Hyperglycemia is associated with altered myocardial substrate use, a condition that has been hypothesized to contribute to impaired cardiac performance. The goals of this study were to determine whether changes in cardiac metabolism, gene expression, and function precede or follow the onset of hyperglycemia in two mouse models of obesity, insulin resistance, and diabetes (ob/ob and db/db mice). Ob/ob and db/db mice were studied at 4, 8, and 15 wk of age. Four-week-old mice of both strains were normoglycemic but hyperinsulinemic. Hyperglycemia develops in db/db mice between 4 and 8 wk of age and in ob/ob mice between 8 and 15 wk. In isolated working hearts, rates of glucose oxidation were reduced by 28-37% at 4 wk and declined no further at 15 wk in both strains. Fatty acid oxidation rates and myocardial oxygen consumption were increased in 4-wk-old mice of both strains. Fatty acid oxidation rates progressively increased in db/db mice in parallel with the earlier onset and greater duration of hyperglycemia. In vivo, cardiac catheterization revealed significantly increased left ventricular contractility and relaxation (positive and negative dP/dt) in both strains at 4 wk of age. dP/dt declined over time in db/db mice but remained elevated in ob/ob mice at 15 wk of age. Increased beta-myosin heavy chain isoform expression was present in 4-wk-old mice and persisted in 15-wk-old mice. Increased expression of peroxisomal proliferator-activated receptor-alpha regulated genes was observed only at 15 wk in both strains. These data indicate that altered myocardial substrate use and reduced myocardial efficiency are early abnormalities in the hearts of obese mice and precede the onset of hyperglycemia. Obesity per se does not cause contractile dysfunction in vivo, but loss of the hypercontractile phenotype of obesity and up-regulation of peroxisomal proliferator-activated receptor-alpha regulated genes occur later and are most pronounced in the presence of longstanding hyperglycemia.

Diabetes alters cardiac substrate metabolism. The cardiac phenotype in insulin-resistant states has not been comprehensively characterized. The goal of these studies was to determine whether the hearts of leptindeficient 8-week-old ob/ob mice were able to modulate cardiac substrate utilization in response to insulin or to changes in fatty acid delivery. Ob/ob mice were insulin resistant and glucose intolerant. Insulin signal transduction and insulin-stimulated glucose uptake were markedly impaired in ob/ob cardiomyocytes. Insulinstimulated rates of glycolysis and glucose oxidation were 1.5-and 1.8-fold higher in wild-type hearts, respectively, versus ob/ob, and glucose metabolism in ob/ob hearts was unresponsive to insulin. Increasing concentrations of palmitate from 0.4 mmol/l (low) to 1.2 mmol/l (high) led to a decline in glucose oxidation in wild-type hearts, whereas glucose oxidation remained depressed and did not change in ob/ob mouse hearts. In contrast, fatty acid utilization in ob/ob hearts was 1.5-to 2-fold greater in the absence or presence of 1 nmol/l insulin and rose with increasing palmitate concentrations. Moreover, the ability of insulin to reduce palmitate oxidation rates was blunted in the hearts of ob/ob mice. Under low-palmitate and insulin-free conditions, cardiac performance was significantly greater in wildtype hearts. However, in the presence of high palmitate and 1 nmol/l insulin, cardiac performance in ob/ob mouse hearts was relatively preserved, whereas function in wild-type mouse hearts declined substantially. Under all perfusion conditions, myocardial oxygen consumption was higher in ob/ob hearts, ranging from 30% higher in low-palmitate conditions to greater than twofold higher under high-palmitate conditions. These data indicate that although the hearts of glucose-intolerant ob/ob mice are capable of maintaining their function under conditions of increased fatty acid supply and hyperinsulinemia, they are insulin-resistant, metabolically inefficient, and unable to modulate substrate utilization in response to changes in insulin and fatty acid supply. Diabetes 53: 2366 -2374, 2004 D iabetes is associated with a switch in myocardial substrate utilization that results in increased fatty acid utilization and decreased glucose utilization (1,2). Most studies of myocardial energy metabolism in diabetes have been performed in models of insulin deficiency. Fewer studies in insulin-resistant animals with type 2 diabetes have also revealed that glucose and/or lactate oxidation rates are decreased and that palmitate oxidation rates are increased (3-5). Moreover, this metabolic profile is associated with reduced myocardial function. Most studies in insulinresistant mouse models have been performed at fatty acid concentrations that are similar to those seen in lean controls. Furthermore, some studies have been performed in the presence of added insulin, whereas others have been performed in the absence of insulin. Thus, it is possible that the experimental conditions might not reflect insulin ...

OBJECTIVE-Fatty acid-induced mitochondrial uncoupling and oxidative stress have been proposed to reduce cardiac efficiency and contribute to cardiac dysfunction in type 2 diabetes. We hypothesized that mitochondrial uncoupling may also contribute to reduced cardiac efficiency and contractile dysfunction in the type 1 diabetic Akita mouse model (Akita). RESEARCH DESIGN AND METHODS-Cardiac function andsubstrate utilization were determined in isolated working hearts and in vivo function by echocardiography. Mitochondrial function and coupling were determined in saponin-permeabilized fibers, and proton leak kinetics was determined in isolated mitochondria. Hydrogen peroxide production and aconitase activity were measured in isolated mitochondria, and total reactive oxygen species (ROS) were measured in heart homogenates. RESULTS-Resting cardiac function was normal in Akita mice, and myocardial insulin sensitivity was preserved. Although Akita hearts oxidized more fatty acids, myocardial O 2 consumption was not increased, and cardiac efficiency was not reduced. ADP-stimulated mitochondrial oxygen consumption and ATP synthesis were decreased, and mitochondria showed grossly abnormal morphology in Akita. There was no evidence of oxidative stress, and despite a twofold increase in uncoupling protein 3 (UCP3) content, ATP-to-O ratios and proton leak kinetics were unchanged, even after perfusion of Akita hearts with 1 mmol/l palmitate.

The Arabidopsis wall-associated receptor kinase, Wak1, is a member of the Wak family (Wak1-5) that links the plasma membrane to the extracellular matrix. By the yeast two-hybrid screen, we found that a glycinerich extracellular protein, AtGRP-3, binds to the extracellular domain of Wak1. Further in vitro binding studies indicated that AtGRP-3 is the only isoform among the six tested AtGRPs that specifically interacts with Waks, and the cysteine-rich carboxyl terminus of AtGRP-3 is essential for its binding to Wak1. We also show that Wak1 and AtGRP-3 form a complex with a molecular size of ϳ500 kDa in vivo in conjunction with the kinaseassociated protein phosphatase, KAPP, that has been shown to interact with a number of plant receptor-like kinases. Binding of AtGRP-3 to Wak1 is shown to be crucial for the integrity of the complex. Wak1 and At-GRP-3 are both induced by salicylic acid treatment. Moreover, exogenously added AtGRP-3 up-regulates the expression of Wak1, AtGRP-3, and PR-1 (for pathogenesis-related) in protoplasts. Taken together, our data suggest that AtGRP-3 regulates Wak1 function through binding to the cell wall domain of Wak1 and that the interaction of Wak1 with AtGRP-3 occurs in a pathogenesis-related process in planta.

The mSin3A corepressor complex contains 7 to 10 tightly associated polypeptides and is utilized by many transcriptional repressors. Much of the corepressor function of mSin3A derives from associations with the histone deacetylases HDAC1 and HDAC2; however, the contributions of the other mSin3A-associated polypeptides remain largely unknown. We have purified an mSin3A complex from K562 erythroleukemia cells and identified three new mSin3A-associated proteins (SAP): SAP180, SAP130, and SAP45. SAP180 is 40% identical to a previously identified mSin3A-associated protein, RBP1. SAP45 is identical to mSDS3, the human ortholog of the SDS3p component of the Saccharomyces cerevisiae Sin3p-Rpd3p corepressor complex. SAP130 does not have detectable homology to other proteins. Coimmunoprecipitation and gel filtration data suggest that the new SAPs are, at the very least, components of the same mSin3A complex. Each new SAP repressed transcription when tethered to DNA. Furthermore, repression correlated with mSin3A binding, suggesting that the new SAPs are components of functional mSin3A corepressor complexes. SAP180 has two repression domains: a C-terminal domain, which interacts with the mSin3A-HDAC complex, and an N-terminal domain, which functions independently of mSin3A-HDAC. SAP130 has a repression domain at its C terminus that interacts with the mSin3A-HDAC complex and an N-terminal domain that probably mediates an interaction with a transcriptional activator. Together, our data suggest that these novel SAPs function in the assembly and/or enzymatic activity of the mSin3A complex or in mediating interactions between the mSin3A complex and other regulatory complexes. Finally, all three SAPs bind to the HDAC-interaction domain (HID) of mSin3A, suggesting that the HID functions as the assembly interface for the mSin3A corepressor complex.