A Volumetric Method for Quantifying Atherosclerosis in Mice by Using MicroCT: Comparison to En Face

Precise quantification of atherosclerotic plaque in preclinical models of atherosclerosis requires the volumetric assessment of the lesion(s) while maintaining in situ architecture. Here we use micro-computed tomography (microCT) to detect ex vivo aortic plaque established in three dyslipidemic mouse models of atherosclerosis. All three models lack the low-density lipoprotein receptor (Ldlr−/−), each differing in plaque severity, allowing the evaluation of different plaque volumes using microCT technology. From clearly identified lesions in the thoracic aorta from each model, we were able to determine plaque volume (0.04–3.1 mm3), intimal surface area (0.5–30 mm2), and maximum plaque (intimal-medial) thickness (0.1–0.7 mm). Further, quantification of aortic volume allowed calculation of vessel occlusion by the plaque. To validate microCT for future preclinical studies, we compared microCT data to intimal surface area (by using en face methodology). Both plaque surface area and plaque volume were in excellent correlation between microCT assessment and en face surface area (r2 = 0.99, p<0.0001 and r2 = 0.95, p<0.0001, respectively). MicroCT also identified internal characteristics of the lipid core and fibrous cap, which were confirmed pathologically as Stary type III-V lesions. These data validate the use of microCT technology to provide a more exact empirical measure of ex vivo plaque volume throughout the entire intact aorta in situ for the quantification of atherosclerosis in preclinical models

FGF21 Promotes Metabolic Homeostasis via White Adipose and Leptin in Mice

<div><p>Fibroblast growth factor 21 (FGF21) is a potent metabolic regulator, and pharmacological administration elicits glucose and lipid lowering responses in mammals. To delineate if adipose tissue is the predominant organ responsible for anti<b>-</b>diabetic effects of FGF21, we treated mice with reduced body fat (lipodystrophy mice with adipose specific expression of active sterol regulatory element binding protein 1c; Tg) with recombinant murine FGF21 (rmuFGF21). Unlike wildtype (WT) mice, Tg mice were refractory to the beneficial effects of rmuFGF21 on body weight, adipose mass, plasma insulin and glucose tolerance. To determine if adipose mass was critical for these effects, we transplanted WT white adipose tissue (WAT) into Tg mice and treated the mice with rmuFGF21. After transplantation, FGF21 responsiveness was completely restored in WAT transplanted Tg mice compared to sham Tg mice. Further, leptin treatment alone was sufficient to restore the anti-diabetic effects of rmuFGF21 in Tg mice. Molecular analyses of Tg mice revealed normal adipose expression of <em>Fgfr1</em>, <em>Klb</em> and an 8-fold over-expression of <em>Fgf21</em>. Impaired FGF21-induced signaling indicated that residual adipose tissue of Tg mice was resistant to FGF21, whilst normal FGF21 signaling was observed in Tg livers. Together these data suggest that adipose tissue is required for the triglyceride and glucose, but not the cholesterol lowering efficacy of FGF21, and that leptin and FGF21 exert additive anti-diabetic effects in Tg mice.</p> </div

Lipodystrophy mice are resistant to anti-diabetic effects of recombinant FGF21 treatment.

<p>Recombinant muFGF21 (1 or 10 mg/kg, pink and red symbols respectively) or vehicle (green symbols) was injected BID in wildtype (WT - open bars) and aP2-nSrebp1c lipodystrophy (Tg - hatched bars) male mice as described in Animal Study Design I (Methodology). (A–B) Body weight was monitored daily. Dramatic drop in body weight on day 17 was caused by the 12 h fast for GTT. (C–D) Adipose and lean mass was recorded in WT and Tg mice at 0, 7 and 14 days of treatment. (E–F) Fed blood glucose (OneTouch Basic glucometer) and plasma insulin were measured in WT and Tg mice at the start (PRE) and the end (POST) of the treatment. (G–H) Glucose tolerance was measured on day 18 of treatment (OneTouch Basic glucometer). (I) Area under the curve (AUC) was calculated in WT and Tg mice. (J–L) Plasma lipids (cholesterol, triglycerides, NEFA – non-esterified fatty acids) were measured in WT and Tg mice at the start (PRE) and the end (POST) of the treatment. WT groups N = 8; Tg vehicle group N = 8; Tg FGF21 treatment group N = 7. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 <i>vs.</i> vehicle-treated genotype-matched mice at same time point, ns – not statistically significant. ++ <i>P</i><0.01, +++ <i>P</i><0.001 <i>vs.</i> WT mice (T-Test).</p

Adipose tissue transplantation restores anti-diabetic effects of recombinant FGF21 treatment in lipodystrophy mice.

<p>Approximately 2 g of wildtype (WT) adipose tissue was transplanted subcutaneously into aP2-nSrebp1c lipodystrophy (Tg) male mice (Animal Study Design III – Methods). Following a 2-week recovery period, WT sham (green bars/symbols), Tg sham (blue bars/symbols) and Tg transplanted (red bars/symbols) mice were treated with vehicle (open bars/solid line) or 10 mg/kg rmuFGF21 (hatched bars/dashed line) BID for 21 days. (A) Adipose mass was measured in WT and Tg mice before (−3 days), immediately after (1 day) and 7, 14, 21 and 28 days following adipose tissue transplantation. Adipose tissue mass was increased in the Tg transplanted mice after implantation and although declined by MRI analysis was still intact at necropsy. Fourteen days following adipose tissue transplantation, mice were treated with vehicle or rmuFGF21. (B) Plasma leptin was measured prior to, and at necropsy. (C–E) Percent change in body weight was calculated daily from the start of rmuFGF21 treatment. Days 17–21 were excluded due to dramatic weight changes following 12 hr fast for the GTT. (F–H) Glucose tolerance was measured after 18 days of rmuFGF21 treatment (AlphaTRAK glucose monitor). (I–J) Insulin and adiponectin levels were measured 3 days before adipose tissue transplantation (Pre-transplant), 14 days following sham or implantation surgery (Post-transplant Pre-treatment) and 32 days following surgery including 14 days of rmuFGF21 or vehicle injections (Post-treatment). (K–L) Plasma lipids (cholesterol and triglycerides) were measured in the same samples described in (I). N = 6–7 group. * <i>P</i><0.05, ** <i>P</i><0.01, ** <i>P</i><0.001 <i>vs.</i> vehicle-treated genotype and surgery-matched mice at same time point;. + P<0.05, ++ P<0.01, +++ P<0.001 <i>vs.</i> comparator groups as indicated. # P<0.05. <i>vs.</i> the same mice Post-transplant Pre-treatment (all analysis by T-Test); ns – not statistically significant.</p

Impaired FGF21 signaling in adipose tissue from lipodystrophy mice.

<p>Wildtype (WT) and aP2-nSrebp1c lipodystrophy (Tg) male mice were treated with vehicle (−) or a single rhuFGF21 (+) injection at 1 mg/kg (Animal Study Design II – Methodology). White adipose tissue (WAT) samples were subjected to immunoblot analysis with antibodies directed against (A) ERK (extracellular signal-regulated kinase), (B) FRS2 (Fibroblast growth factor receptor substrate (C) SHP2 (Src homology domain 2 containing protein tyrosine phosphatase), 2), (D) ERK signaling was analyzed in liver samples. Antibodies directed against phosphorylated (p-) or total (T-) proteins of specific resides are noted alongside each panel.</p

Small Molecule Disruptors of the Glucokinase–Glucokinase Regulatory Protein Interaction: 3. Structure–Activity Relationships within the Aryl Carbinol Region of the <i>N</i>‑Arylsulfonamido‑<i>N</i>′‑arylpiperazine Series

We have recently reported a novel approach to increase cytosolic glucokinase (GK) levels through the binding of a small molecule to its endogenous inhibitor, glucokinase regulatory protein (GKRP). These initial investigations culminated in the identification of 2-(4-((2<i>S</i>)-4-((6-amino-3-pyridinyl)­sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)­phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (<b>1</b>, AMG-3969), a compound that effectively enhanced GK translocation and reduced blood glucose levels in diabetic animals. Herein we report the results of our expanded SAR investigations that focused on modifications to the aryl carbinol group of this series. Guided by the X-ray cocrystal structure of compound <b>1</b> bound to hGKRP, we identified several potent GK–GKRP disruptors bearing a diverse set of functionalities in the aryl carbinol region. Among them, sulfoximine and pyridinyl derivatives <b>24</b> and <b>29</b> possessed excellent potency as well as favorable PK properties. When dosed orally in <i>db</i>/<i>db</i> mice, both compounds significantly lowered fed blood glucose levels (up to 58%)

Small Molecule Disruptors of the Glucokinase–Glucokinase Regulatory Protein Interaction: 1. Discovery of a Novel Tool Compound for in Vivo Proof-of-Concept

Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide <b>25</b> (AMG-1694). When dosed in ZDF rats, <b>25</b> showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats