Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Transporter Activity Changes in Nonalcoholic Steatohepatitis: Assessment with Plasma Coproporphyrin I and III.

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.

MGAT2 inhibitor decreases liver fibrosis and inflammation in murine NASH models and reduces body weight in human adults with obesity.

Monoacylglycerol acyltransferase 2 (MGAT2) is an important enzyme highly expressed in the human small intestine and liver for the regulation of triglyceride absorption and homeostasis. We report that treatment with BMS-963272, a potent and selective MGAT2 inhibitor, decreased inflammation and fibrosis in CDAHFD and STAM, two murine nonalcoholic steatohepatitis (NASH) models. In high-fat-diet-treated cynomolgus monkeys, in contrast to a selective diacylglycerol acyltransferase 1 (DGAT1) inhibitor, BMS-963272 did not cause diarrhea. In a Phase 1 multiple-dose trial of healthy human adults with obesity (NCT04116632), BMS-963272 was safe and well tolerated with no treatment discontinuations due to adverse events. Consistent with the findings in rodent models, BMS-963272 elevated plasma long-chain dicarboxylic acid, indicating robust pharmacodynamic biomarker modulation; increased gut hormones GLP-1 and PYY; and decreased body weight in human subjects. These data suggest MGAT2 inhibition is a promising therapeutic opportunity for NASH, a disease with high unmet medical needs.

Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).

Gene expression analysis in rats treated with experimental acetyl-coenzyme A carboxylase inhibitors suggests interactions with the peroxisome proliferator-activated receptor alpha pathway.

Acetyl CoA carboxylase (ACC) 2, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, has been identified as a potential target for type 2 diabetes and obesity. Small-molecule inhibitors of ACC2 would be expected to reduce de novo lipid synthesis and increase lipid oxidation. Treatment of ob/ob mice with compound A-908292 (S) ({(S)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), a small-molecule inhibitor with an IC(50) of 23 nM against ACC2, resulted in a reduction of serum glucose and triglyceride levels. However, compound A-875400 (R) ({(R)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), an inactive enantiomer of A-908292 (S) with approximately 50-fold less activity against ACC2, also caused a similar reduction in glucose and triglycerides, suggesting that the glucose-lowering effects in ob/ob mice may be mediated by other metabolic pathways independent of ACC2 inhibition. To characterize the pharmacological activity of these experimental compounds at a transcriptional level, rats were orally dosed for 3 days with either A-908292 (S) or A-875400 (R), and gene expression analysis was performed. Gene expression analysis of livers showed that treatment with A-908292 (S) or A-875400 (R) resulted in gene expression profiles highly similar to known peroxisome proliferator-activated receptor (PPAR)-alpha activators. The results suggest that, in vivo, both A-908292 (S) and A-875400 (R) stimulated the PPAR-alpha-dependent signaling pathway. These results were further supported by both an in vitro genomic evaluation using rat hepatocytes and immunohistochemical evaluation using 70-kDa peroxisomal membrane protein. Overall, the gene expression analysis suggests a plausible mechanism for the similar pharmacological findings with active and inactive enantiomers of an ACC2 inhibitor.

Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists.

G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to promote glucose-stimulated insulin secretion. Herein, we report our efforts to develop highly selective and potent GPR40 agonists with a dual mechanism of action, promoting both glucose-dependent insulin and incretin secretion. Employing strategies to increase polarity and the ratio of sp3/sp2 character of the chemotype, we identified BMS-986118 (compound 4), which showed potent and selective GPR40 agonist activity in vitro. In vivo, compound 4 demonstrated insulinotropic efficacy and GLP-1 secretory effects resulting in improved glucose control in acute animal models.

Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode.

A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes. The initial pyrrolidine hit was modified by moving the position of the carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gs-coupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors.

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC50<20 nM and >1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.

Discovery of ((4R,5S)-5-amino-4-(2,4,5- trifluorophenyl)cyclohex-1-enyl)-(3- (trifluoromethyl)-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (ABT-341), a highly potent, selective, orally efficacious, and safe dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341), a highly potent, selective, and orally bioavailable DPP4 inhibitor. When dosed orally, 25 dose-dependently reduced glucose excursion in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests and may represent a new therapeutic agent for the treatment of type 2 diabetes.

Discovery, structure-activity relationship, and pharmacological evaluation of (5-substituted-pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidines as potent dipeptidyl peptidase IV inhibitors.

A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.

Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-pyrrolidinyl]-2-oxoethyl]amino]- 4-methyl-1-piperidinyl]-4-pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of diabetes.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.

Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo.

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (K(i) = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses approximately 2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.

Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in ob/ob mice.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin receptor (IR) signal transduction and a drug target for treatment of type 2 diabetes. Using PTP1B antisense oligonucleotides (ASOs), effects of decreased PTP1B levels on insulin signaling in diabetic ob/ob mice were examined. Insulin stimulation, prior to sacrifice, resulted in no significant activation of insulin signaling pathways in livers from ob/ob mice. However, in PTP1B ASO-treated mice, in which PTP1B protein was decreased by 60% in liver, similar stimulation with insulin resulted in increased tyrosine phosphorylation of the IR and IR substrate (IRS)-1 and -2 by threefold, fourfold, and threefold, respectively. IRS-2-associated phosphatidylinositol 3-kinase activity was also increased threefold. Protein kinase B (PKB) serine phosphorylation was increased sevenfold in liver of PTP1B ASO-treated mice upon insulin stimulation, while phosphorylation of PKB substrates, glycogen synthase kinase (GSK)-3alpha and -3beta, was increased more than twofold. Peripheral insulin signaling was increased by PTP1B ASO, as evidenced by increased phosphorylation of PKB in muscle of insulin-stimulated PTP1B ASO-treated animals despite the lack of measurable effects on muscle PTP1B protein. These results indicate that reduction of PTP1B is sufficient to increase insulin-dependent metabolic signaling and improve insulin sensitivity in a diabetic animal model.

Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis.

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor gamma. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85alpha) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.

Endothelin antagonism improves hepatic insulin sensitivity associated with insulin signaling in Zucker fatty rats.

In the present study, we investigated the effects of long-term treatment with the endothelin (ET) antagonist atrasentan, an ET(A)-selective antagonist, on whole body glucose metabolism and insulin signaling in a commonly used model of insulin resistance, the Zucker fatty rat. Zucker lean and fatty rats were maintained for 6 weeks on either control or atrasentan-treated water. Euglycemic-hyperinsulinemic clamps (4 mU/kg per minute) were performed at the end of the 6-week treatment on a subset of rats (n=10/treatment). In another subset (n=5/treatment), an insulin tolerance test was performed; liver and muscle tissues were harvested 10 minutes following the challenge for further analysis. Results of the clamps demonstrated that long-term atrasentan treatment significantly increased whole body glucose metabolism in fatty rats compared with vehicle control subjects. Insulin-induced insulin receptor substrate 1 tyrosine and protein kinase B serine phosphorylation were significantly reduced in the liver and muscle of fatty animals compared with their lean littermates. This reduction was overcome with atrasentan treatment in the liver but not in the muscle. There was no difference between lean and fatty animals, however, in insulin receptor substrate 1 and protein kinase B protein expression in the liver and muscle and no effect by atrasentan. In contrast, expression of the regulatory subunit of PI-3 kinase (p85alpha) was significantly increased in the liver but not in the muscle of fatty animals compared with their lean littermates and this was normalized to levels of lean animals with atrasentan treatment. These findings indicate that long-standing ET antagonism improves whole body glucose metabolism in Zucker fatty rats through improvements in insulin signaling in the liver. These results indicate that therapeutic ET antagonism may assist in correcting the insulin-resistant state.

Metabolic responses with endothelin antagonism in a model of insulin resistance.

Atrasentan, an endothelin antagonist, would have beneficial effects on metabolic responses in a model of insulin resistance. Zucker lean or fatty rats were maintained either on regular (lean and fatty control, n = 12) or atrasentan-treated water (5 mg/kg/d, fatty atrasentan, n = 13) for 6 weeks. There was no significant difference in water intake and body weight with the atrasentan-treated group compared with fatty controls. Although atrasentan had no effect on 3-hour fasting glucose levels, it reduced fasting insulin levels between weeks 2 and 4 of treatment by 53% (fatty control vs fatty atrasentan, P < .01). Atrasentan decreased the incremental area under the plasma glucose response curve ( Delta AUC) after a nutritionally complete meal tolerance test (MTT), by 28% in the atrasentan-treated group compared with fatty controls ( P < .05), and decreased the MTT-induced insulin Delta AUC by 63% in treated animals compared with the fatty control group ( P < .01). In addition, atrasentan significantly decreased the MTT-induced glucose-insulin index Delta AUC by 58% in treated rats compared with fatty controls ( P < .01). In summary, in the Zucker fatty rat, atrasentan significantly reduces (1) 3-hour fasting insulin levels at 4 weeks, (2) glucose and insulin MTT-induced Delta AUCs, and (3) the MTT-induced glucose-insulin index Delta AUC. These results demonstrate an improvement in hyperinsulinemia as well as in glucose tolerance and insulin sensitivity with chronic endothelin antagonism in a model of insulin resistance and suggest that chronic endothelin antagonism may have benefits in the treatment of insulin resistance and/or diabetes.

Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice.

Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver-to below lean littermate levels-and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNFalpha protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38-MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNFalpha and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.

Discovery and structure-activity relationship of oxalylarylaminobenzoic acids as inhibitors of protein tyrosine phosphatase 1B.

Protein Tyrosine phosphatase 1B (PTP1B) has been implicated as a key negative regulator of both insulin and leptin signaling pathways. Using an NMR-based screening approach with 15N- and 13C-labeled PTP1B, we have identified 2,3-dimethylphenyloxalylaminobenzoic acid (1) as a general, reversible, and competitive PTPase inhibitor. Structure-based approach guided by X-ray crystallography facilitated the development of 1 into a novel series of potent and selective PTP1B inhibitors occupying both the catalytic site and a portion of the noncatalytic, second phosphotyrosine binding site. Interestingly, oral biovailability has been observed in rats for some compounds. Furthermore, we demonstrated in vivo plasma glucose lowering effects with compound 12d in ob/ob mice.

PTP1B antisense-treated mice show regulation of genes involved in lipogenesis in liver and fat.

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.

Metabolic responses in a model of insulin resistance: comparison between oral glucose and meal tolerance tests.

The purpose of this investigation was to compare the benefits of a meal tolerance test (MTT) against those of an oral glucose tolerance test (OGTT) in one of the most commonly used models of insulin resistance, the Zucker fatty rat. Comparison of these two oral challenges will facilitate determination of the most effective means of inducing both glucose and insulin responses in this particular model and allow for possible therapeutic benefits to be examined more effectively. Eight-week-old Zucker fatty rats (n = 7 or 8) were used to perform either an OGTT or a MTT following an overnight fast. The OGTT contained a final amount of carbohydrate (CHO) of 1.2 g/kg body weight (BW). The MTT (commercially available liquid meal), in addition to having fat and protein, included a final amount of available CHO and volume to match the OGTT. A saline-treated group served as control. A greater glucose excursion was observed following the OGTT compared to the MTT. The maximal change in glucose from baseline was 140 +/- 10 mg/dL (a 2.1-fold rise) for the OGTT compared to 86.3 +/- 6.1 mg/dL (a 1.7-fold rise) for the MTT (P <.05). The MTT induced a greater change from baseline in insulin response compared to the OGTT (7.5 +/- 1.1 v 3.9 +/- 0.5 ng/mL, MTT v OGTT, respectively; P <.05). The saline challenge induced only minimal glucose and insulin responses in comparison to the other treatments. These results suggest that, in a model of insulin resistance, the MTT is a more potent insulin stimulator than glucose alone. A mixed meal, such as a MTT, provides a complete nutrient challenge (CHO, fat, and protein) that will induce both glucose and insulin responses, enabling a better capacity to detect differences in one of the most often used models of insulin resistance, the Zucker fatty rat.