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1.
Peptides ; 120: 170116, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31348991

RESUMO

Insulin-like peptide 5 (INSL5) is a member of the insulin-like family of peptides. It has been reported to be orexigenic in rodent models of obesity with impaired glucose metabolism. We attempted to confirm this property as a first step in establishing the ability of INSL5 to successfully integrate with other agents more proven in their ability to reverse obesity and improve metabolism. INSL5 was chemically synthesized by two alternative methods to a native form and one that was site-specifically conjugated to a 20 KDa polyethylene glycol (PEG) polymer. The pharmacology of each peptide was assessed by high-dose chronic administration in normal and obese mice. INSL5 failed to produce pharmacologically relevant effects on food intake, body weight or glucose control indicative of a negligible role of the peptide in the control of feeding and glucose metabolism.

2.
ACS Chem Biol ; 14(8): 1829-1835, 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31343157

RESUMO

Insulin is the principal hormone involved in the regulation of metabolism and has served a seminal role in the treatment of diabetes. Building upon advances in insulin synthetic methodology, we have developed a straightforward route to novel insulins containing a fourth disulfide bond in a [3 + 1] fashion establishing the first disulfide scan of the hormone. All the targeted analogs accommodated the constraint to demonstrate an unexpected conformational flexibility of native insulin. The bioactivity was established for the constrained (4-DS) and unconstrained (3-DS) analogs by in vitro methods, and extended to in vivo study for select peptides. We also identified residue B10 as a preferred anchor to introduce a tether that would regulate insulin bioactivity. We believe that the described [3 + 1] methodology might constitute the preferred approach for performing similar disulfide scanning in peptides that contain multiple disulfides.

3.
Cell Rep ; 27(8): 2399-2410.e6, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31116984

RESUMO

The melanocortin system is a brain circuit that influences energy balance by regulating energy intake and expenditure. In addition, the brain-melanocortin system controls adipose tissue metabolism to optimize fuel mobilization and storage. Specifically, increased brain-melanocortin signaling or negative energy balance promotes lipid mobilization by increasing sympathetic nervous system input to adipose tissue. In contrast, calorie-independent mechanisms favoring energy storage are less understood. Here, we demonstrate that reduction of brain-melanocortin signaling actively promotes fat mass gain by activating the lipogenic program and adipocyte and endothelial cell proliferation in white fat depots independently of caloric intake via efferent nerve fibers conveyed by the common hepatic branch of the vagus nerve. Those vagally regulated obesogenic signals also contribute to the fat mass gain following chronic high-fat diet feeding. These data reveal a physiological mechanism whereby the brain controls energy stores that may contribute to increased susceptibility to obesity.

4.
Trends Endocrinol Metab ; 30(4): 232-233, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30792083

RESUMO

Leptin promotes adequate caloric intake and glycemia in healthy lean individuals, harnessing the benefits of the ideal therapy against metabolic syndrome. Yet, new evidence demonstrates an unexpected causal role for leptin in obesity-associated hyperglycemia. Like the betrayal of Julius Caesar by Brutus, insulin did not see that coming from leptin.

5.
Mol Metab ; 20: 51-62, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30578168

RESUMO

OBJECTIVE: Structurally-improved GIP analogs were developed to determine precisely whether GIP receptor (GIPR) agonism or antagonism lowers body weight in obese mice. METHODS: A series of peptide-based GIP analogs, including structurally diverse agonists and a long-acting antagonist, were generated and characterized in vitro using functional assays in cell systems overexpressing human and mouse derived receptors. These analogs were characterized in vivo in DIO mice following acute dosing for effects on glycemic control, and following chronic dosing for effects on body weight and food intake. Pair-feeding studies and indirect calorimetry were used to survey the mechanism for body weight lowering. Congenital Gipr-/- and Glp1r-/- DIO mice were used to investigate the selectivity of the agonists and to ascribe the pharmacology to effects mediated by the GIPR. RESULTS: Non-acylated, Aib2 substituted analogs derived from human GIP sequence showed full in vitro potency at human GIPR and subtly reduced in vitro potency at mouse GIPR without cross-reactivity at GLP-1R. These GIPR agonists lowered acute blood glucose in wild-type and Glp1r-/- mice, and this effect was absent in Gipr-/- mice, which confirmed selectivity towards GIPR. Chronic treatment of DIO mice resulted in modest yet consistent, dose-dependent decreased body weight across many studies with diverse analogs. The mechanism for body weight lowering is due to reductions in food intake, not energy expenditure, as suggested by pair-feeding studies and indirect calorimetry assessment. The weight lowering effect was preserved in DIO Glp-1r-/- mice and absent in DIO Gipr-/- mice. The body weight lowering efficacy of GIPR agonists was enhanced with analogs that exhibit higher mouse GIPR potency, with increased frequency of administration, and with fatty-acylated peptides of extended duration of action. Additionally, a fatty-acylated, N-terminally truncated GIP analog was shown to have high in vitro antagonism potency for human and mouse GIPR without cross-reactive activity at mouse GLP-1R or mouse glucagon receptor (GcgR). This acylated antagonist sufficiently inhibited the acute effects of GIP to improve glucose tolerance in DIO mice. Chronic treatment of DIO mice with high doses of this acylated GIPR antagonist did not result in body weight change. Further, co-treatment of this acylated GIPR antagonist with liraglutide, an acylated GLP-1R agonist, to DIO mice did not result in increased body weight lowering relative to liraglutide-treated mice. Enhanced body weight lowering in DIO mice was evident however following co-treatment of long-acting selective individual agonists for GLP-1R and GIPR, consistent with previous data. CONCLUSIONS: We conclude that peptide-based GIPR agonists, not peptide-based GIPR antagonists, that are suitably optimized for receptor selectivity, cross-species activity, and duration of action consistently lower body weight in DIO mice, although with moderate efficacy relative to GLP-1R agonists. These preclinical rodent pharmacology results, in accordance with recent clinical results, provide definitive proof that systemic GIPR agonism, not antagonism, is beneficial for body weight loss.


Assuntos
Fármacos Antiobesidade/farmacologia , Polipeptídeo Inibidor Gástrico/análogos & derivados , Obesidade/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Perda de Peso/efeitos dos fármacos , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/uso terapêutico , Glicemia/análise , Dieta Hiperlipídica/efeitos adversos , Células HEK293 , Humanos , Liraglutida/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo
6.
Mol Metab ; 17: 28-38, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30170980

RESUMO

OBJECTIVE: Mice with congenital loss of the glucagon receptor gene (Gcgr-/- mice) remain normoglycemic in insulinopenic conditions, suggesting that unopposed glucagon action is the driving force for hyperglycemia in Type-1 Diabetes Mellitus (T1DM). However, chronic loss of GCGR results in a neomorphic phenotype that includes hormonal signals with hypoglycemic activity. We combined temporally-controlled GCGR deletion with pharmacological treatments to dissect the direct contribution of GCGR signaling to glucose control in a common mouse model of T1DM. METHODS: We induced experimental T1DM by injecting the beta-cell cytotoxin streptozotocin (STZ) in mice with congenital or temporally-controlled Gcgr loss-of-function using tamoxifen (TMX). RESULTS: Disruption of Gcgr expression, using either an inducible approach in adult mice or animals with congenital knockout, abolished the response to a long-acting Gcgr agonist. Mice with either developmental Gcgr disruption or inducible deletion several weeks before STZ treatment maintained normoglycemia. However, mice with inducible knockout of the Gcgr one week after the onset of STZ diabetes had only partial correction of hyperglycemia, an effect that was reversed by GLP-1 receptor blockade. Mice with Gcgr deletion for either 2 or 6 weeks had similar patterns of gene expression, although the changes were generally larger with longer GCGR knockout. CONCLUSIONS: These findings demonstrate that the effects of glucagon to mitigate diabetic hyperglycemia are not through acute signaling but require compensations that take weeks to develop.

7.
Diabetes ; 67(11): 2157-2166, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30150304

RESUMO

Glucagon receptor (GCGR) agonists cause hyperglycemia but also weight loss. However, GCG-like peptide 1 receptor (GLP1R)/GCGR mixed agonists do not exhibit the diabetogenic effects often attributed to GCGR activity. Thus, we sought to investigate the effect of glucagon agonism on insulin action and glucose homeostasis. Acute GCGR agonism induced immediate hyperglycemia, followed by improved glucose tolerance and enhanced glucose-stimulated insulin secretion. Moreover, acute GCGR agonism improved insulin tolerance in a dose-dependent manner in both lean and obese mice. Improved insulin tolerance was independent of GLP1R, FGF21, and hepatic glycogenolysis. Moreover, we observed increased glucose infusion rate, disposal, uptake, and suppressed endogenous glucose production during euglycemic clamps. Mice treated with insulin and GCGR agonist had enhanced phosphorylation of hepatic AKT at Ser473; this effect was reproduced in isolated mouse primary hepatocytes and resulted in increased AKT kinase activity. These data reveal that GCGR agonism enhances glucose tolerance, in part, by augmenting insulin action, with implications for the use of GCGR agonism in therapeutic strategies for diabetes.

8.
Cell Rep ; 23(12): 3607-3620, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29925002

RESUMO

Exposure to cold temperature is well known to upregulate heat shock protein (Hsp) expression and recruit and/or activate brown adipose tissue and beige adipocytes in humans and animals. However, whether and how Hsps regulate adipocyte function for energy homeostatic responses is poorly understood. Here, we demonstrate a critical role of Hsp20 as a negative regulator of adipocyte function. Deletion of Hsp20 enhances non-shivering thermogenesis and suppresses inflammatory responses, leading to improvement of glucose and lipid metabolism under both chow diet and high-fat diet conditions. Mechanistically, Hsp20 controls adipocyte function by interacting with the subunit of the ubiquitin ligase complex, F-box only protein 4 (FBXO4), and regulating the ubiquitin-dependent degradation of peroxisome proliferation activated receptor gamma (PPARγ). Indeed, Hsp20 deficiency mimics and enhances the pharmacological effects of the PPARγ agonist rosiglitazone. Together, our findings suggest a role of Hsp20 in mediating adipocyte function by linking ß-adrenergic signaling to PPARγ activity.

9.
Diabetes ; 67(9): 1773-1782, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29925501

RESUMO

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss, in part through potentiation of fibroblast growth factor 21 (FGF21) secretion. However, FGF21 is only a partial mediator of metabolic actions ensuing from glucagon receptor (GCGR) activation, prompting us to search for additional pathways. Intriguingly, chronic GCGR agonism increases plasma bile acid levels. We hypothesized that GCGR agonism regulates energy metabolism, at least in part, through farnesoid X receptor (FXR). To test this hypothesis, we studied whole-body and liver-specific FXR-knockout (Fxr∆liver) mice. Chronic GCGR agonist (IUB288) administration in diet-induced obese (DIO) Gcgr, Fgf21, and Fxr whole-body or liver-specific knockout (∆liver) mice failed to reduce body weight when compared with wild-type (WT) mice. IUB288 increased energy expenditure and respiration in DIO WT mice, but not Fxr∆liver mice. GCGR agonism increased [14C]palmitate oxidation in hepatocytes isolated from WT mice in a dose-dependent manner, an effect blunted in hepatocytes from Fxr∆liver mice. Our data clearly demonstrate that control of whole-body energy expenditure by GCGR agonism requires intact FXR signaling in the liver. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GCGR agonism in the therapy of metabolic disorders.


Assuntos
Fármacos Antiobesidade/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glucagon/agonistas , Adiposidade/efeitos dos fármacos , Animais , Calorimetria Indireta , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Especificidade de Órgãos , Fosforilação Oxidativa/efeitos dos fármacos , Peptídeos/uso terapêutico , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ganho de Peso/efeitos dos fármacos
10.
Mol Metab ; 13: 45-55, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29789271

RESUMO

OBJECTIVE: To signal, FGF19 and FGF21 require co-receptor ßKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. METHODS: We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. RESULTS: C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. CONCLUSIONS: We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.

11.
Endocrinology ; 159(1): 388-399, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29155981

RESUMO

Ghrelin is a 28-amino acid polypeptide that regulates feeding, glucose metabolism, and emotionality (stress, anxiety, and depression). Plasma ghrelin circulates as desacyl ghrelin (DAG) or, in an acylated form, acyl ghrelin (AG), through the actions of ghrelin O-acyltransferase (GOAT), exhibiting low or high affinity, respectively, for the growth hormone secretagogue receptor (GHSR) 1a. We investigated the role of endogenous AG, DAG, and GHSR1a signaling on anxiety and stress responses using ghrelin knockout (Ghr KO), GOAT KO, and Ghsr stop-floxed (Ghsr null) mice. Behavioral and hormonal responses were tested in the elevated plus maze and light/dark (LD) box. Mice lacking both AG and DAG (Ghr KO) increased anxiety-like behaviors across tests, whereas anxiety reactions were attenuated in DAG-treated Ghr KO mice and in mice lacking AG (GOAT KO). Notably, loss of GHSR1a (Ghsr null) did not affect anxiety-like behavior in any test. Administration of AG and DAG to Ghr KO mice with lifelong ghrelin deficiency reduced anxiety-like behavior and decreased phospho-extracellular signal-regulated kinase phosphorylation in the Edinger-Westphal nucleus in wild-type mice, a site normally expressing GHSR1a and involved in stress- and anxiety-related behavior. Collectively, our data demonstrate distinct roles for endogenous AG and DAG in regulation of anxiety responses and suggest that the behavioral impact of ghrelin may be context dependent.


Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Núcleo de Edinger-Westphal/efeitos dos fármacos , Grelina/uso terapêutico , Neurônios/efeitos dos fármacos , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Ansiedade/etiologia , Ansiedade/metabolismo , Ansiedade/patologia , Comportamento Animal/efeitos dos fármacos , Corticosterona/sangue , Núcleo de Edinger-Westphal/metabolismo , Núcleo de Edinger-Westphal/patologia , Grelina/genética , Grelina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Restrição Física/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , Estresse Psicológico/fisiopatologia
13.
Endocrinology ; 159(1): 400-413, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29077838

RESUMO

In response to an acute threat to homeostasis or well-being, the hypothalamic-pituitary-adrenocortical (HPA) axis is engaged. A major outcome of this HPA axis activation is the mobilization of stored energy, to fuel an appropriate behavioral and/or physiological response to the perceived threat. Importantly, the extent of HPA axis activity is thought to be modulated by an individual's nutritional environment. In this study, we report that nutritional manipulations signaling a relative depletion of dietary carbohydrates, thereby inducing nutritional ketosis, acutely and chronically activate the HPA axis. Male rats and mice maintained on a low-carbohydrate high-fat ketogenic diet (KD) exhibited canonical markers of chronic stress, including increased basal and stress-evoked plasma corticosterone, increased adrenal sensitivity to adrenocorticotropin hormone, increased stress-evoked c-Fos immunolabeling in the paraventricular nucleus of the hypothalamus, and thymic atrophy, an indicator of chronic glucocorticoid exposure. Moreover, acutely feeding medium-chain triglycerides (MCTs) to rapidly induce ketosis among chow-fed male rats and mice also acutely increased HPA axis activity. Lastly, and consistent with a growing literature that characterizes the hepatokine fibroblast growth factor-21 (FGF21) as both a marker of the ketotic state and as a key metabolic stress hormone, the HPA response to both KD and MCTs was significantly blunted among mice lacking FGF21. We conclude that dietary manipulations that induce ketosis lead to increased HPA axis tone, and that the hepatokine FGF21 may play an important role to facilitate this effect.


Assuntos
Dieta Cetogênica/efeitos adversos , Fatores de Crescimento de Fibroblastos/metabolismo , Sistema Hipotálamo-Hipofisário/fisiopatologia , Cetose/etiologia , Sistema Hipófise-Suprarrenal/fisiopatologia , Animais , Atrofia , Comportamento Animal , Biomarcadores/sangue , Corticosterona/sangue , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Humanos , Sistema Hipotálamo-Hipofisário/patologia , Infusões Intraventriculares , Cetose/sangue , Cetose/patologia , Cetose/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Tamanho do Órgão , Núcleo Hipotalâmico Paraventricular/metabolismo , Núcleo Hipotalâmico Paraventricular/patologia , Sistema Hipófise-Suprarrenal/patologia , Ratos Long-Evans , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Timo/patologia
14.
Cell Metab ; 26(4): 620-632.e6, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28943448

RESUMO

Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity.


Assuntos
Dexametasona/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Glucocorticoides/uso terapêutico , Incretinas/uso terapêutico , Inflamação/tratamento farmacológico , Obesidade/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Dexametasona/análogos & derivados , Metabolismo Energético/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Glucocorticoides/química , Glucose/metabolismo , Células HEK293 , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Incretinas/química , Inflamação/complicações , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/metabolismo
15.
Endocrinology ; 158(5): 1085-1094, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28200100

RESUMO

The incidence of obesity, with its impact on the development of comorbidities, including diabetes and cardiovascular disease, represents one of the greatest global health threats of the 21st century. This is particularly damning considering the vast progress that has been made in understanding the factors and molecular mechanisms playing a role in the control of energy balance by the central nervous system, especially during the past 3 decades. Despite the wealth of newfound knowledge, effective therapies for prevention of and/or intervention in obesity have not been forthcoming. That said, recent technological advances and the revisiting of previously discarded concepts have identified novel neural mechanisms involved in the control of energy homeostasis, thereby providing potential new targets and experimental approaches that may bring us closer to effective therapies to improve metabolic control. This review summarizes some of the most recent findings, with special emphasis on the role of neural circuits of the hypothalamus.


Assuntos
Peso Corporal/fisiologia , Hipotálamo/fisiologia , Vias Neurais/fisiologia , Obesidade/fisiopatologia , Animais , Metabolismo Energético , Homeostase , Humanos , Obesidade/terapia
16.
ACS Chem Biol ; 11(12): 3412-3420, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27797473

RESUMO

Peptide-based therapeutics commonly suffer from biophysical properties that compromise pharmacology and medicinal use. Structural optimization of the primary sequence is the usual route to address such challenges while trying to maintain as much native character and avoiding introduction of any foreign element that might evoke an immunological response. Glucagon serves a seminal physiological role in buffering against hypoglycemia, but its low aqueous solubility, chemical instability, and propensity to self-aggregate severely complicate its medicinal use. Selective amide bond replacement with metastable ester bonds is a preferred approach to the preparation of peptides with biophysical properties that otherwise inhibit synthesis. We have recruited such chemistry in the design and development of unique glucagon prodrugs that have physical properties suitable for medicinal use and yet rapidly convert to native hormone upon exposure to slightly alkaline pH. These prodrugs demonstrate in vitro and in vivo pharmacology when formulated in physiological buffers that are nearly identical to native hormone when solubilized in conventional dilute hydrochloric acid. This approach provides the best of both worlds, where the pro-drug delivers chemical properties supportive of aqueous formulation and the native biological properties.


Assuntos
Fármacos Gastrointestinais/química , Fármacos Gastrointestinais/farmacologia , Glucagon/química , Glucagon/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Desenho de Drogas , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Hipoglicemia/tratamento farmacológico , Hipoglicemia/metabolismo , Modelos Moleculares , Ratos , Solubilidade
17.
Cell ; 167(3): 843-857.e14, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27720451

RESUMO

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.


Assuntos
Glucagon/uso terapêutico , Doenças Metabólicas/tratamento farmacológico , Tri-Iodotironina/efeitos dos fármacos , Animais , Aterosclerose/tratamento farmacológico , Peso Corporal/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Engenharia Química/métodos , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Glucagon/efeitos adversos , Glucagon/química , Glucagon/farmacologia , Hiperglicemia/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Terapia de Alvo Molecular , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Tri-Iodotironina/efeitos adversos , Tri-Iodotironina/química , Tri-Iodotironina/farmacologia
18.
Mol Metab ; 5(10): 1015-1024, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27689014

RESUMO

OBJECTIVE: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice. METHODS: To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity. RESULTS: TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. CONCLUSIONS: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.

19.
J Med Chem ; 59(17): 8061-7, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27509198

RESUMO

Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogues of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analogue 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.


Assuntos
Alanina/análogos & derivados , Glucagon/análogos & derivados , Glucagon/química , Hipoglicemia/tratamento farmacológico , Piridinas/química , Alanina/química , Animais , AMP Cíclico/biossíntese , Estabilidade de Medicamentos , Glucagon/síntese química , Glucagon/farmacologia , Células HEK293 , Humanos , Masculino , Ratos , Técnicas de Síntese em Fase Sólida , Solubilidade , Suínos
20.
J Appl Physiol (1985) ; 121(3): 687-98, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27445299

RESUMO

Exercise is an effective therapy against the metabolic syndrome. However, the molecular pathways underlying the advantageous effects of exercise are elusive. Glucagon receptor signaling is essential for exercise benefits, and recent evidence indicates that a downstream effector of glucagon, fibroblast growth factor 21 (FGF21), is implicated in this response. Therefore, we tested the hypothesis that FGF21 action is necessary in mediating metabolic effects of exercise. We utilized acute exhaustive treadmill exercise in Wistar rats to identify a putative, concomitant increase in plasma glucagon and FGF21 with the increase in glucose and lactate following exercise. To test the necessity of FGF21 action in the exercise response, we exposed FGF21 congenitally deficient mice (Fgf21(-/-)) and their wild-type (Wt) littermates to chronic high-fat (HF) feeding and inoperable (sedentary) or operable (exercise) voluntary running wheels. Physiological tests were performed to assess the role of FGF21 in the beneficial effect of exercise on glucose metabolism. Wt and Fgf21(-/-) littermates exhibited similar running behavior, and exercise was effective in suppressing weight and fat mass gain and dyslipidemia independently of genotype. However, exercise failed to positively affect hepatic triglyceride content and glucose tolerance in HF diet-fed Fgf21(-/-) mice. Furthermore, Fgf21(-/-) mice exhibited an impaired adaptation to exercise training, including reduced AMP-activated protein kinase activity in skeletal muscle. This study demonstrates that FGF21 action is necessary to achieve the full metabolic benefits of exercise during chronic HF feeding.


Assuntos
Glicemia/metabolismo , Dieta Hiperlipídica , Terapia por Exercício/métodos , Fatores de Crescimento de Fibroblastos/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Condicionamento Físico Animal/métodos , Animais , Peso Corporal , Doença Crônica , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Wistar , Resultado do Tratamento
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