal a new site of action of insulin on glucose production and suggest that hypothalamic insulin resistance can contribute to hyperglycemia in type 2 diabetes mellitus.     

Circumventing central leptin resistance: lessons from central leptin and POMC gene delivery

We identified that leptin resistance in aged-obese rats has both peripheral and central components. The central resistance is characterized by diminished hypothalamic leptin receptors and impaired leptin signal transduction. We developed a new model of leptin-induced leptin resistance in which application of the central leptin gene delivery produces unabated hypothalamic leptin over-expression. The chronic central elevation of leptin precipitates leptin resistance in young animals devoid of obesity and exacerbates it in mature or aged animals with obesity. Despite leptin resistance, our aged obese, DIO, and leptin-induced leptin resistant rats were fully responsive to central pharmacological melanocortin activation. We propose that the central leptin resistance resides between leptin receptor and melanocortin receptor activation. Our central POMC gene therapy overcame leptin resistance, producing weight and fat loss and improved insulin sensitivity in obese Zucker and aged rats. This success highlights the central melanocortin system as a useful drug target for combating obesity.