To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20–30% due to reduced cardiomyocyte size and had persistent expression of the fetal β-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial β-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erba␣ gene (rev-erba␣), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20 -24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 M) for 2 h reinitiates rhythmic expression of circadian clock genes in a serumindependent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.heart; metabolism; neurohumoral; rat; zeitgebers CIRCADIAN CLOCKS are controlled by a set of genes that generate self-sustained positive and negative transcriptional feedback loops with a free-running period of 24 h (7,11,40). This molecular mechanism is intrinsic to the cell, persisting in cultured cells such as fibroblasts, vascular smooth muscle cells, and various cell lines (3,13,20,23). Circadian clocks confer the selective advantage of anticipation, conditioning the cell to changes in its environment, such that it can respond rapidly to a specific extracellular signal at an appropriate time of the day. For example, our laboratory (30) has recently hypothesized that the circadian clock within cardiac and skeletal muscle allows the myocytes to anticipate diurnal variations in circulating fatty acids through modulation of fatty acid oxidative capacity at a transcriptional level. However, the existence of an intracellular circadian clock mechanism that is intrinsic to either the cardiomyocyte or...

To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20-30% due to reduced cardiomyocyte size and had persistent expression of the fetal beta-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial beta-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.

by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-␣ (PPAR␣)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR␣-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR␣ activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR␣ activity and FAO rates, repressed cardiac mte1 expression.

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity in rodents. Here, we examined the effect of resistin on glucose uptake in isolated adult mouse cardiomyocytes. Murine resistin reduced insulinstimulated glucose uptake, establishing the heart as a resistin target tissue. Notably, human resistin also impaired insulin action in mouse cardiomyocytes, providing the first evidence that human and mouse resistin homologs have similar functions. Resistin is a cysteinerich molecule that circulates as a multimer of a dimeric form dependent upon a single intermolecular disulfide bond, which, in the mouse, involves Cys 26 ; mutation of this residue to alanine (C26A) produces a monomeric molecule that appears to be bioactive in the liver. Remarkably, unlike native resistin, monomeric C26A resistin had no effect on basal or insulin-stimulated glucose uptake in mouse cardiomyocytes. Resistin impairs glucose uptake in cardiomyocytes by mechanisms that involve altered vesicle trafficking. Thus, in cardiomyocytes, both mouse and human resistins directly impair glucose transport; and in contrast to effects on the liver, these actions of resistin require oligomerization.