Ingestion of whey protein and ß-conglycinin exerts opposite effects on intestinal FGF15 and serotonin secretion in mice.

Farnesoid X receptor (FXR) and Takeda G protein-coupled Receptor 5 (TGR5), the intestinal bile acid (BA) receptors, regulate the gut-derived hormones including fibroblast growth factor 15/19 (FGF15/19) and serotonin (5-hydrooxytryptamine, 5-HT). Here we show that ingestion of whey protein isolate, a milk protein, significantly decreased expression of heteromeric organic solute transporter Ostα and Ostß, which is the basolateral BA transporter in the enterocyte, and increased the expression of FXR and FGF15 in C57BL6J mouse ileum and plasma FGF15 levels. In addition, the ingestion of whey protein isolate significantly suppressed expression of hepatic cholesterol-7α hydroxylase (CYP7A1), which induces the primary BA synthesis, bile salt export pump (BSEP) and sodium-taurocholate cotransporting polypeptide (NTCP), which are the key transporters for the BA excretion and uptake in the liver, and genes involved in gluconeogenesis, and decreased the primary BAs including cholic acid, taurocholic acid, glycocholic acid, and taurochenodeoxycholic acid in the liver compared with controls. Moreover, ingestion of whey protein isolate significantly decreased the expression of TGR5, glucagon-like peptide 1 (GLP-1), and tryptophan hydroxylase1 (Tph1) in the small intestine, leading to decreases in plasma 5-HT and insulin levels. On the other hand, ingestion of the soy protein ß-conglycinin significantly increased the expression of Ostα and Ostß, and decreased the expression of FGF15 in the ileum and plasma FGF15 levels, leading to the increases in expression of hepatic CYP7A1, BSEP, NTCP, and genes involved in gluconeogenesis, and the primary BAs in the liver. Moreover, ingestion of ß-conglycinin significantly increased the expression of intestinal TGR5, GLP-1, and Tph1, leading to increases in plasma 5-HT and insulin levels. These findings suggest that whey protein and ß-conglycinin have opposite effects on intestinal FGF15 and 5-HT secretion in mice.

The Regulatory Role of the Central and Peripheral Serotonin Network on Feeding Signals in Metabolic Diseases.

Central and peripheral serotonin (5-hydroxytryptamine, 5-HT) regulate feeding signals for energy metabolism. Disruption of central 5-HT signaling via 5-HT2C receptors (5-HT2CRs) induces leptin-independent hyperphagia in mice, leading to late-onset obesity, insulin resistance, and impaired glucose tolerance. 5-HT2CR mutant mice are more responsive than wild-type mice to a high-fat diet, exhibiting earlier-onset obesity and type 2 diabetes. High-fat and high-carbohydrate diets increase plasma 5-HT and fibroblast growth factor-21 (FGF21) levels. Plasma 5-HT and FGF21 levels are increased in rodents and humans with obesity, type 2 diabetes, and non-alcohol fatty liver diseases (NAFLD). The increases in plasma FGF21 and hepatic FGF21 expression precede hyperinsulinemia, insulin resistance, hyperglycemia, and weight gain in mice fed a high-fat diet. Nutritional, pharmacologic, or genetic inhibition of peripheral 5-HT synthesis via tryptophan hydroxylase 1 (Tph1) decreases hepatic FGF21 expression and plasma FGF21 levels in mice. Thus, perturbing central 5-HT signaling via 5-HT2CRs alters feeding behavior. Increased energy intake via a high-fat diet and/or high-carbohydrate diet can upregulate gut-derived 5-HT synthesis via Tph1. Peripheral 5-HT upregulates hepatic FGF21 expression and plasma FGF21 levels, leading to metabolic diseases such as obesity, insulin resistance, type 2 diabetes, and NAFLD. The 5-HT network in the brain-gut-liver axis regulates feeding signals and may be involved in the development and/or prevention of metabolic diseases.

Pharmacologic inhibition of serotonin htr2b ameliorates hyperglycemia and the altered expression of hepatic FGF21, Sdf2l1, and htr2a in db/db mice and KKAy mice.

Plasma fibroblast growth factor 21 (FGF21) levels and hepatic FGF21, serotonin 2a receptor (htr2a), and stromal cell-derived factor 2 like 1 (Sdf2l1) expression are increased in insulin-resistant C57BL6J mice fed a high-fat diet. Here we show that plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression were decreased in 6-week-old db/db mice compared with C57BL6J mice, whereas they were increased in 6-week-old KKAy mice compared with KK mice. Expression of hepatic htr2b was increased in db/db mice and KKAy mice compared with controls. Treatment with the selective htr2b antagonist SB204741 suppressed the hyperglycemia in either db/db mice or KKAy mice. Treatment with SB20471 reversed the decreases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in db/db mice, whereas it suppressed the increases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in KKAy mice. Moreover, treatment with SB204741 increased plasma FGF21 levels and expression of hepatic FGF21, htr2a, and Sdf2l1 in C57BL6J mice, whereas it decreased plasma FGF21 levels and hepatic FGF21 expression in KK mice. These findings suggest that pharmacologic inhibition of htr2b ameliorates the hyperglycemia and altered expression of hepatic FGF21, Sdf2l1 and htr2a in obese and diabetic db/db and KKAy mice.

Whey protein isolate inhibits hepatic FGF21 production, which precedes weight gain, hyperinsulinemia and hyperglycemia in mice fed a high-fat diet.

Insufficient expression of hepatic fibroblast growth factor 21 (FGF21) and stromal cell-derived factor 2 like 1 (Sdf2l1) reportedly leads to insulin resistance and hepatosteatosis in obesity and type 2 diabetes. On the other hand, increased expression of hepatic serotonin receptor 2a (htr2a) in diet-induced obesity contributes to hepatosteatosis. Here we show that increases in circulating FGF21 levels and expression of hepatic FGF21 preceded weight gain, hyperinsulinemia, and hyperglycemia in C57BLJ6 mice fed a high-fat diet. Expression of hepatic htr2a and Sdf2l1 increased in insulin-resistant mice fed a high-fat diet. Intake of whey protein isolate decreased plasma FGF21 levels and expression of hepatic FGF21 in mice fed either a high-fat diet or a chow diet, whereas it only suppressed the overexpression of hepatic Sdf2 and htr2a in insulin-resistant mice fed a high-fat diet. Moreover, intake of whey protein isolate decreased plasma serotonin levels in mice fed either a high-fat diet or a chow diet. Genetic inhibition of tryptophan hydroxylase 1 decreased hepatic FGF21 expression and plasma FGF21 levels in mice. These findings suggest that increased hepatic FGF21 production precedes diet-induced weight gain, hyperinsulinemia, and hyperglycemia, and that intake of whey protein isolate could inhibit hepatic FGF21 production by suppressing peripheral serotonin synthesis.

Low-frequency and low-intensity ultrasound increases cardiac parasympathetic neural activity and decreases clinic hypertension in elderly hypertensive subjects with type 2 diabetes.

BACKGROUND: The aims of the present study were to determine the effects of an ultrasound irradiation on clinic hypertension and the heart rate variability in elderly hypertensive subjects with type 2 diabetes. METHODS: We examined the effects of ultrasound (800 kHz, 25 mW/cm2) applied to the forearm for 10 min on the autonomic nerve activity and the difference between BP at home and at a clinic visit in Japanese subjects with type 2 diabetes and hypertension. RESULTS: In 108 subjects who displayed systolic BP (SBP) >140 mm Hg at a clinic visit, 75 subjects (69%) had a mean SBP <135 mm Hg at home and 33 subjects (31%) had a mean SBP >135 mm Hg at home in the morning for 14 days. SBP, pulse rate, and pulse pressure in the ultrasound treatment group were significantly lower than the baseline values in these hypertensive subjects with type 2 diabetes, and lower than those of placebo controls independently of SBP at home. In 31 subjects who displayed systolic BP >140 mm Hg at a clinic, standard deviation of all RR intervals and the root mean square of successive differences were significantly higher in the ultrasound treatment group than the baseline values in these hypertensive subjects with type 2 diabetes, and lower than those of placebo controls. CONCLUSIONS: The ultrasound treatment increases the cardiac parasympathetic neural activity and decreases the differences between SBP at home and at a clinic visit in elderly hypertensive subjects with type 2 diabetes.

Liraglutide, a GLP-1 Receptor Agonist, Which Decreases Hypothalamic 5-HT2A Receptor Expression, Reduces Appetite and Body Weight Independently of Serotonin Synthesis in Mice.

A recent report suggested that brain-derived serotonin (5-HT) is critical for maintaining weight loss induced by glucagon-like peptide-1 (GLP-1) receptor activation in rats and that 5-HT2A receptors mediate the feeding suppression and weight loss induced by GLP-1 receptor activation. Here, we show that changes in daily food intake and body weight induced by intraperitoneal administration of liraglutide, a GLP-1 receptor agonist, over 4 days did not differ between mice treated with the tryptophan hydroxylase (Tph) inhibitor p-chlorophenylalanine (PCPA) for 3 days and mice without PCPA treatment. Treatment with PCPA did not affect hypothalamic 5-HT2A receptor expression. Despite the anorexic effect of liraglutide disappearing after the first day of treatment, the body weight loss induced by liraglutide persisted for 4 days in mice treated with or without PCPA. Intraperitoneal administration of liraglutide significantly decreased the gene expression of hypothalamic 5-HT2A receptors 1 h after injection. Moreover, the acute anorexic effects of liraglutide were blunted in mice treated with the high-affinity 5-HT2A agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide 14 h or 24 h before liraglutide injection. These findings suggest that liraglutide reduces appetite and body weight independently of 5-HT synthesis in mice, whereas GLP-1 receptor activation downregulates the gene expression of hypothalamic 5-HT2A receptors.

Low-frequency and low-intensity ultrasound irradiation to the forearm improves an index of arterial stiffness in subjects with type 2 diabetes and hypertension.

OBJECTIVES: The arterial pressure-volume index (API) is a non-invasive assessment of arterial stiffness, and is suggested as a useful predictor of future cardiovascular events. The aim of the present study was to determine the effects of low-frequency and low-intensity ultrasound applied to the forearm for 10 min on the API in Japanese subjects with type 2 diabetes and hypertension. METHODS: We examined the effects of low-frequency and low-intensity ultrasound (800 kHz, 25 mW/cm2) applied to the forearm for 10 min on the API, blood pressure (BP) and pulse rate in 40 Japanese subjects (13 men and 27 women; mean age ± SE, 70 ± 2 years) with type 2 diabetes and hypertension, who had the API > 30 and systolic BP > 140 mmHg at a clinic visit. We also examined the effects of the ultrasound irradiation for 10 min on the API, BP and pulse rate in 33 Japanese subjects (11 men and 22 women; mean age ± SE, 65 ± 2 years) with type 2 diabetes and hypertension, who had the API > 30 and systolic BP (SBP) < 140 mmHg. RESULTS: The API, systolic BP and pulse rate in the ultrasound treatment group was significantly lower than the baseline values in the subjects who had the API > 30 and either the baseline of systolic BP > 140 mmHg or systolic BP < 140 mmHg. CONCLUSIONS: The low-frequency and low-intensity ultrasound irradiation to the forearm for 10 min might be useful as a preventive application for arterial stiffness in subjects with type 2 diabetes and hypertension.

α1-Adrenergic receptor downregulates hepatic FGF21 production and circulating FGF21 levels in mice.

Fibroblast growth factor 21 (FGF21) is primarily secreted by the liver as an endocrine hormone and is suggested as a promising target for the treatment of metabolic diseases. FGF21 acts centrally to exert its effects on energy expenditure and body weight via the sympathetic nervous system in mice. Here we show that intraperitoneal injection of phentolamine (an α-adrenergic receptor antagonist, 5mg/kg) significantly increased plasma FGF21 levels compared with the saline controls in C57BL6J mice, whereas alprenolol (a ß-adrenergic receptor antagonist, 6mg/kg) had no effect. In addition, intraperitoneal injection of prazosin (an α1-adrenergic receptor antagonist, 5mg/kg) significantly increased plasma FGF21 levels compared with the controls, whereas yohimbine (an α2-adrenergic receptor antagonist, 5mg/kg) had no effect. Moreover, the treatment with prazosin significantly increased the expression of hepatic FGF21, while having no effect on the expression of hepatic PPARα and PPARγ. After a 5-h fast, intraperitoneal injection of prazosin significantly increased plasma FGF21 levels and impaired glucose tolerance compared with controls. These findings suggest that α1-adrenergic receptor downregulates the expression of hepatic FGF21 and plasma FGF21 levels independently of feeding and hepatic PPARα and PPARγ expression in mice, and that the increases in circulating FGF21 levels might be related to impaired glucose tolerance.

The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, does not require functional leptin receptor, serotonin, and hypothalamic POMC and CART activities in mice.

The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, did not require functional leptin receptor, serotonin, and hypothalamic proopiomelanocortin and cocaine amphetamine regulated transcript activities in mice, although decrease in functional hypothalamic orexin activity might be involved in the acute anorexic effect of liraglutide.

Low-frequency and very low-intensity ultrasound decreases blood pressure in hypertensive subjects with type 2 diabetes.

BACKGROUND: Despite lifestyle interventions and various types of anti-hypertension agents, hypertension remains difficult to control in some patients with type 2 diabetes. As a noninvasive device-based approach for the treatment of clinic hypertension, we examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on blood pressure (BP) and pulse rate in Japanese subjects with type 2 diabetes and hypertension. METHODS: We examined the effects of low-frequency and low-intensity ultrasound (500 or 800kHz, 25mW/cm(2)) applied to the forearm on BP, pulse rate, and pulse pressure in 212 Japanese subjects (82 men and 130 women; mean age±SE, 65±1years) with type 2 diabetes and hypertension (systolic BP>140mmHg). The subjects were treated with anti-hypertension agents. RESULTS: Systolic and diastolic BP, pulse rate, pulse pressure in the 800-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and lower than those of placebo controls. In addition, systolic and diastolic BP, pulse rate, and pulse pressure in the 500-kHz ultrasound treatment group were significantly lower than the baseline values in hypertensive subjects with type 2 diabetes, and systolic BP, pulse rate, and pulse pressure were significantly lower than those of placebo controls. CONCLUSIONS: Low-frequency (800kHz or 500kHz) and low-intensity (25mW/cm(2)) ultrasound irradiation to the forearm might have potential usefulness as a therapeutic application for clinic hypertension in subjects with type 2 diabetes.

Pharmacologic stimulation of central GLP-1 receptors has opposite effects on the alterations of plasma FGF21 levels induced by feeding and fasting.

Fibroblast growth factor 21 (FGF21) functions as an endocrine hormone to regulate energy metabolism. Circulating FGF21 is derived from the liver and is produced in response to alterations of nutritional status. Here we show the effects of liraglutide, a human glucagon-like-peptide-1 (GLP-1) receptor agonist, injected into the third cerebral ventricle on body weight and plasma FGF21 levels in free-feeding mice, food-deprived mice, and mice provided 1g after the injection. In free-feeding mice, liraglutide (5-100µg/kg) injected into the third cerebral ventricle suppressed food intake and body weight after 24h in a dose-dependent manner. Liraglutide (50 and 100µg/kg) significantly increased plasma FGF21 levels and hepatic FGF21 expression, whereas smaller doses (5 and 10µg/kg) had no effect. In food-deprived mice, body weight did not differ significantly between the saline control and liraglutide-treated groups, but liraglutide (100µg/kg) significantly decreased plasma FGF21 levels at 24h compared with the saline control. In mice provided 1g food, body weight did not differ significantly between the saline control and liraglutide-treated groups, but liraglutide (50µg/kg) significantly decreased plasma FGF21 levels at 24h compared with the saline control. These findings suggest that intracerebral injection of liraglutide decreases body weight by inhibiting food intake and increases plasma FGF21 levels in free-feeding mice, whereas it suppresses the elevations of plasma FGF21 levels induced by fasting or the restricted feeding. Thus, pharmacologic stimulation of central GLP-1 receptors has opposite effects on the alterations of plasma FGF21 levels induced by feeding and fasting.

Mosapride, a selective serotonin 5-HT4 receptor agonist, and alogliptin, a selective dipeptidyl peptidase-4 inhibitor, exert synergic effects on plasma active GLP-1 levels and glucose tolerance in mice.

Pharmacologic stimulation of serotonin 5-HT4 receptors increased plasma active glucagon-like-peptide-1 (GLP-1) levels independent of feeding, and that pharmacologic stimulation of 5-HT4 receptors and pharmacologic inhibition of dipeptidyl peptidase-4 exerted synergic effects on plasma active GLP-1 levels and glucose tolerance in mice.

Ingestion of eicosapentaenoic acid in the early stage of social isolation reduces a fibroblast growth factor 21 resistant state independently of body weight in KKA(y) mice.

Fibroblast growth factor (FGF) 21 is a mediator of glucose and lipid metabolism. Although exogenous administration of FGF21 exerts beneficial effects on glucose and lipid metabolism, circulating FGF21 levels are elevated in ob/ob and db/db mice, diet-induced obese mice and obese human. Here we show that ingestion of eicosapentaenoic acid (EPA) for 6 days after individually-housing significantly suppressed the hyperglycemia and hypertriglyceridemia associated with decreases in plasma insulin and FGF21 levels in KKA(y) mice while having no effects on food intake, body weight or plasma active GLP-1 levels. The ingestion of EPA had no significant effects on the expression of FGF21 in the liver, epididymal white adipose tissue and skeletal muscle. Moreover, the ingestion of EPA significantly decreased the expression of hepatic peroxisome sterol regulatory element-binding protein (SREBP1c), carbohydrate response element-binding protein (ChREBP), stearoyl-CoA deaturase and periostin, which are involved in hepatic lipogenesis and hepatosteaotosis, in KKA(y) mice. On the other hand, the ingestion of EPA had no significant effects on expression of hepatic gp78, Notch, forkhead box protein O1 or glucose-6-phosphatase. These findings suggest that EPA ingestion in the early stage of social isolation suppresses hyperglycemia and hypertriglyceridemia associated with reduced FGF21 and insulin resistance without altering food intake and body weight, and that the EPA ingestion suppresses hepatic lipogenesis by suppressing Notch- and gp78-independent SEREBP1c and ChREBP pathways in KKA(y) mice.

Liraglutide suppresses obesity and hyperglycemia associated with increases in hepatic fibroblast growth factor 21 production in KKAy mice.

Social isolation contributes to the development of obesity and insulin-independent diabetes in KKA(y) mice. Here we show that systemic administration of liraglutide, a long-acting human glucagon-like peptide-1 (GLP-1) analog, significantly decreased food intake, body weight, and blood glucose levels at 24 h after its administration while having no significant effects on plasma insulin and glucagon levels in individually housed KKA(y) mice. In addition, the systemic administration of liraglutide significantly increased plasma fibroblast growth factor (Fgf) 21 levels (1.8-fold increase) associated with increases in the expression of hepatic Fgf21 (1.9-fold increase) and Pparγ (1.8-fold increase), while having no effects on the expression of hepatic Pparα and Fgf21 in white adipose tissue. Moreover, systemic administration of liraglutide over 3 days significantly suppressed food intake, body weight gain, and hyperglycemia in KKA(y) mice. On the other hand, despite remarkably increased plasma active GLP-1 levels (4.2-fold increase), the ingestion of alogliptin, a selective dipeptidyl peptidase-4 inhibitor, over 3 days had no effects on food intake, body weight, blood glucose levels, and plasma Fgf21 levels in KKA(y) mice. These findings suggest that systemic administration of liraglutide induces hepatic Fgf21 production and suppresses the social isolation-induced obesity and diabetes independently of insulin, glucagon, and active GLP-1 in KKA(y) mice.

Liraglutide suppresses the plasma levels of active and des-acyl ghrelin independently of active glucagon-like Peptide-1 levels in mice.

Glucagon-like peptide-1 (GLP-1), an insulinotropic gastrointestinal peptide that is primarily produced by intestinal endocrine L-cells, stimulates satiety. Ghrelin, a hormone that is produced predominantly by the stomach stimulates hunger. There are two forms of ghrelin: active ghrelin and inactive des-acyl ghrelin. After depriving mice of food for 24 h, we demonstrated that the systemic administration of liraglutide (100 µ g/kg), a human GLP-1 analog that binds to the GLP-1 receptor, increased (1.4-fold) the plasma levels of active GLP-1 and suppressed the plasma levels of active and des-acyl ghrelin after 1 h. Despite the elevated plasma levels of active GLP-1 (11-fold), liraglutide had no effect on the plasma levels of active or des-acyl ghrelin after 12 h. These findings demonstrated that liraglutide suppresses the plasma levels of active and des-acyl ghrelin independently of active GLP-1 levels in fasted mice, suggesting a novel in vivo biological effect of liraglutide beyond regulating plasma GLP-1.

Role of homeobox genes in the hypothalamic development and energy balance.

Homeobox genes contribute to the regionalization, patterning and cell differentiation during embryogenesis and organ development. During mammalian embryonic development, homeobox genes, including orthopedia (Otp), a brain-specific homeobox transcription factor (Bsx) and a thyroid transcription factor-1 (TTF-1), are expressed in the hypothalamus. The genetic ablation of these genes indicated that Otp and TTF-1 are essential for the normal morphological development of the hypothalamus, including the arcuate nucleus (ARC), whereas Bsx is not required. In the adult stage, Bsx and TTF-1 continue to be expressed in the hypothalamus, including the ARC, and serve as transcription factors of neuropeptide Y and agouti-related protein. The expression of hypothalamic Bsx and TTF-1 can be altered by the feeding state and appetite regulatory hormones such as ghrelin and leptin. Although Bsx and TTF-1 are essential for normal feeding behavior in adult mice, they exert different effects on the expression of hypothalamic pro-opiomelanocortin (POMC) and body weight homeostasis. Thus, the hypothalamic homeobox genes may contribute to the dissociation of food intake and body weight via AgRP-POMC neurons.

Serotonin conflict in sleep-feeding.

Short sleep duration has been suggested to be a risk factor for weight gain and adiposity. Serotonin (5-HT) substantially contributes to the regulation of sleep and feeding behavior. Although 5-HT predominately promotes waking and satiety, the effects of 5-HT depend on 5-HT receptor function. The 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT6, and 5-HT7 receptors reportedly contribute to sleep-waking regulation, whereas the 5-HT1B and 5-HT2C receptors contribute to the regulation of satiety. The 5-HT1B and 2C receptors may therefore be involved in the regulation of sleep-feeding. In genetic studies, 5-HT1B receptor mutant mice display greater amounts of rapid eye movement sleep (REMS) than wild-type mice, while displaying no effects on waking or slow wave sleep (SWS). On the other hand, 5-HT2C receptor mutant mice exhibit increased wakefulness and decreased SWS, without any effect on REMS. Moreover, the 5-HT2C receptor mutants display leptin-independent hyperphagia, leading to a middle-aged onset of obesity, whereas 5-HT1B receptor mutants do not display any effect on food intake. Thus, the genetic deletion of 5-HT2C receptors results in sleep loss-associated hyperphagia, leading to the late onset of obesity. This is a quite different pattern of sleep-feeding behavior than is observed in disturbed leptin signaling, which displays an increase in sleep-associated hyperphagia. In pharmacologic studies, 5-HT1B and 5-HT2C receptors upregulate wakefulness and downregulate SWS, REMS, and food intake. These findings suggest that 5-HT1B/2C receptor stimulation induces sleep loss-associated anorexia. Thus, the central 5-HT regulation of sleep-feeding can be dissociated. Functional hypothalamic proopiomelanocortin and orexin activities may contribute to the dissociated 5-HT regulation.