Role of PPARα in inflammatory response of C2C12 myotubes.

Recent studies have shown a role of inflammation in muscle atrophy and sarcopenia. However, no anti-inflammatory pharmacotherapy has been established for the treatment of sarcopenia. Here, we investigate the potential role of PPARα and its ligands on inflammatory response and PGC-1α gene expression in LPS-treated C2C12 myotubes. Knockdown of PPARα, whose expression was upregulated upon differentiation, augmented IL-6 or TNFα gene expression. Conversely, PPARα overexpression or its activation by ligands suppressed 2-h LPS-induced cytokine expression, with pemafibrate attenuating NF-κB or STAT3 phosphorylation. Of note, reduction of PGC-1α gene expression by LPS treatment for 24 hours was partially reversed by fenofibrate. Our data demonstrate a critical inhibitory role of PPARα in inflammatory response of C2C12 myotubes and suggest a future possibility of PPARα ligands as a candidate for anti-inflammatory therapy against sarcopenia.

Maturity-onset diabetes of the young type 5, presenting as diabetic ketoacidosis with alkalemia: A report of a case.

A 34-year-old man visited our Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, because of dry mouth and weight loss. His plasma glucose level was 32.8 mmol/L and serum levels of ketone bodies were increased, but with metabolic alkalemia. He was also suffering from renal tubular hypomagnesemia and hypokalemia. Abdominal computed tomography showed bilateral renal cysts. These findings were suggestive of maturity-onset diabetes of the young type 5. Genetic testing showed heterozygous hepatocyte nuclear factor 1 beta gene deletion. In the present case, it seemed reasonable to view hepatocyte nuclear factor 1 beta gene deletion as the common cause of maturity-onset diabetes of the young type 5-associated diabetic ketoacidosis and tubular malfunction-induced hypokalemic alkalosis. This case exemplifies the importance of hepatocyte nuclear factor 1 beta gene abnormality as a potential cause of diabetic ketoacidosis with alkalemia.

A mouse model of weight gain after nicotine withdrawal.

Smoking cessation increases body weight. The underlying mechanisms, however, have not been fully understood. We here report an establishment of a mouse model that exhibits an augmented body weight gain after nicotine withdrawal. High fat diet-fed mice were infused with nicotine for two weeks, and then with vehicle for another two weeks using osmotic minipumps. Body weight increased immediately after nicotine cessation and was significantly higher than that of mice continued on nicotine. Mice switched to vehicle consumed more food than nicotine-continued mice during the first week of cessation, while oxygen consumption was comparable. Elevated expression of orexigenic agouti-related peptide was observed in the hypothalamic appetite center. Pair-feeding experiment revealed that the accelerated weight gain after nicotine withdrawal is explained by enhanced energy intake. As a showcase of an efficacy of pharmacologic intervention, exendin-4 was administered and showed a potent suppression of energy intake and weight gain in mice withdrawn from nicotine. Our current model provides a unique platform for the investigation of the changes of energy regulation after smoking cessation.

Impaired leptin responsiveness in the nucleus accumbens of leptin-overexpressing transgenic mice with dysregulated sucrose and lipid preference independent of obesity.

While hypothalamic leptin resistance can occur prior to establishment of obesity, clarification is needed as to whether the impaired response to leptin in the reward-related nuclei occurs independently of obesity. To answer this question, we attempted to dissociate the normally coexisting leptin resistance from obesity. We investigated phenotypes of leptin-overexpressing transgenic mice fed for 1 week with 60 % high-fat diet (HFD) (LepTg-HFD1W mice). After 1 week, we observed that LepTg-HFD1W mice weighed as same as wild type (WT) mice fed standard chow diet (CD) for 1 week (WT-CD1W mice). However, compared to WT-CD1W mice, LepTg-HFD1W mice exhibited attenuated leptin-induced anorexia, decreased leptin-induced c-fos immunostaining in nucleus accumbens (NAc), one of important site of reward system, decreased leptin-stimulated pSTAT3 immunostaining in hypothalamus. Furthermore, neither sucrose nor lipid preference was suppressed by leptin in LepTg-HFD1W mice. On the contrary, leptin significantly suppressed both preferences in WT mice fed HFD (WT-HFD1 W mice). These results indicate that leptin responsiveness decreases in NAc independently of obesity. Additionally, in this situation, suppressive effect of leptin on the hedonic feeding results in impaired regulation. Such findings suggest the impaired leptin responsiveness in NAc partially contributes to dysregulated hedonic feeding behavior independently of obesity.

Pemafibrate, a selective PPARα modulator, and fenofibrate suppress microglial activation through distinct PPARα and SIRT1-dependent pathways.

Pemafibrate, a selective peroxisome proliferator-activated receptor (PPAR) α modulator, is a new drug that specifically modulates PPARα conformation and co-activator recruitment, thereby lowers plasma triglycerides with less off-target effects. Classical PPARα ligands such as fenofibrate suppress inflammatory cells including microglia. However, effects of pemafibrate on microglia have never been addressed. Here we show that pemafibrate, like other PPARα ligands, potently suppressed NF-κB phosphorylation and cytokine expression in microglial cells. PPARα knockdown significantly amplified LPS-induced cytokine expression. Pemafibrate-induced suppression of IL-6 expression was reversed by PPARα knockdown. However, suppression by fenofibrate was not reversed by PPARα knockdown but by Sirtuin 1 (SIRT1) knockdown. In conclusion, pemafibrate and fenofibrate similarly suppresses microglial activation but through distinct PPARα and SIRT1-dependet pathways.

A combination of dietary fat intake and nicotine exposure enhances CB1 endocannabinoid receptor expression in hypothalamic nuclei in male mice.

BACKGROUND: Cannabinoid receptor 1 (CB1R) is a GPCR expressed widely in the brain as well as in peripheral metabolic organs. Although pharmacological blockade of CB1R has been effective for the treatment of obesity and tobacco addiction, precise distribution of CB1R within the brain and potential changes by obesity or nicotine exposure have not been thoroughly addressed. METHODS: To examine CB1R distribution within the central energy center, we performed immunostaining and qPCR analysis of micro-dissected hypothalamic nuclei from male C57BL/6 mice. To address the effect of nicotine on food intake and body weight, and on potential changes of CB1R levels in the hypothalamus, mice kept on a high fat diet (HFD) for four weeks were challenged with nicotine intraperitoneally. RESULTS: Validity of the micro-dissected samples was confirmed by the expression of established nucleus-enriched genes. The expression levels of CB1R in the arcuate and lateral nuclei of the hypothalamus were higher than paraventricular and ventral-dorsal medial nuclei. Nicotine administration led to a significant suppression of food intake and body weight either under standard or high fat diet. Neither HFD nor nicotine alone altered CB1R levels in any nucleus tested. By contrast, treatment of HFD-fed mice with nicotine led to a significant increase in CB1R levels in the arcuate, paraventricular and lateral nuclei. CONCLUSIONS: CB1R was widely distributed in multiple hypothalamic nuclei. The expression of CB1R was augmented only when mice were treated with HFD and nicotine in combination. These data suggest that the exposure to nicotine may provoke an enhanced endocannabinoid response in diet-induced obesity.

Effect of Asfotase Alfa on Muscle Weakness in a Japanese Adult Patient of Hypophosphatasia with Low ALP Levels.

A 40-year-old Japanese woman presented to our hospital with general fatigue and muscle weakness. She had a history of premature loss of deciduous teeth at 4 years old, her serum alkaline phosphatase (ALP) activity was as low as 91 U/L, and radiologic studies revealed thoracic deformity and sacroiliac calcification. Genetic sequencing revealed a heterozygous c.1559delT mutation in the tissue non-specific alkaline phosphatase gene (ALPL). Based on these findings, she was diagnosed with hypophosphatasia (HPP), and treatment with asfotase alfa, a recombinant human tissue-nonspecific alkaline phosphatase (TNSALP), was initiated. After six months of treatment with asfotase alfa, improvements were observed in the SF-36 score, six-minute walk distance, and grasping power. Although the overdiagnosis needs to be avoided, HPP should be considered in patients with undiagnosed musculoskeletal symptoms and a low serum ALP activity.

Clinical characteristics of HNF1B-related disorders in a Japanese population.

BACKGROUND: Hepatocyte nuclear factor 1ß (HNF1B), located on chromosome 17q12, causes renal cysts and diabetes syndrome (RCAD). Moreover, various phenotypes related to congenital anomalies of the kidney and urinary tract (CAKUT) or Bartter-like electrolyte abnormalities can be caused by HNF1B variants. In addition, 17q12 deletion syndrome presents with multi-system disorders, as well as RCAD. As HNF1B mutations are associated with different phenotypes and genotype-phenotype relationships remain unclear, here, we extensively studied these mutations in Japan. METHODS: We performed genetic screening of RCAD, CAKUT, and Bartter-like syndrome cases. Heterozygous variants or whole-gene deletions in HNF1B were detected in 33 cases (19 and 14, respectively). All deletion cases were diagnosed as 17q12 deletion syndrome, confirmed by multiplex ligation probe amplification and/or array comparative genomic hybridization. A retrospective review of clinical data was also conducted. RESULTS: Most cases had morphological abnormalities in the renal-urinary tract system. Diabetes developed in 12 cases (38.7%). Hyperuricemia and hypomagnesemia were associated with six (19.3%) and 13 cases (41.9%), respectively. Pancreatic malformations were detected in seven cases (22.6%). Ten patients (32.3%) had liver abnormalities. Estimated glomerular filtration rates were significantly lower in the patients with heterozygous variants compared to those in patients harboring the deletion (median 37.6 vs 58.8 ml/min/1.73 m2; p = 0.0091). CONCLUSION: We present the clinical characteristics of HNF1B-related disorders. To predict renal prognosis and complications, accurate genetic diagnosis is important. Genetic testing for HNF1B mutations should be considered for patients with renal malformations, especially when associated with other organ involvement.

CRISPR/Cas9-mediated Angptl8 knockout suppresses plasma triglyceride concentrations and adiposity in rats.

Angiopoietin-like protein (ANGPTL)8 is a liver- and adipocyte-derived protein that controls plasma triglyceride (TG) levels. Most animal studies have used mouse models. Here, we generated an Angptl8 KO rat model using a clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system to clarify the roles of ANGPTL8 in glucose and lipid metabolism. Compared with WT rats, Angptl8 KO rats had lower body weight and fat content, associated with impaired lipogenesis in adipocytes; no differences existed between the groups in food intake or rectal temperature. Plasma TG levels in both the fasted and refed states were significantly lower in KO than in WT rats, and an oral fat tolerance test showed decreased plasma TG excursion in Angptl8 KO rats. Higher levels of lipase activity in the heart and greater expression of genes related to ß-oxidation in heart and skeletal muscle were observed in Angptl8 KO rats. However, there were no significant differences between KO and WT rats in glucose metabolism or the histology of pancreatic ß-cells on both standard and high-fat diets. In conclusion, we demonstrated that Angptl8 KO in rats resulted in lower body weight and plasma TG levels without affecting glucose metabolism. ANGPTL8 might be an important therapeutic target for obesity and dyslipidemia.

Development of ghrelin transgenic mice for elucidation of clinical implication of ghrelin.

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of ß-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

Role of leptin in conditioned place preference to high-fat diet in leptin-deficient ob/ob mice.

Leptin is an adipocyte-derived anorexic hormone that exerts its effects via the hypothalamus and other brain regions, including the reward system. Leptin-deficient ob/ob mice that present morbid obesity, hyperphagia, insulin resistance, and infertility are one of the most investigated mouse models of obesity. Conditioned place preference (CPP) paradigm is a standard behavioral model to evaluate the rewarding value of substrates. While leptin is reported to decrease the CPP of lean mice for high fat diet (HFD), it is unknown how CPP toward HFD is affected by leptin replacement in the pathophysiological condition of ob/ob mice. In the present study, we performed the CPP test in order to clarify the effect of leptin on the preference of ob/ob mice for HFD. Ob/ob mice had a significantly higher HFD preference in CPP test when compared with wild-type (WT) mice and this preference was suppressed to the levels comparable to the WT mice by leptin replacement with or without normalization of body weight. These results demonstrate that leptin decreases the reward value of HFD independently of obesity, suggesting that leptin reduces food intake by suppressing the hedonic feeding pathway in ob/ob mice.

Reevaluation of anti-obesity action of mazindol and elucidation of its effect on the reward system.

In this study, we evaluated the preventive effect of mazindol on the development of obesity and sought to elucidate the drug's effects on the reward system. In mice, body weight gain and hyperphagia induced by high-fat diet (HFD) were decreased by 38.6% and 13.9%, respectively, by subcutaneous infusion of mazindol (1.5mg/kg/day) for 28days. A single intraperitoneal administration of mazindol (1.5mg/kg) significantly reduced lipid preference, as assessed using the two-bottle preference paradigm (vehicle, 89.98±1.66%; mazindol, 75.65±5.47%; p<0.05). In addition, the conditioned place preference (CPP) test demonstrated that mazindol (1.5mg/kg) significantly decreased CPP score for HFD as compared with vehicle (vehicle, 330.44±58.61s; mazindol, 144.72±43.02s; p<0.05). Moreover, at the dose required for these effects, mazindol did not elicit abuse potential or induce psychostimulant-like behavior. These results confirm that mazindol prevents diet-induced obesity without addictive behavior and demonstrate that its action is mediated at least in part via the reward system, advancing our understanding of mazindol in clinical practice.

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.

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 ß-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 ß-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.

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.

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 ß-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.