Megumi Aizawa-Abe

Pathophysiological role of leptin in obesity-related hypertension

To explore the pathophysiological role of leptin in obesity-related hypertension, we examined cardiovascular phenotypes of transgenic skinny mice whose elevated plasma leptin concentrations are comparable to those seen in obese subjects. We also studied genetically obese KKA(y) mice with hyperleptinemia, in which hypothalamic melanocortin system is antagonized by ectopic expression of the agouti protein. Systolic blood pressure (BP) and urinary catecholamine excretion are elevated in transgenic skinny mice relative to nontransgenic littermates. The BP elevation in transgenic skinny mice is abolished by alpha(1)-adrenergic, beta-adrenergic, or ganglionic blockers at doses that do not affect BP in nontransgenic littermates. Central administration of an alpha-melanocyte-stimulating hormone antagonist causes a marked increase in cumulative food intake but no significant changes in BP. The obese KKA(y) mice develop BP elevation with increased urinary catecholamine excretion relative to control KK mice. After a 2-week caloric restriction, BP elevation is reversed in nontransgenic littermates with the A(y) allele, in parallel with a reduction in plasma leptin concentrations, but is sustained in transgenic mice overexpressing leptin with the A(y) allele, which remain hyperleptinemic. This study demonstrates BP elevation in transgenic skinny mice and obese KKA(y) mice that are both hyperleptinemic, thereby suggesting the pathophysiological role of leptin in some forms of obesity-related hypertension.

Accelerated puberty and late-onset hypothalamic hypogonadism in female transgenic skinny mice overexpressing leptin

Excess or loss of body fat can be associated with infertility, suggesting that adequate fat mass is essential for proper reproductive function. Leptin is an adipocyte-derived hormone that is involved in the regulation of food intake and energy expenditure, and its synthesis and secretion are markedly increased in obesity. Short-term administration of leptin accelerates the onset of puberty in normal mice and corrects the sterility of leptin-deficient ob/ob mice. These findings suggest a role for leptin as an endocrine signal between fat depots and the reproductive axis, but the effect of hyperleptinemia on the initiation and maintenance of reproductive function has not been elucidated. To address this issue, we examined the reproductive phenotypes of female transgenic skinny mice with elevated plasma leptin concentrations comparable to those in obese subjects. With no apparent adipose tissue, female transgenic skinny mice exhibit accelerated puberty and intact fertility at younger ages followed by successful delivery of healthy pups. However, at older ages, they develop hypothalamic hypogonadism characterized by prolonged menstrual cycles, atrophic ovary, reduced hypothalamic gonadotropin releasing hormone contents, and poor pituitary luteinizing hormone secretion. This study has demonstrated for the first time to our knowledge that accelerated puberty and late-onset hypothalamic hypogonadism are associated with chronic hyperleptinemia, thereby leading to a better understanding of the pathophysiological and therapeutic implication of leptin.

Leptin Activates Hepatic 5′-AMP-activated Protein Kinase through Sympathetic Nervous System and α1-Adrenergic Receptor A POTENTIAL MECHANISM FOR IMPROVEMENT OF FATTY LIVER IN LIPODYSTROPHY BY LEPTIN

Background: AMPK activation promotes glucose and lipid metabolism. Results: Hepatic AMPK activities were decreased in fatty liver from lipodystrophic mice, and leptin activated the hepatic AMPK via the α-adrenergic effect. Conclusion: Leptin improved the fatty liver possibly by activating hepatic AMPK through the central and sympathetic nervous systems. Significance: Hepatic AMPK plays significant roles in the pathophysiology of lipodystrophy and metabolic action of leptin. Leptin is an adipocyte-derived hormone that regulates energy homeostasis. Leptin treatment strikingly ameliorates metabolic disorders of lipodystrophy, which exhibits ectopic fat accumulation and severe insulin-resistant diabetes due to a paucity of adipose tissue. Although leptin is shown to activate 5′-AMP-activated protein kinase (AMPK) in the skeletal muscle, the effect of leptin in the liver is still unclear. We investigated the effect of leptin on hepatic AMPK and its pathophysiological relevance in A-ZIP/F-1 mice, a model of generalized lipodystrophy. Here, we demonstrated that leptin activates hepatic AMPK through the central nervous system and α-adrenergic sympathetic nerves. AMPK activities were decreased in the fatty liver of A-ZIP/F-1 mice, and leptin administration increased AMPK activities in the liver as well as in skeletal muscle with significant reduction in triglyceride content. Activation of hepatic AMPK with A769662 also led to a decrease in hepatic triglyceride content and blood glucose levels in A-ZIP/F-1 mice. These results indicate that the down-regulation of hepatic AMPK activities plays a pathophysiological role in the metabolic disturbances of lipodystrophy, and the hepatic AMPK activation is involved in the therapeutic effects of leptin.

Functional Magnetic Resonance Imaging Analysis of Food-Related Brain Activity in Patients with Lipodystrophy Undergoing Leptin Replacement Therapy

CONTEXT Lipodystrophy is a disease characterized by a paucity of adipose tissue and low circulating concentrations of adipocyte-derived leptin. Leptin-replacement therapy improves eating and metabolic disorders in patients with lipodystrophy. OBJECTIVE The aim of the study was to clarify the pathogenic mechanism of eating disorders in lipodystrophic patients and the action mechanism of leptin on appetite regulation. SUBJECTS AND INTERVENTIONS We investigated food-related neural activity using functional magnetic resonance imaging in lipodystrophic patients with or without leptin replacement therapy and in healthy controls. We also measured the subjective feelings of appetite. RESULTS Although there was little difference in the enhancement of neural activity by food stimuli between patients and controls under fasting, postprandial suppression of neural activity was insufficient in many regions of interest including amygdala, insula, nucleus accumbens, caudate, putamen, and globus pallidus in patients when compared with controls. Leptin treatment effectively suppressed postprandial neural activity in many of these regions of interest, whereas it showed little effect under fasting in patients. Consistent with these results, postprandial formation of satiety feeling was insufficient in patients when compared with controls, which was effectively reinforced by leptin treatment. CONCLUSIONS This study demonstrated the insufficiency of postprandial suppression of food-related neural activity and formation of satiety feeling in lipodystrophic patients, which was effectively restored by leptin. The findings in this study emphasize the important pathological role of leptin in eating disorders in lipodystrophy and provide a clue to understanding the action mechanism of leptin in human, which may lead to development of novel strategies for prevention and treatment of obesity.

Seipin is necessary for normal brain development and spermatogenesis in addition to adipogenesis

Seipin, encoded by BSCL2 gene, is a protein whose physiological functions remain unclear. Mutations of BSCL2 cause the most-severe form of congenital generalized lipodystrophy (CGL). BSCL2 mRNA is highly expressed in the brain and testis in addition to the adipose tissue in human, suggesting physiological roles of seipin in non-adipose tissues. Since we found BSCL2 mRNA expression pattern among organs in rat is similar to human while it is not highly expressed in mouse brain, we generated a Bscl2/seipin knockout (SKO) rat using the method with ENU (N-ethyl-N-nitrosourea) mutagenesis. SKO rats showed total lack of white adipose tissues including mechanical fat such as bone marrow and retro-orbital fats, while physiologically functional brown adipose tissue was preserved. Besides the lipodystrophic phenotypes, SKO rats showed impairment of spatial working memory with brain weight reduction and infertility with azoospermia. We confirmed reduction of brain volume and number of sperm in human patients with BSCL2 mutation. This is the first report demonstrating that seipin is necessary for normal brain development and spermatogenesis in addition to white adipose tissue development.

Amylin improves the effect of leptin on insulin sensitivity in leptin-resistant diet-induced obese mice.

Leptin enhances insulin sensitivity in addition to reducing food intake and body weight. Recently, amylin, a pancreatic β-cell-derived hormone, was shown to restore a weight-reducing effect of leptin in leptin-resistant diet-induced obesity. However, whether amylin improves the effect of leptin on insulin sensitivity in diet-induced obesity is unclear. Diet-induced obese (DIO) mice were infused with either saline (S), leptin (L; 500 μg·kg⁻¹·day⁻¹), amylin (A; 100 μg·kg⁻¹·day⁻¹), or leptin plus amylin (L/A) for 14 days using osmotic minipumps. Food intake, body weight, metabolic parameters, tissue triglyceride content, and AMP-activated protein kinase (AMPK) activity were examined. Pair-feeding and weight-matched calorie restriction experiments were performed to assess the influence of food intake and body weight reduction. Continuous L/A coadministration significantly reduced food intake, increased energy expenditure, and reduced body weight, whereas administration of L or A alone had no effects. L/A coadministration did not affect blood glucose levels during ad libitum feeding but decreased plasma insulin levels significantly (by 48%), suggesting the enhancement of insulin sensitivity. Insulin tolerance test actually showed the increased effect of insulin in L/A-treated mice. In addition, L/A coadministration significantly decreased tissue triglyceride content and increased AMPKα2 activity in skeletal muscle (by 67%). L/A coadministration enhanced insulin sensitivity more than pair-feeding and weight-matched calorie restriction. In conclusion, this study demonstrates the beneficial effect of L/A coadministration on glucose and lipid metabolism in DIO mice, indicating the possible clinical usefulness of L/A coadministration as a new antidiabetic treatment in obesity-associated diabetes.

Pathophysiogical role of leptin in lifestyle-related diseases: Studies with transgenic skinny mice overexpressing leptin

Leptin is a major adipocyte-derived hormone that is involved in the regulation of food intake and energy expenditure. Plasma leptin concentrations are elevated in obese subjects, suggesting its pathophysiological role in obesity-related lifestyle-related diseases. We have recently succeeded in the generation of transgenic skinny mice overexpressing leptin. They exhibit increased glucose metabolism and insulin sensitivity accompanied by a significant increase in insulin signaling for glucose utilization in the skeletal muscle and liver. They also show blood pressure elevation through the sympathetic activation. Introduction of the lethal yellow agouti (A(y)) allele into transgenic skinny mice results in late-onset obesity and diabetes with blood pressure elevation similar to those found in nontransgenic agouti mice (A(y)/+ mice). After caloric restriction, blood pressure elevation is reversed but insulin resistance still remains in A(y)/+ mice in parallel with a reduction of plasma leptin concentrations. By contrast, blood pressure elevation is sustained but insulin resistance is reversed in transgenic mice overexpressing leptin with the A(y) allele (Tg/+:A(y)/+ mice), which remain hyperleptinemic. Collectively, our data suggest the pathophysiologic and therapeutic implication of leptin in obesity-related insulin resistance and hypertension.

The hepatokine FGF21 is crucial for peroxisome proliferator-activated receptor-α agonist-induced amelioration of metabolic disorders in obese mice

Obesity causes excess fat accumulation in white adipose tissues (WAT) and also in other insulin-responsive organs such as the skeletal muscle, increasing the risk for insulin resistance, which can lead to obesity-related metabolic disorders. Peroxisome proliferator-activated receptor-α (PPARα) is a master regulator of fatty acid oxidation whose activator is known to improve hyperlipidemia. However, the molecular mechanisms underlying PPARα activator-mediated reduction in adiposity and improvement of metabolic disorders are largely unknown. In this study we investigated the effects of PPARα agonist (fenofibrate) on glucose metabolism dysfunction in obese mice. Fenofibrate treatment reduced adiposity and attenuated obesity-induced dysfunctions of glucose metabolism in obese mice fed a high-fat diet. However, fenofibrate treatment did not improve glucose metabolism in lipodystrophic A-Zip/F1 mice, suggesting that adipose tissue is important for the fenofibrate-mediated amelioration of glucose metabolism, although skeletal muscle actions could not be completely excluded. Moreover, we investigated the role of the hepatokine fibroblast growth factor 21 (FGF21), which regulates energy metabolism in adipose tissue. In WAT of WT mice, but not of FGF21-deficient mice, fenofibrate enhanced the expression of genes related to brown adipocyte functions, such as Ucp1, Pgc1a, and Cpt1b. Fenofibrate increased energy expenditure and attenuated obesity, whole body insulin resistance, and adipocyte dysfunctions in WAT in high-fat-diet-fed WT mice but not in FGF21-deficient mice. These findings indicate that FGF21 is crucial for the fenofibrate-mediated improvement of whole body glucose metabolism in obese mice via the amelioration of WAT dysfunctions.

Leptin restores the insulinotropic effect of exenatide in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet

Leptin may reduce pancreatic lipid deposition, which increases with progression of obesity and can impair β-cell function. The insulinotropic effect of glucagon-like peptide-1 (GLP-1) and the efficacy of GLP-1 receptor agonist are reduced associated with impaired β-cell function. In this study, we examined whether leptin could restore the efficacy of exenatide, a GLP-1 receptor agonist, in type 2 diabetes with increased adiposity. We chronically administered leptin (500 μg·kg⁻¹·day⁻¹) and/or exenatide (20 μg·kg⁻¹·day⁻¹) for 2 wk in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet (STZ/HFD mice). The STZ/HFD mice exhibited hyperglycemia, overweight, increased pancreatic triglyceride level, and reduced glucose-stimulated insulin secretion (GSIS); moreover, the insulinotropic effect of exenatide was reduced. However, leptin significantly reduced pancreatic triglyceride level, and adding leptin to exenatide (LEP/EX) remarkably enhanced GSIS. These results suggested that the leptin treatment restored the insulinotropic effect of exenatide in the mice. In addition, LEP/EX reduced food intake, body weight, and triglyceride levels in the skeletal muscle and liver, and corrected hyperglycemia to a greater extent than either monotherapy. The pair-feeding experiment indicated that the marked reduction of pancreatic triglyceride level and enhancement of GSIS by LEP/EX occurred via mechanisms other than calorie restriction. These results suggest that leptin treatment may restore the insulinotropic effect of exenatide associated with the reduction of pancreatic lipid deposition in type 2 diabetes with increased adiposity. Combination therapy with leptin and exenatide could be an effective treatment for patients with type 2 diabetes with increased adiposity.

Generation of leptin-deficient Lepmkyo/Lepmkyo rats and identification of leptin-responsive genes in the liver

Leptin is one of the key molecules in maintaining energy homeostasis. Although genetically leptin-deficient Lep(ob)/Lep(ob) mice have greatly contributed to elucidating leptin physiology, the use of more than one species can improve the accuracy of analysis results. Using the N-ethyl-N-nitrosourea mutagenesis method, we generated a leptin-deficient Lep(mkyo)/Lep(mkyo) rat that had a nonsense mutation (Q92X) in leptin gene. Lep(mkyo)/Lep(mkyo) rats showed obese phenotypes including severe fatty liver, which were comparable to Lep(ob)/Lep(ob) mice. To identify genes that respond to leptin in the liver, we performed microarray analysis with Lep(mkyo)/Lep(mkyo) rats and Lep(ob)/Lep(ob) mice. We sorted out genes whose expression levels in the liver of Lep(mkyo)/Lep(mkyo) rats were changed from wild-type (WT) rats and were reversed toward WT rats by leptin administration. In this analysis, livers were sampled for 6 h, a relatively short time after leptin administration to avoid the secondary effect of metabolic changes such as improvement of fatty liver. We did the same procedure in Lep(ob)/Lep(ob) mice and selected genes whose expression patterns were common in rat and mouse. We verified their gene expressions by real-time quantitative PCR. Finally, we identified eight genes that primarily respond to leptin in the liver commonly in rat and mouse. These genes might be important for the effect of leptin in the liver.