ABSTRACT
OBJECTIVE: Standardized, reproducible, and feasible quantification of ß-cell function (BCF) is necessary for the evaluation of interventions to improve insulin secretion and important for comparison across studies. We therefore characterized the responses to, and reproducibility of, standardized methods of in vivo BCF across different glucose tolerance states. RESEARCH DESIGN AND METHODS: Participants classified as having normal glucose tolerance (NGT; n = 23), prediabetes (PDM; n = 17), and type 2 diabetes mellitus (T2DM; n = 22) underwent two standardized mixed-meal tolerance tests (MMTT) and two standardized arginine stimulation tests (AST) in a test-retest paradigm and one frequently sampled intravenous glucose tolerance test (FSIGT). RESULTS: From the MMTT, insulin secretion in T2DM was >86% lower compared with NGT or PDM (P < 0.001). Insulin sensitivity (Si) decreased from NGT to PDM (â¼50%) to T2DM (93% lower [P < 0.001]). In the AST, insulin secretory response to arginine at basal glucose and during hyperglycemia was lower in T2DM compared with NGT and PDM (>58%; all P < 0.001). FSIGT showed decreases in both insulin secretion and Si across populations (P < 0.001), although Si did not differ significantly between PDM and T2DM populations. Reproducibility was generally good for the MMTT, with intraclass correlation coefficients (ICCs) ranging from â¼0.3 to â¼0.8 depending on population and variable. Reproducibility for the AST was very good, with ICC values >0.8 across all variables and populations. CONCLUSIONS: Standardized MMTT and AST provide reproducible and complementary measures of BCF with characteristics favorable for longitudinal interventional trials use.
Subject(s)
Arginine , Blood Glucose/metabolism , Diabetes Mellitus, Type 2/metabolism , Insulin Resistance , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Meals , Prediabetic State/metabolism , Adult , Case-Control Studies , Diabetes Mellitus, Type 2/diagnosis , Female , Glucose , Glucose Tolerance Test , Humans , Insulin Secretion , Male , Middle Aged , National Institutes of Health (U.S.) , Prediabetic State/diagnosis , Reference Standards , Reproducibility of Results , United StatesABSTRACT
FGF21 plays a central role in energy, lipid, and glucose homeostasis. To characterize the pharmacologic effects of FGF21, we administered a long-acting FGF21 analog, PF-05231023, to obese cynomolgus monkeys. PF-05231023 caused a marked decrease in food intake that led to reduced body weight. To assess the effects of PF-05231023 in humans, we conducted a placebo-controlled, multiple ascending-dose study in overweight/obese subjects with type 2 diabetes. PF-05231023 treatment resulted in a significant decrease in body weight, improved plasma lipoprotein profile, and increased adiponectin levels. Importantly, there were no significant effects of PF-05231023 on glycemic control. PF-05231023 treatment led to dose-dependent changes in multiple markers of bone formation and resorption and elevated insulin-like growth factor 1. The favorable effects of PF-05231023 on body weight support further evaluation of this molecule for the treatment of obesity. Longer studies are needed to assess potential direct effects of FGF21 on bone in humans.
Subject(s)
Anti-Obesity Agents/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Fibroblast Growth Factors/pharmacology , Obesity/drug therapy , Adolescent , Adult , Aged , Animals , Anti-Obesity Agents/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Blood Glucose , Body Weight/drug effects , Diabetes Mellitus, Type 2/blood , Drug Evaluation, Preclinical , Female , Fibroblast Growth Factors/therapeutic use , Gene Expression/drug effects , Humans , Insulin/blood , Lipid Metabolism/drug effects , Macaca fascicularis , Male , Middle Aged , Obesity/blood , Subcutaneous Fat/drug effects , Subcutaneous Fat/metabolism , Weight Loss , Young AdultABSTRACT
PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Drugs, Investigational/pharmacokinetics , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/pharmacology , Hypoglycemic Agents/pharmacokinetics , Hypolipidemic Agents/pharmacokinetics , Immunoglobulin G/chemistry , Models, Biological , Recombinant Proteins/pharmacokinetics , Amino Acid Substitution , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/blood , Antibodies, Monoclonal, Humanized/chemistry , Drug Evaluation, Preclinical , Drugs, Investigational/administration & dosage , Drugs, Investigational/analysis , Drugs, Investigational/chemistry , Fibroblast Growth Factors/administration & dosage , Fibroblast Growth Factors/blood , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Half-Life , Humans , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/blood , Hypoglycemic Agents/chemistry , Hypolipidemic Agents/administration & dosage , Hypolipidemic Agents/blood , Hypolipidemic Agents/chemistry , Immunoglobulin G/blood , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin kappa-Chains/blood , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/metabolism , Injections, Intravenous , Injections, Subcutaneous , Macaca fascicularis , Male , Metabolic Clearance Rate , Mutant Proteins/administration & dosage , Mutant Proteins/blood , Mutant Proteins/chemistry , Mutant Proteins/pharmacokinetics , Peptide Fragments/blood , Proteolysis , Rats , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Recombinant Proteins/chemistryABSTRACT
Alterations in fat metabolism, in particular elevated plasma concentrations of free fatty acids and triglycerides (TG), have been implicated in the pathogenesis of Type 2 diabetes, obesity, and cardiovascular disease. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a member of the large family of membrane-bound O-acyltransferases, catalyzes the final step in triacylglycerol formation. In the intestine, DGAT1 is one of the acyltransferases responsible for the reesterficiation of dietary TG. Following a single dose of a selective pharmacological inhibitor of DGAT1, PF-04620110, a dose-dependent inhibition of TG and vitamin A absorption postprandially was demonstrated in rodents and human subjects. In C57/BL6J mice, acute DGAT1 inhibition alters the temporal and spatial pattern of dietary lipid absorption. To understand the impact of DGAT1 inhibition on enterocyte lipid metabolism, lipomic profiling was performed in rat intestine and plasma as well as human plasma. DGAT1 inhibition causes an enrichment of polyunsaturated fatty acids within the TG class of lipids. This pharmacological intervention gives us insight as to the role of DGAT1 in human dietary lipid absorption.
Subject(s)
Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Intestinal Absorption/drug effects , Oxazepines/pharmacology , Adolescent , Adult , Animals , Case-Control Studies , Diacylglycerol O-Acyltransferase/genetics , Diacylglycerol O-Acyltransferase/metabolism , Dietary Fats/blood , Dietary Fats/metabolism , Dose-Response Relationship, Drug , Enterocytes/metabolism , Enzyme Inhibitors/pharmacokinetics , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/metabolism , Female , Humans , Intestinal Mucosa/metabolism , Lipid Metabolism/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Oxazepines/pharmacokinetics , Postprandial Period , Rats , Rats, Sprague-Dawley , Triglycerides/blood , Triglycerides/metabolism , Vitamin A/metabolismABSTRACT
Based on previous data demonstrating activation of phospholipase D (PLD) in response to angiotensin II (AngII), we have hypothesized a role for PLD in mediating aldosterone secretion from bovine adrenal glomerulosa cells. In this study we demonstrate that a PLD-generated signal(s) is required for the AngII-elicited secretory response, since interfering with lipid second messenger formation using a primary alcohol inhibited AngII-induced aldosterone secretion, but not that elicited by incubation with a hydrophilic cholesterol analog, 22(R)-hydroxycholesterol, which bypasses signaling pathways. Three mechanisms for hormonal activation of PLD have been described in other systems: direct receptor coupling, activation through protein kinase C (PKC) and a combination of these two mechanisms. Our results indicate that the PKC activator, phorbol 12-myristic 13-acetate (PMA), is able to activate PLD, and that receptor engagement is apparently not necessary for PLD activation in response to this agent. Maximal doses of AngII and PMA produced no additive effect on PLD activation, suggesting that these two agents function through a common PKC pathway. This interpretation was confirmed by the ability of a PKC inhibitor, Gö 6976, to inhibit partially AngII-induced PLD activation. Finally, treatment with the calcium ionophores A23187 or ionomycin or the calcium channel agonist BAY K8644 had no effect on PLD activity. Likewise, inhibiting calcium influx with high-dose nitrendipine affected neither basal PLD activity nor that stimulated by AngII. Thus, our results suggest a role for PKC, independent of calcium influx, in mediating AngII-induced PLD activation in glomerulosa cells.