Eriika Savontaus

Metabolic regulation in progression to autoimmune diabetes

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.

MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13 −/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13 −/− mice. Granulation tissue in Mmp13 −/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13 −/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13 −/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13 −/− mice compared to WT mice. Mmp13 −/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

Transgenic Mice Overexpressing Neuropeptide Y in Noradrenergic Neurons: A Novel Model of Increased Adiposity and Impaired Glucose Tolerance

OBJECTIVE—A functional polymorphism leucine 7 proline in the human neuropeptide Y (NPY) gene leading to increased NPY release from sympathetic nerves is associated with traits of metabolic syndrome. Although hypothalamic NPY neurons play an established role in promoting positive energy balance, the role of NPY colocalized with norepinephrine in sympathetic nervous system and brain noradrenergic neurons remains obscure. RESEARCH DESIGN AND METHODS—To clarify the role of NPY in noradrenergic neurons, we generated a transgenic mouse overexpressing NPY under dopamine–β-hydroxylase promoter and characterized the metabolic phenotype of the OE-NPYDβH mouse. RESULTS—NPY levels are increased by 1.3-fold in adrenal glands and 1.8-fold in the brainstem but not in the hypothalamus in OE-NPYDβH mice. They display increased white adipose tissue mass and cellularity and liver triglyceride accumulation without hyperphagia or increased body weight. Hyperinsulinemia and impaired glucose tolerance develop by the age of 6 months in the OE-NPYDβH mice. Furthermore, circulating ghrelin is significantly increased in comparison with wild-type mice. CONCLUSIONS—The present study shows that even a moderate increase in NPY levels in noradrenergic neurons leads to disturbances in glucose and lipid metabolism. The OE-NPYDβH mouse is an interesting new model to investigate the pathophysiology of some key components of the cluster of abnormalities characterizing the metabolic syndrome.

Stress-induced hypertension and increased sympathetic activity in mice overexpressing neuropeptide Y in noradrenergic neurons.

Background and Aims: Neuropeptide Y (NPY) is a sympathetic neurotransmitter co-stored and co-released with noradrenaline and adrenaline. We have constructed a novel NPY transgenic mouse model (OE-NPYDBH mouse) where targeted overexpression results in increased levels of NPY in the brainstem and adrenal glands. The present study was aimed to understand the role of NPY released from sympathetic nerves and brain noradrenergic neurons in regulation of blood pressure, and behavioral responses to stress. Methods: Blood pressure was measured by radiotelemetry in conscious male OE-NPYDBH and wild-type mice during surgical stress and in baseline conditions. Plasma and adrenal gland catecholamine levels were measured at baseline. Acute immobilization and cold exposure were used to study the plasma levels of NPY and corticosterone in stress, and brown adipose tissue thermogenic activity was measured with [3H]GDP binding after cold. Results: Here, we demonstrate that sympathoadrenal activity is enhanced in the OE-NPYDBH mice. Blood pressure during surgical stress was significantly increased in comparison with wild-type controls. Furthermore, OE-NPYDBH mice showed sexually dimorphic NPY responses to stress, and an anxiolytic-like behavior in elevated plus-maze and light-dark tests. Conclusion: This study shows that the overactive noradrenergic NPY system plays a role in regulation of blood pressure and adaptive responses to stress, and may be a link between chronic stress and adiposity-associated disturbances in metabolism.

The effects of equal caloric high fat and western diet on metabolic syndrome, oxidative stress and vascular endothelial function in mice

Nutrition contributes to increased adiposity, but it remains to be determined whether high fat rather than Western diet exacerbates the development of obesity and other characteristics of metabolic syndrome and vascular function.

Anti-obesity effect of MPV-1743 A III, a novel imidazoline derivative, in genetic obesity

MPV-1743 A III ((+/-)-4-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a novel imidazoline derivative. In this study, it was shown to bind with high affinity to alpha2-adrenoceptor subtypes alpha2A (IC50) = 0.66 +/- 0.06 nM), alpha2B (IC50) = 3.8 +/- 0.53 nM), alpha2C (IC50) = 3.1 +/- 0.61 nM) in the recombinant S115 cells and to alpha2D (IC50 = 0.94 +/- 0.10 nM) in the rat submandibular gland. MPV-1743 A III also showed remarkably high affinity to alpha1-adrenoceptors (IC50 = 150 +/- 12 nM) in the rat cerebral cortex and to imidazoline I2b-binding sites (IC50) = 150 +/- 5.0 nM) in the rat liver. The functional alpha2-adrenoceptor antagonistic effect of MPV-1743 A III was demonstrated by studying the ability of orally administered MPV-1743 A III to reverse and prevent the alpha2-adrenoceptor agonist detomidine-induced mydriasis in rat. The anti-obesity effect of MPV-1743 A III was investigated in genetically obese (fa/fa) Zucker rats in two different phases of obesity. Chronic treatment with MPV-1743 A III (0.3 3 mg/kg per day p.o. for 3 weeks) dose dependently decreased weight gain in early-phase obesity. In fully established obesity, GDP binding to mitochondria and expression of uncoupling protein mRNA were increased in brown adipose tissue by MPV-1743 A III indicating an activation of non-shivering thermogenesis. The present study shows that MPV- 1743 A III has a modest anti-obesity effect in the genetic rodent model of obesity. The relative importance of alpha2- and alpha1-adrenoceptors and imidazoline I2b-binding sites in mediating the effects of MPV-1743 A III needs further evaluation.

Neuropeptide Y Attenuates Stress-Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons.

α-Melanocyte-stimulating hormone regulates vascular NO availability and protects against endothelial dysfunction

AIMS α-Melanocyte-stimulating hormone (α-MSH), derived from the precursor molecule pro-opiomelanocortin, exerts potent anti-inflammatory actions in the vasculature, but its role in circulatory regulation remains unclear. Therefore, we sought to investigate whether α-MSH could regulate the local control of blood vessel tone. METHODS AND RESULTS Using in vivo and ex vivo methods to assess vascular reactivity, we found that α-MSH improved endothelium-dependent vasodilatation in the mouse aorta and coronary circulation without directly contracting or relaxing blood vessels. α-MSH promoted vasodilatation by enhancing endothelial nitric oxide (NO) formation and by improving sensitivity to endothelium-independent blood vessel relaxation. Using cultured human endothelial cells to elucidate the involved molecular mechanisms, we show that α-MSH increased the expression and phosphorylation of endothelial NO synthase in these cells. The observed effects were regulated by melanocortin 1 (MC1) receptors expressed in the endothelium. In keeping with the vascular protective role of α-MSH, in vivo treatment with stable analogues of α-MSH ameliorated endothelial dysfunction associated with aging and diet-induced obesity in mice. CONCLUSION The present study identifies α-MSH and endothelial MC1 receptors as a new signalling pathway contributing to the regulation of NO availability and vascular function. These findings suggest applicability of α-MSH analogues for therapeutic use in pathological conditions that are characterized by vascular dysfunction.

Diet-Induced Obesity in Mice Overexpressing Neuropeptide Y in Noradrenergic Neurons

Neuropeptide Y (NPY) is a neurotransmitter associated with feeding and obesity. We have constructed an NPY transgenic mouse model (OE-NPYDBH mouse), where targeted overexpression leads to increased levels of NPY in noradrenergic and adrenergic neurons. We previously showed that these mice become obese on a normal chow. Now we aimed to study the effect of a Western-type diet in OE-NPYDBH and wildtype (WT) mice, and to compare the genotype differences in the development of obesity, insulin resistance, and diabetes. Weight gain, glucose, and insulin tolerance tests, fasted plasma insulin, and cholesterol levels were assayed. We found that female OE-NPYDBH mice gained significantly more weight without hyperphagia or decreased activity, and showed larger white and brown fat depots with no difference in UCP-1 levels. They also displayed impaired glucose tolerance and decreased insulin sensitivity. OE-NPYDBH and WT males gained weight robustly, but no difference in the degree of adiposity was observed. However, 40% of OE-NPYDBH but none of the WT males developed hyperglycaemia while on the diet. The present study shows that female OE-NPYDBH mice were not protected from the obesogenic effect of the diet suggesting that increased NPY release may predispose females to a greater risk of weight gain under high caloric conditions.

Effects of estrous cycle and steroid replacement on the expression of leptin and uncoupling proteins in adipose tissue in the rat

Among other actions ,leptin has been suggested to increase energy expenditure and to modulate the menstrual cycle. In fact ,the main effect of leptin seems to be modulating the sympathetic nervous system and gonadotropin-releasing hormone pulsatility. We investigated whether changes in the plasma steroid concentrations during the estrous cycle and after ovariectomy and steroid replacement can modulate plasma leptin levels ,adipose tissue leptin mRNA expression ,and some of the candidates for mediating energy expenditure (uncoupling proteins (UCP) 1 ,2 ,and 3 mRNA) in white and brown adipose tissue. Rats in estrous cycle or ovariectomized rats with or without estradiol or progesterone replacement therapy for 18 days were studied. Plasma leptin ,insulin ,estradiol and progesterone were measured with radio-immunoassays. Leptin mRNA expression was measured in subcutaneous ,periovarian and mesenteric white adipose tissue and in interscapular brown adipose tissue. Expression of UCP 1 ,2 ,and 3 mRNA in periovarian white and brown adipose tissue was analyzed. Plasma leptin levels were significantly decreased in the estrous (1.1 ± 0.4 ng/ml) compared with the pro-estrous (1.7 ± 0.4 ng/ml ,F = 3.0 ,p = 0.046) phase of cycle. UCP1 mRNA levels in brown adipose tissue were more elevated during pro-estrus than during metestrus (F = 3.17 ,p = 0.039). Gene expressions of leptin ,UCP2 or UCP3 mRNA did not change significantly during the cycle. In ovariectomized rats ,estradiol and/or progesterone treatment had no effect on plasma leptin levels. Gene expression analysis of leptin and UCP1 ,2 and 3 in adipose tissue was not affected by steroid replacement. In conclusion ,the estrous cycle appears to have a minor effect on modulation of leptin and uncoupling proteins. Only plasma leptin levels and expression of UCP1 mRNA are modestly elevated during the estrous cycle in the rat. Since estrogen and/or progesterone substitution in ovariectomized rats does not affect circulating leptin concentration or expression of leptin and UCPs in adipose tissue ,it is unlikely that steroids play a major role in their regulation.