Sustained insulin treatment restoring metabolic status, body weight, and cognition in an anorexia nervosa-like animal model in mice.

INTRODUCTION: Anorexia Nervosa (AN) is a psycho-socio-biological disease characterized by severe weight loss as result of dieting and hyperactivity. Effective treatments are scarce, despite its significant prevalence and mortality. AN patients show lower basal insulin levels and increased metabolic clearance, leading to weight loss, cognitive deficits, and hormonal imbalances. Low-dose polymer insulin could potentially reverse these effects by restoring brain function, reducing fear of weight gain, encouraging food intake, and restoring fat depots. This study evaluates an insulin delivery system designed for sustained release and AN treatment. METHODS: AN-like model was established through dietary restriction (DR). On days 1-25, mice were on DR, and on days 26-31 they were on ad libitum regimen. An insulin-loaded delivery system was administered subcutaneously (1% w/w insulin). The impact of insulin treatment on gene expression in the hippocampus (cognition, regulation of stress, neurogenesis) and hypothalamus (eating behavior, mood) was assessed. Behavioral assays were conducted to evaluate motor activity and cognitive function. RESULTS: The delivery system demonstrated sustained insulin release, maintaining therapeutic plasma levels. Diet restriction mice treated with the insulin delivery system showed body weight restoration. Gene expression analysis revealed enhanced expression of CB1 and CB2 genes associated with improved eating behavior and cognition, while POMC expression was reduced. Insulin-polymer treatment restored cognitive function and decreased hyperactivity in the AN-like model. CONCLUSION: The PSA-RA-based insulin delivery system effectively restores metabolic balance, body weight, and cognitive function in the AN model. Its ability to steadily release insulin makes it a promising candidate for AN treatment."

Novel glucagon- and OXM-based peptides acting through glucagon and GLP-1 receptors with body weight reduction and anti-diabetic properties.

Oxyntomodulin (OXM) is an endogenous gastrointestinal hormone, which activates both the Glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR). However, OXM has shortcomings including poor GLP-1R agonism to control glycemia, short half-life and others. Inspired from the sequence relationship between OXM and glucagon, in this study, we introduced different C-terminus residues of GLP-1, exenatide and OXM to glucagon to get a series of hybrid peptides with enhanced GLP-1R activation. The formed glucagon-exenatide hybrid peptide shows higher GLP-1R activation properties than OXM. Then the peptides based on the glucagon-exenatide hybrid peptide were coupled with fatty acid side chains to prolong their half-lives. As a result, the most potent compound 16a could stimulate insulin secretion and maintain blood glucose in normal level for ~42.6 h in diabetic mice. 16a exhibited reduced HbA1c level in diabetic mice, lowered body weight significantly in obesity mice on chronic treatment assay. 16a, combined efficient GCGR/GLP-1R activity, is potential as novel treatment for obesity and diabetes. This finding provides new insights into balancing GLP-1/GCGR potency of glucagon-exenatide hybrid peptide and is helpful for discovery of novel anti-diabetic and bodyweight-reducing drugs.

Beta-carotene as a novel therapy for the treatment of "Autistic like behavior" in animal models of Autism.

Autism-affected individuals are characterized by lower plasma oxytocin and its ectoenzyme regulator CD38. Oxytocin, a hypothalamic hormone secreted upon the release of CD38, plays a role in social behavior and bonding. All-trans retinoic acid is a potent inducer of CD38 and can be used as a novel therapeutic strategy in autism. We investigated the role of beta-carotene in rescuing autistic-like behavior in BALB/c and BTBR mice. Beta-carotene derivatives are preferred as they are neither toxic nor teratogenic. Beta-carotene at 0.1-5.0 mg/kg was administered orally to BALB/c and BTBR newborn mice on days 1-7. They were tested at age 2-3 months for five behavioral tests for "autism"; in addition, brain CD38, oxytocin, oxytocin receptor, Brain Derived Neurotrophic Factor (BDNF) and retinoic acid receptor gene expression, serum oxytocin levels, and neurological score were evaluated. Beta-carotene administered at birth significantly increased T-maze alternations and led to longer time spent with an unfamiliar mouse in the "three-chamber test" and less time spent in the empty chamber. Furthermore, enhanced activity in the open field test; increased time spent in the reciprocal social interaction test; decreased grooming and bedding behaviors; and enhanced brain CD38, oxytocin, oxytocin receptor, BDNF, retinoic acid gene expression, and serum oxytocin levels. No changes in neurological score were observed. Beta-carotene oral supplementation to BALB/c and BTBR mice at birth significantly reduced restricted and stereotyped behaviors and interests, increased social interactions and communication, CD38, and oxytocin, probably by enhancing brain neuroplasticity without toxicity. Thus, beta-carotene administered after birth to newborns of families predisposed to "autism" has the potential to prevent/ameliorate" autistic like behavior". These results support further clinical studies.

Novel fatty acid chain modified GLP-1 derivatives with prolonged in vivo glucose-lowering ability and balanced glucoregulatory activity.

Glucagon-like peptide-1 is a potent hypoglycemic hormone with beneficial properties for the treatment of diabetes. However, its half-life is short because the rapid metabolic degradation. This study aims to prolong the half-life of glucagon-like peptide-1 through conjugation with the fatty acid side chain which helps the conjugates to interact with the albumin. Firstly, we chose two optimized polypeptide chains which have tremendous hypoglycemic effect named Cys17-Gly8-GLP-1(7-36)-NH2 and Cys37-Gly8-GLP-1(7-37)-NH2, and various fatty acid chains were modified. All conjugates preserved relatively strong GLP-1R activation and I-6 behaved best in glucose-lowering ability. The prolonged antidiabetic effects of I-6 were further confirmed by hypoglycemic efficacy test in vivo. Meanwhile, once daily injection of I-6 to diabetic mice achieved long-term beneficial effects on glucose tolerance, body weight and blood chemistry. It is concluded that I-6 is a promising agent for further investigation of its potential to treat obese patients with diabetes.

Leptin resistance and desensitization of hypophagia during prolonged inflammatory challenge.

Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of 2-arachidonoylglycerol (2-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high 2-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, 2-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on 2-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and 2-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

Fish oil promotes survival and protects against cognitive decline in severely undernourished mice by normalizing satiety signals.

Severe malnutrition resulting from anorexia nervosa or involuntary starvation leads to low weight, cognitive deficits and increased mortality rates. In the present study, we examined whether fish oil supplementation, compared with that of canola oil, would ameliorate the morbidity and mortality associated with these conditions by normalizing endocannabinoid and monoaminergic systems as well as other systems involved in satiety and cognitive function within the hypothalamus and hippocampus. Female Sabra mice restricted to 40% of their daily food intake exhibited decreased body weight, were sickly in appearance, displayed cognitive deficits and had increased mortality rates. Strikingly, fish oil supplementation that contains high omega-3 fatty acids levels decreased mortality and morbidity, and normalized the expression of genes and neurotransmitters in the hippocampus and hypothalamus. Fish oil supplementation, but not canola oil, increased survival rates, improved general appearance and prevented cognitive decline, despite the facts that both diets contained an equivalent number of calories and that there were no differences in weight between mice maintained on the two diets in 100% but decrease in the 40%. In the hypothalamus, the beneficial effects of fish oil supplementation were related to normalization of the endocannabinoid 2-arachidonylglycerol, serotonin (5-HT) (P<.056), dopamine, neuropeptide Y (NPY) and Ca(2+)/calmodulin (CaM)-dependent protein kinase (Camkk2). In the hippocampus, fish oil supplementation normalized 5-HT, Camkk2, silent mating type information regulation 1 and brain-derived neurotrophic factor. In conclusion, dietary supplements of fish oil, as source of omega-3 fatty acids, may alleviate cognitive impairments associated with severe diet restriction and prolong survival independently of weight gain by normalizing neurochemical systems.

Cannabidiol ameliorates cognitive and motor impairments in mice with bile duct ligation.

BACKGROUND/AIMS: The endocannabinoid system in mice plays a role in models of human cirrhosis and hepatic encephalopathy (HE), induced by a hepatotoxin. We report now the therapeutic effects of cannabidiol (CBD), a non-psychoactive constituent of Cannabis sativa, on HE caused by bile duct ligation (BDL), a model of chronic liver disease. METHODS: CBD (5mg/kg; i.p.) was administered over 4weeks to mice that had undergone BDL. RESULTS: Cognitive function in the eight arm maze and the T-maze tests, as well as locomotor function in the open field test were impaired by the ligation and were improved by CBD. BDL raised hippocampal expression of the TNF-alpha-receptor 1 gene, which was reduced by CBD. However, BDL reduced expression of the brain-derived neurotrophic factor (BDNF) gene, which was increased by CBD. The effects of CBD on cognition, locomotion and on TNF-alpha receptor 1 expression were blocked by ZM241385, an A(2)A adenosine receptor antagonist. BDL lowers the expression of this receptor. CONCLUSIONS: The effects of BDL apparently result in part from down-regulation of A(2)A adenosine receptor. CBD reverses these effects through activation of this receptor, leading to compensation of the ligation effect.

2-Arachidonoylglycerol, an endogenous cannabinoid receptor agonist, in various rat tissues during the evolution of experimental cholestatic liver disease.

BACKGROUND/AIM: Changes in tissue levels of 2-arachidonoylglycerol (2-AG), an endocannabinoid, during the evolution of bile duct ligation (BDL) may indicate that endocannabinoids have a role in the hemodynamic changes that occur in this condition. METHODS: 2-AG levels, in various organs and vascular beds of BDL rats, 2 and 4 weeks post surgery, were determined. Untouched and sham-operated (SO) rats were used as controls. RESULTS: 2-AG content of a specific organ was not a static finding and depended on the rat's age, the time from the surgical procedure and the type of procedure. The most pronounced changes were observed in BDL rats 4 weeks post surgery. In these rats, hepatic, pulmonary, cardiac and renal medullary and papillary 2-AG levels were highest observed. No changes in splenic, aortic and renal cortical 2-AG levels were observed. In addition a stepwise increase in 2-AG levels from the cortex to the papilla was detected and was followed by a decrease in creatinine clearance. CONCLUSIONS: 2-AG probably has a role in the pathophysiologic changes in the liver, heart, lung and kidney that follows BDL.

The synthetic cannabinoid HU-210 attenuates neural damage in diabetic mice and hyperglycemic pheochromocytoma PC12 cells.

Diabetic neuropathy (DN) is a common complication of diabetes mellitus resulting in cognitive dysfunction and synaptic plasticity impairment. Hyperglycemia plays a critical role in the development and progression of DN, through a number of mechanisms including increased oxidative stress. Cannabinoids are a diverse family of compounds which can act as antioxidative agents and exhibit neuroprotective properties. We investigated the effect of the synthetic cannabinoid HU-210 on brain function of streptozotocin (STZ)-induced diabetic mice. These animals exhibit hyperglycemia, increased cerebral oxidative stress and impaired brain function. HU-210, through a receptor independent pathway, alleviates the oxidative damage and cognitive impairment without affecting glycemic control. To study the neuroprotective mechanism(s) involved, we cultured PC12 cells under hyperglycemic conditions. Hyperglycemia enhanced oxidative stress and cellular injuries were all counteracted by HU-210-in a dose dependent manner. These results suggest cannabinoids might have a therapeutic role in the management of the neurological complications of diabetes.

Nutritional status, cognition, and survival: a new role for leptin and AMP kinase.

Although adequate nutrition is essential for optimal neural activity and survival, mild energy restriction may improve cognition and prolong longevity. Energy status is monitored by the cellular AMP-activated protein kinase (AMPK) system, whereas leptin regulates total energy balance. We investigated the roles of AMPK and leptin in cognition and survival under diet restriction (DR). Hippocampal AMPK activity increases with energy restriction. Modest activation (DR to 60%) induces neurogenesis and improves cognition. However, DR to 40% augmented AMPK activity, reduced cognition and catecholamines, and increased neural apoptosis and mortality. Leptin signaling is preserved only in DR to 60%, countering the effects of AMPK "overactivation" by preventing neuroapoptosis, restoring noradrenergic activity and behavioral performance, and increasing longevity. The balance between leptin and AMPK is crucial in determining neuronal fate, cognitive ability, and survival. Should these findings extend to Man, then controlled activation of AMPK may improve neurodegenerative diseases, and leptin may have a new role in treating stress-associated malnutrition.

Effects of the endocannabinoid noladin ether on body weight, food consumption, locomotor activity, and cognitive index in mice.

We have investigated the effect of 2-arachidonylglyceryl-ether (Noladin) on food consumption, weight, activity, and cognitive function in mice during diet restriction for 17 days and subsequent ad libitum feeding for 32 days. Female Sabra mice were given food for 2.5 h/day (equal to 60% diet restriction), received Noladin (0.001, 0.01, 0.1 mg/(kg day) intraperitonially (i.p.)) with or without the CB1 antagonist SR141716A (1 mg/kg i.p.) during days 3-17. Noladin (0.001 mg/kg) significantly increased food consumption without a change in body weight, probably due to increased activity and there was no change in cognitive function. A higher dose (0.1 mg/kg) did not affect food consumption, but increased activity and slightly decreased weight 32 days after termination of Noladin administration; however, cognitive deterioration was observed. At all doses tested, Noladin did not affect weight during the diet-restriction period, whereas the CB1 antagonist (with or without Noladin) caused a very significant decline in weight in this phase. Weight catch-up was observed 1 month after administration of Noladin was discontinued. Weight at day 32 after the termination of Noladin (0.1 mg/(kg day)) treatment was 5% less than control. Female C57BL/6 mice (same protocol, with 0.001 mg/(kg day) Noladin) gave similar results to 0.1 mg/kg in Sabra mice as regards weight. CB1 antagonist treatment caused very significant decline in both weight and food consumption; cognition and activity were unchanged. These results indicate that Noladin has a significant dose-dependent effect on food consumption, cognition and weight maintenance after weight loss. Low doses of Noladin may possibly allow an increase in food intake without a gain in weight after dieting. Thus, Noladin could be of potential clinical benefit in treating disorders of body weight. Noladin seems to signal food consumption and weight through CB1 receptors based on effects observed with the CB1 antagonist, while the cognition and activity are probably mediated by non-cannabinoid receptors.

Immunomodulation of experimental colitis via caloric restriction: role of Nk1.1+ T cells.

Inflammatory bowel diseases are immune-mediated disorders. Dietary restriction and NK1.1+ liver-associated lymphocytes (LAL) are considered to be involved in immunomodulation of autoimmune diseases. Our aim was to evaluate the effect of caloric restriction on experimental colitis and to determine NK1.1+ LAL function in immunoregulation. Experimental colitis was induced in C57 black mice by intracolonic instillation of trinitrobenzene sulfonic acid. Caloric restriction to 60% of the daily requirement was started 2 weeks prior to, or simultaneously with, colitis induction and continued throughout the study. Control mice were fed ad libitum. Colitis was assessed by standard clinical and macroscopic scores. To determine the mechanism involved in immunomodulation, liver lymphocytes were isolated and analyzed for NK1.1+ T-cell markers by FACS. T-cell function was evaluated by T-cell proliferation. Serum cytokines were measured by ELISA. Dietary restriction to 60% markedly ameliorated experimental colitis in both groups. These mice gained weight and showed improved macroscopic parameters of colitis. NK1.1+ LAL numbers increased fourfold and NKT cytotoxicity twofold in caloric-restricted mice. The antigen-specific T-cell proliferation index decreased (from 4.45 in controls to 1.15), and IFN-gamma and IL-12 serum levels decreased (from 290 to 200 pg and from 122 to 53 pg, respectively) in caloric-restricted mice. Our conclusion was that dietary restriction induced immunomodulation of experimental colitis and ameliorated the disease. This effect was mediated via an increase in NK1.1+ T lymphocytes, which may play a critical role in keeping the T-cell balance in immunoregulation.

Hypothalamic-pituitary-adrenal responses to weight loss in mice following diet restriction, activity or separation stress: effects of tyrosine.

We have studied three different types of weight-loss stress caused by Diet restriction, Activity or Separation, for their effects on the hypothalamic-pituitary axis in young female mice and their responses to tyrosine 100 mg/kg/day. Plasma was assayed for ACTH and glucocorticoid determinations, and brain catecholamine concentrations were measured by HPLC/ECD. A similar weight loss of 24-28% was observed in the models despite significant differences in food intake. Diet restriction to 60% and Separation models produced a significant increase in hypothalamic noradrenaline (p < 0.01), while there was a significant decrease (p < 0.05) in the Diet restriction to 40% that was restored after tyrosine. After Activity, noradrenaline levels did not change. ACTH concentrations decreased following Diet restriction (p < 0.05) but were unaffected by Separation or Activity. The peripheral glucocorticoid response increased significantly after Activity and Diet restriction (p < 0.001), but decreased significantly after Separation (p < 0.001). Tyrosine increased glucocorticoid concentrations in the Activity and Separation models (p < 0.05), but not after Diet restriction. Despite similar weight loss in the three models there were no predictable associations between hypothalamic noradrenaline metabolism and plasma ACTH or glucocorticoid concentrations. Tyrosine might alleviate some of the different pathophysiological problems associated with the stress of weight loss.