OXPAT/PAT-1 is a PPAR-Induced Lipid Droplet Protein that Promotes Fatty Acid Utilization

Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as OXPAT. Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARα overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARγ agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes

Exenatide Improves Glucose Homeostasis and Prolongs Survival in a Murine Model of Dilated Cardiomyopathy

There is growing awareness of secondary insulin resistance and alterations in myocardial glucose utilization in congestive heart failure. Whether therapies that directly target these changes would be beneficial is unclear. We previously demonstrated that acute blockade of the insulin responsive facilitative glucose transporter GLUT4 precipitates acute decompensated heart failure in mice with advanced dilated cardiomyopathy. Our current objective was to determine whether pharmacologic enhancement of insulin sensitivity and myocardial glucose uptake preserves cardiac function and survival in the setting of primary heart failure.The GLP-1 agonist exenatide was administered twice daily to a murine model of dilated cardiomyopathy (TG9) starting at 56 days of life. TG9 mice develop congestive heart failure and secondary insulin resistance in a highly predictable manner with death by 12 weeks of age. Glucose homeostasis was assessed by measuring glucose tolerance at 8 and 10 weeks and tissue 2-deoxyglucose uptake at 75 days. Exenatide treatment improved glucose tolerance, myocardial GLUT4 expression and 2-deoxyglucose uptake, cardiac contractility, and survival over control vehicle-treated TG9 mice. Phosphorylation of AMP kinase and AKT was also increased in exenatide-treated animals. Total myocardial GLUT1 levels were not different between groups. Exenatide also abrogated the detrimental effect of the GLUT4 antagonist ritonavir on survival in TG9 mice.In heart failure secondary insulin resistance is maladaptive and myocardial glucose uptake is suboptimal. An incretin-based therapy, which addresses these changes, appears beneficial

Western blot analysis and protein quantification of left ventricular myocardium harvested from 70-day-old TG9 mice.

<p><b>A</b>. GLUT4 <b>B</b>. GLUT1 <b>C</b>. pan-AKT and phospho-AKT. <b>D</b>. AMPKα and phospho-AMPK. For each protein, the top panel represents expression as determined using antibody recognizing the protein of interest. Bottom panel: Data is represented as the mean protein intensity normalized to GAPDH from 4–6 independent mice. Each western blot was performed in triplicate. Values are expressed as the mean ± SEM; * indicates p<0.03, student's t-test.</p

Insulin levels were obtained on age matched female TG9 mice at 10 weeks following a 5 hour fast at 0, 15 and 30 minutes after an intraperitoneal glucose dose (1 g/kg).

<p>Data shown as the mean ± SEM (n = 4 per group). *, p<0.05 compared to 0 minute insulin level.</p

Intraperitoneal glucose tolerance tests (1 g/kg) on age-matched female TG9 mice following a 5 hour fast.

<p><b>A</b>. Baseline responses at 8 weeks of age. <b>B</b>. Following 2 weeks of treatment with either vehicle or exenatide (Age 10 weeks). Data is shown as the mean ± SEM (n = 4-6). *p<0.01, ANOVA.</p

Echocardiography of TG9 mice.

<p>Transthoracic echocardiography performed on 70 day old TG9 mice. Exenatide (or vehicle) was administered subcutaneously at a dose of 40 µg/kg/day (divided b.i.d.) starting at 56 days of age. Hypothermic animals were warmed to 36°C immediately prior to obtaining echocardiograms. LVPWd, left ventricular posterior wall diastolic dimension. LVIDs, left ventricular internal diameter in systole. LVIDd, left ventricular internal diameter in diastole. Data is shown as the average +/− SEM.</p

Kaplan-Meier survival curves of female TG9 mice treated with 40 µg/kg/day of exenatide subcutaneously (divided b.i.d.) or vehicle beginning at 56 days of life and continuing until the time of death.

<p>Panel A: Exenatide-treated mice (n = 9) versus vehicle (n = 6), p-value <0.01. Panel B: Exenatide and vehicle treated mice given ritonavir daily by intraperitoneal injection (10 mg/kg) beginning at 75 days of age and continuing until the time of death (n = 7 and 12 respectively per group).</p

RT- PCR for brain naturetic peptide measurement in the TG9 left ventricular tissue harvested at 70 days of life.

<p>Mice were treated with exenatide or vehicle (40 µg/kg/day divided b.i.d.) starting at 56 days. n = 4 per group, p-value  = 0.02.</p